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EFFECT OF DIETARY SUPPLEMENTATION
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Juliana Perez Laspiur

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EFFECT OF DIETARY SUPPLEMENTATION OF L-ARGININE TO LACTATING
SOWS UNDER HEAT STRESS

By

Juliana Pérez Laspiur

AN ABSTRACT OF A THESIS
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
MASTER IN SCIENCE
Department of Animal Science
2001

Professor Nathalie L. Trottier

ABSTRACT

EFFECT OF DIETARY SUPPLEMENTATION OF L—ARGININE TO LACTATING
SOWS UNDER HEAT STRESS

By
Juliana Pérez Laspiur

The objective of this study was to determine if dietary L-arginine supplementation
improves lactation performance of sows exposed to heat stress. Sixty-six multiparous
lactating sows were arranged in a 2 x 3 factorial design with two environments, i.e.,
thermoneutral (20 °C) and hot (29.4 °C) and three dietary treatments, i.e., control (0.96 %
arg), medium (1.34 % arg), and high (1.73 % arg). Heat exposure increased respiration
rate, rectal temperature, but decreased heart rate of the sow. Sow feed intake was
decreased, weight loss was increased and litter weight gain was depressed in sows
exposed to heat. Heat exposure increased plasma glucose and growth hormone, and did
not affect insulin, cortisol, or prolactin concentration. Arginine supplementation
decreased weight loss, increased insulin, and decreased growth hormone concentration in
heat stressed sows. Litter growth rate was not improved by arginine supplementation.
Heat exposure decreased plasma and milk amino acid concentration compared to
thermoneutral exposure but did not increase nitric oxide production. Nitric oxide
production was not increased with arginine supplementation. Proline concentration in
milk was increased with arginine supplementation. Nursing behavior was not altered

during heat stress.

To

Marni y Papi who have dedicated their life to mine

iii

ACKNOWLEDGEMENTS

First, I would like to acknowledge Nutri Quest, Inc. for providing partial funding
for this project. I would like to acknowledge my mentor, Dr. Nathalie L. Trottier, for her
continuous support and encouragement, patience, and good heart. I would also like to
acknowledge Dr. Adroaldo J. Zanella for his enthusiasm in our discussions and with this
project. To Dr. Barbara Straw, Dr. Dale Rozeboom, and Dr. Mike Orth, my committee
members for the guidance and advice. I would like to thank Dr. Pao Ku for his great
ideas in the laboratory and his great attitude in life. To Dr. Chantal Farmer for her input
on hormone metabolism and sample analysis. To Dr. Gretchen Hill and Dr. Melvin
Yokoyama for their help and support from the beginning. I would also like to
acknowledge Dr. Robert Tempelman, for his willingness to answer all my questions
anytime. To Dr. Kent Ames, for his great help in caring for sick animals. I would like to
give a special thank you to Barb Sweeney, for her smile everyday, her patience, and her
guidance. To Allan Snedegar, for teaching me in his own special way, great things about
pigs, life, and also for his charm. To Brian Story and the MSU Swine Farm staff, for all
their help and assistance with the sows and the projects. To Donna Barnes for being such
a hard worker and for her positive attitude. To Marta M. Pintos, Marcelo Pérez Laspiur,
Marcos Pérez Laspiur, you are my roots. To my friends here, and for those back home,

for all the good times and the great times.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... vii
LIST OF FIGURES ................................................................................ viii
CHAPTER 1
1. Introduction ............................................................................. l
2. Heat stress .............................................................................. 2
2.1 Introduction .................................................................. 2
2.2 Physiological adaptations to heat stress .................................. 2
2.3 Lactation performance ...................................................... 4
2.3.1 Basal metabolism ................................................. 5
2.3.2 Mammary blood ﬂow ............................................ 6
2.3.3 Hormonal and nutrient regulation .............................. 7
2.4 Sow and piglet behavior ................................................... 13
3. Role of arginine in nutrition and metabolism ..................................... 16
3.1 Introduction .................................................................. 16
3.2 Arginine metabolism ........................................................ 16
3.3 Arginine and mammary blood ﬂow ....................................... 17
3.4 Arginine and hormonal regulation ......................................... 19
3.4.1 Amino acids ....................................................... 19
3.4.2 Insulin and glucose ............................................... 19
3.4.3 Growth hormone .................................................. 20
3.4.4 Prolactin ........................................................... 23
3.5 Heat stress ..................................................................... 23
3.6 Effects of arginine on feed efﬁciency and performance ................ 24

CHAPTER 2: EFFECT OF DIETARY L-ARGININE SUPPLEMENTATION AND
ENVIRONMENTAL TEMPERATURE ON SOW LACTATION PERFORMANCE

Abstract ..................................................................................... 38
Introduction ................................................................................. 40
Materials and methods .................................................................... 41
Results ....................................................................................... 42
Discussion .................................................................................. 44
Implications ................................................................................. 47
Literature cited ............................................................................. 54

CHAPTER 3: INSULIN, GLUCOSE, CORTISOL, GROWTH HORMONE AND
PROLACTIN RESPONSES TO ORAL L-ARGININE SUPPLEMENTATION TO
LACTATING SOWS UNDER HEAT STRESS

Abstract ..................................................................................... 57
Introduction ................................................................................. 58
Materials and methods .................................................................... 59
Results and discussion .................................................................. 63
Implications ................................................................................. 69
Literature cited ............................................................................. 87

CHAPTER 4: SUPPLEMENTAL L-ARGININE IN DIETS OF LACTATING SOWS
UNDER HEAT STRESS: EFFECT ON PLASMA NITRIC OXIDE
CONCENTRATION AND MILK AMINO ACID CONCENTRATION

Abstract ..................................................................................... 93
Introduction ................................................................................. 94
Materials and methods ..................................................................... 95
Results and discussion ..................................................................... 98
Implications ............................................................................... 103
Literature cited ............................................................................ 110

CHAPTER 5: EFFECT OF HEAT STRESS ON SOW NURSING BEHAVIOR AND
CONSEQUENT LITTER PERFORMANCE

Abstract .................................................................................... 113
Introduction ................................................................................ 1 14
Materials and methods ................................................................... 117
Results ...................................................................................... 119
Discussion ................................................................................. 122
Implications ................................................................................ 126
Literature cited ............................................................................ 135
SUMMARY AND CONCLUSIONS ........................................................... 138

vi

LIST OF TABLES
Table 2.1 Ingredient composition of experimental diets on as fed basis .................... 48
Table 2.2 Nutrient composition of experimental diets on as fed basis ...................... 49

Table 2.3.1 Effect of environmental temperature and dietary arginine supplementation on
sow lactation performance ........................................................................ 50

Table 2.3.2 Effect of environmental temperature and dietary arginine supplementation on
sow lactation performance ........................................................................ 51

Table 3.1 Effect of L-arginine supplementation on metabolic hormones and glucose
concentration ........................................................................................ 71

Table 3.2 Effect of L-arginine supplementation on metabolic hormones ................... 72

Table 4.1 Effect of environmental temperature and arginine supplementation on plasma
indispensable amino acid concentration ........................................................ 104

Table 4.2 Effect of environmental temperature and arginine supplementation on plasma
dispensable amino acid concentration .......................................................... 105

Table 4.3 Effect of environmental temperature and arginine supplementation on milk
indispensable amino acid concentration ........................................................ 106

Table 4.4 Effect of environmental temperature and arginine supplementation on milk
dispensable amino acid concentration .......................................................... 107

vii

LIST OF FIGURES

Figure 2.1 Effect of environmental temperature and dietary arginine supplementation on
sow weight change during a 21-d lactation period ............................................. 53

Figure 3.1 Effect of environmental temperature on sow body weight loss during a 21-day
lactation period ..................................................................................... 74

Figure 3.2 Effect of environmental temperature on litter average daily gain during a 21-
day lactation period ................................................................................. 76

Figure 3.3 Effect of environmental temperature and arginine supplementation on plasma
insulin concentration ................................................................................ 78

Figure 3.4 Effect of environmental temperature and arginine supplementation on plasma
glucose concentration ............................................................................... 80

Figure 3.5 Effect of environmental temperature and arginine supplementation on salivary
cortisol concentration ............................................................................... 82

Figure 3.6 Effect of environmental temperature and arginine supplementation on plasma
growth hormone concentration .................................................................... 84

Figure 3.7 Effect of environmental temperature and arginine supplementation on plasma
prolactin concentration ............................................................................. 86

Figure 4.1 Effect of environmental temperature and arginine supplementation on plasma
nitric oxide concentration ......................................................................... 109

Figure 5.1 Overall frequency (%) of SS and US is sows exposed to 20° C (TN) or 29.4° C
(HT) .................................................................................................. 128

Figure 5.2 Overall frequency (%) of behavior of litter (80 % of pigs) before a nursing
event by sows exposed to 20° C (TN) or 29.4° C (HT) ..................................... 130

Figure 5.3 Overall frequency (%) of behavior of litter (80 % of pigs) after a nursing event
by sows exposed to 20° C (TN) or 29.4° C (HT) .............................................. 132

Figure 5.4 Overall frequency (%) of behavior of sows exposed to 20° C (TN) or 29.4° C
(HT) after a nursing event ........................................................................ 134

viii

CHAPTER 1: LITERATURE REVIEW
1. Introduction

Lactating sows exposed to high ambient temperatures suffer from heat stress.

' Heat stress depresses milk production and impairs sow performance. Depressed milk
production impacts the nursing pig’s ability to grow, potentially prolonging the time for a
pig to reach market weight. The impact on the sow is also important because depressed
feed intake and increased weight loss during lactation extends the weaning to estrus
interval. Furthermore, subsequent lactation performances may be affected. The
underlying physiological mechanisms involved in lactation performance depression
during heat stress are very poorly understood. Further understanding of these
mechanisms is needed to design managerial and nutritional strategies to improve lactation
performance under hot climatic conditions.

In this thesis, the role of dietary arginine in improving lactation performances of
heat stressed sows is examined. Four questions are addressed. First, does heat stress
translate into decreased performance and does arginine supplementation improves sow
body condition and litter growth rate. Second, does heat stress modulates key metabolic
hormones and metabolites and does arginine supplementation ameliorates the effect of
heat stress on these hormones. Third, does heat exposure decreases amino acid
concentrations in plasma and milk and does arginine supplementation increases nitric

oxide production. Fourth, is the sow’s nursing behavior altered during heat stress.

2. Heat stress
2.]. Introduction

Heat stress is a signiﬁcant economic factor for the swine industry. In sows
exposed to high ambient temperatures, piglet growth rate is decreased from 10 to 35 %
'(Schoenherr et al., 1989; Vidal et al., 1991). The sow’s ability to cope with heat stress is
diminished due to both physiological limitations and managerial constraints. In this
section, the physiological mechanisms involved in adaptation to heat stress and their
effects on milk production will be discussed.

2.2. Physiological adaptations to heat stress

Heat stress is deﬁned as the physiological state by which an animal responds to
high ambient temperatures (review; Hill and Wyse, 1989). Heat stress occurs when the
ambient temperature exceeds the evaporative critical limit, i.e., when the rate of heat
production exceeds the rate of heat dissipation. The evaporative critical limit in the sow
is 22° C and is 37° C in the nursing pig (Pond and Maner, 1974; Black et al., 1993). Basal
metabolic rate (BMR) is constant throughout the thermal comfort zone and increases as
temperature surpasses either limit. The increase in BMR results ﬁ'om an increase in
physiological work required to either conserve or dissipate heat, and thus to maintain a
normal body temperature (review; Hill and Wyse, 1989). The rate of metabolic heat
production must equal the rate of heat loss in order to maintain a normal body
temperature (review; Hill and Wyse, 1989). The normal body temperature is
approximately 383°C for nursing pigs and 397°C for sows.

Heat dissipation in vertebrate animals occurs via evaporative (sweating and panting),

conductive (contact with object), convective (contact with air), and radiation loss (review;

Hill and Wyse, 1989). Evaporative heat loss is minimal in the pig due to the lack of
sweat glands (Srichana et al., 1996). Additionally, the ability of the pig to dissipate heat
through convection is limited by a thick subcutaneous fat layer. Thus, behavioral
therrnoregulation is an important mechanism to dissipate heat in the pig (Bull et al., 1997).
For instance, the pig will instinctively wallow in mud and in its own urine and feces to cool
itself (Pond and Houpt, 1978).

When the rate of metabolic heat production exceeds the rate of heat dissipation,
the animal adjusts the rate of heat production by controlling physical activities such as
eating and exercise (Black et al., 1993). Physiological indicators of heat stress are similar
among species. However, the intensity of the physiological response differs with
duration of exposure to high temperatures and the physiological state of the animal. The
lactating sow physiologically adapts to heat stress by decreasing feed intake and increasing
respiration rate (Schoenberr et al., 1989; Barb et al., 1991; Black et al., 1993; Prunier et al.,
1997; Messias de Braganca et al., 1998; Johnston et al., 1999; Quiniou and Noblet, 1999)
and heart rate (Black et al., 1993). Similar physiological adaptations to heat stress have
been documented in other species. The lactating cow under heat stress experiences
increased body temperature, respiration rate and decreased feed intake and heart rate (Itoh et
al., 1998b). Non-lactating cows under heat stress experience increases in respiration rate
and heart rate (Itoh et al., 1998a). Takahashi et a1. (1986) reported an increased body
temperature, respiration rate and heart rate in calves. In heifers, body temperature increases
while feed intake decreases (Ronchi et al., 2001). Non-pregnant, pregnant and lactating
ewes experience increased rectal temperature, respiration rate, and heart rate while feed

intake decreases (Hooley et al., 1979; Dreiling et al., 1991; Abdalla et al., 1993). Rectal

temperature and respiration rate has been reported to increase in sheep exposed to heat stress
(Achmadi et al., 1993). In pregnant and lactating rabbits, body temperature, respiration rate,
and heart rate increase (Lublin and Wolfenson, 1996). Several cooling systems have been
evaluated in an attempt to ameliorate heat stress in sows exposed to high ambient
temperatures. Water drippers, snout coolers, and different ﬂooring types have been
shown to improve sow appetite (McGlone et al., 1988a), however, improvements in milk
production have been inconsistent.

2.3. Lactation performance

Lactation in the sow is a high energy demanding process with up to 100% of

maternal dietary energy intake required for milk production (Pluske et al., 1995b). Sow
appetite is suppressed by 43% (Messias de Braganca et al., 1998) and milk production
impaired when the ambient temperature is above 27°C (Steinbach, 1973; Black et al., 1993;
Prunier et al., 1997; Messias de Braganca et al., 1998; Johnston et al., 1999; Quiniou and
Noblet, 1999). Litter average daily gain is approximately 10% to 35 % less in a hot
environment compared to daily gain of nursing pigs housed in a thermoneutral zone
(Schoenberr et al., 1989; Vidal et al., 1991). In cows exposed to heat stress, milk yield is
decreased by 20 % and percentage of protein in milk is decreased by 6 % (Itoh et al.,
1998b). A decrease in energy intake accompanied by an increase in energy expenditure by
heat loss mechanisms has negative effects on growth and lactation (F eddes et al., 1996).
The suppressed feed intake of sows under heat stress has been proposed to be the major
factor impairing lactation performance (Prunier et al., 1996). Other factors are now

recognized as possible contributors to the impairment in performance.

2.3.1. Basal metabolism

The lactating sow adapts to high environmental temperatures via reduction in
metabolism rate hence decreasing the amount of heat produced. Reduction of the
metabolism rate is accomplished by a decrease in activities via a decrease in the thyroid
hormones and corticosteroids. These hormones control the rate at which body reserves
are mobilized. Thus a decrease in thyroid hormones and corticosteroids may decrease the
supply of nutrients to the mammary gland and milk synthesis (Prunier et al., 1997). The
decrease in metabolism via hormonal alterations is believed to be a major factor affecting
milk production and litter growth rate (Black et al., 1993; Messias de Braganca et al., 1998;
Prunier et al., 1997).

Endocrine changes have been recorded in pigs under heat stress. A decrease in
plasma cortisol concentrations has been reported under heat stress (Barb et al., 1991;
Messias de Braganca et al., 1998). The thermogenic hormone, cortisol, is reduced by
reducing the adrenocortical activity. This is thought to be a thermoregulatory protective
action preventing increases in heat production.

Extreme weight loss is characteristic of lactating sows under heat stress, as a
result of decreasing voluntary feed intake to reduce metabolism rate and high nutrient
demand for milk production (Schoenberr et al., 1989; Messias de Braganca et al., 1998;
Johnston et al., 1999; Quiniou and Noblet, 1999). However, milk production, estimated by
litter weight gain, is depressed to a higher extent than what can be accounted by the
decrease in feed intake of the sow (Black et al., 1993). Other studies have shown that a
decrease in feed intake due to heat stress has only minor detrimental effects on litter

weight gain (Messias de Braganca et al., 1998). Similarly, the reduction in feed intake of

heifers under heat stress is not the principal cause of depressed reproductive performance
(Ronchi et al., 2001). Messias de Braganca et a1. (1998) reported a greater weight loss in
sows housed in a thermoneutral environment when pair-fed to sows under heat stress.
Reduction in voluntary feed intake and a decrease of the thyroid hormones and
corticosteroids (Fraser et al., 1970) may be important mechanisms contributing to
decreasing heat production for lactation (Prunier et al., 1997). Sows housed at a
thermoneutral environment pair-fed to sows under heat stress loose more weight than sows
under heat stress, and do not show a decrease in thyroid hormones and corticosteroids
plasma concentrations.

2.3.2. Mammary blood ﬂow

Black et a1. (1993) suggested that high ambient temperature depresses litter
growth rate in part by causing a redirection of blood ﬂow ﬁom other organs including the
mammary gland to the skin (Black et al., 1993; Williams et al., 1994). Heat is dissipated
to the environment through cutaneous circulation (Song et al., 1988). Blood ﬂow is
decreased to the mammary gland during early lactation in rabbits (Lublin and Wolfenson,
1996) and to the conceptus in the pregnant sheep (Alexander et al., 1987) exposed to heat
stress. Reduction of blood ﬂowing to the sow udder may decrease nutrient availability to
the sow udder for milk synthesis.

Nutrient uptake by mammary cells is dependent on three factors: blood ﬂow
through the mammary gland, arterial nutrient concentration, and transport systems for
nutrients in blood across mammary cells, all of which regulate the rate of nutrient passage
into the mammary gland (Mepharn, 1982; Baumrucker, 1985). Within many species,

milk yield variation is accompanied by mammary blood ﬂow variation. In the goat,

blood ﬂow increases at parturition and rises steadily as lactation progresses. Mammary
gland blood ﬂow is correlated with milk yield (Linzell, 1960).

2.3.3. Hormonal and nutrient regulation

During lactation the partitioning of nutrients among tissues and organs is
regulated by several metabolic hormones such as insulin, glucagon, growth hormone,
cortisol, and thyroxine (Sartin et al., 1985). In both animals and humans, the synthesis of
milk proteins and mammary enzymes is induced primarily by prolactin and further
stimulated by insulin and cortisol (Worthington-Roberts, 1997). The consistent decrease
in milk yield at high ambient temperatures could potentially be mediated via an alteration
in the normal metabolism of these hormones. Alterations in endocrine metabolism by
heat exposure have been well investigated in ruminant species. Studies in monogastric
species are limited.

2.3.3.1. Insulin and glucose

Insulin stimulates glucose utilization and suppresses endogenous glucose
production, thereby decreasing plasma glucose concentration (review; Cryer, 1981).
Insulin promotes protein synthesis and inhibits lipolysis (review; Porterﬁeld, 1996).
Insulin and glucose concentration decrease with advancement of lactation (Tokach et al.,
1992) and sow body weight loss and litter growth rate are negatively correlated with
plasma insulin concentration (White et al., 1984).

Insulin responses to hot environmental exposure are inconsistent among species.
Under heat exposure, insulin concentration decreased in rats and rabbits (Souza et al.,
1993; Trammell et al., 1988), remained unchanged in broilers (Geraert et al., 1996), and

increased in sheep (Terashima et al., 1996) and growing calves (Takahashi et al., 1986).

In heifers exposed to heat stress, insulin and glucose concentration decreased (Itoh et al.,
1997). In non-lactating cows exposed to heat stress, insulin and glucose concentration
decreased while glucagon concentration was unaffected (Itoh et al., 1998a). Similarly, in
lactating cows, heat exposure tended to decrease glucose concentration with no change in
glucagon concentration; however insulin concentration increased (Itoh et al., 1998b).
The concentration of insulin in the blood reﬂects a balance between the rate of release
from the pancreas and the rate of removal by other tissues including the liver (Itoh et al.,
1998b). It is unclear how heat stress affects insulin release or clearance rate. In lactating
sows exposed to heat stress, glucose concentration increased possibly via a decrease in
glucose clearance rate (Prunier et al., 1997). This has been demonstrated in the lactating
goat exposed to heat stress where whole body glucose turnover decreased (Sano et al.,
1985). In contrast, glucose concentration was not increased in lactating ewes exposed to
heat stress (Abdalla et al., 1993).

In goats exposed to heat stress, glucose uptake by the mammary gland tended to
decrease by 15 to 50 %, decreasing milk yield (Sano et al., 1985). In lactating sows
under heat stress, plasma glucose concentration is unchanged compared to sows housed
under thermoneutral temperatures (Messias de Braganca and Prunier, 1998), or compared
to sows housed under thermoneutral enviromnent but pair-fed to heat stress sows
(Messias de Braganca and Prunier, 1998). Furthermore, insulin concentration was lower

in sows exposed to heat stress and in sows pair-fed to sows under heat stress.

2.3.3.2. Cortisol

Stressors such as crowding, handling, and transportation increase corticosteroids
concentrations (Schonreiter et al., 1999). Heat stress decreases cortisol concentration as a
metabolic adaptation to the environment (Messias de Braganca et al., 1998). Plasma
cortisol concentration is lower in sows under heat stress (Barb et al., 1991). In heifers,
glucocorticoids levels also decreased when exposed to high ambient temperatures
(Ronchi et al., 2001). Cortisol stimulates glucose release from body reserves by
glycogenolysis (review; Cryer, 1981). A decrease in cortisol concentration could
potentially decrease glucose availability to the mammary gland, explaining in part the
decrease in milk production.

Cortisol in blood is predominantly (90 %) bound to cortisol-binding globulin and
albumin (Cook et al., 1997). Cortisol concentration in saliva is independent of ﬂow rate
and is a direct reﬂection of the free fraction of cortisol in blood (Riad-Fahmy et al.,
1982). A small fraction of cortisol is converted to cortisone in the salivary gland, and the
concentration in saliva is 10 % of the serum cortisol concentration (Cook et al., 1996).

2.3.3.3. Growth hormone

Growth hormone (GH) is a species-speciﬁc hormone and its activity overlaps
with prolactin and placental lactogen. Secretion of GH occurs in diurnal rhythms, and is
stimulated by deep sleep with the highest peak occurring in the morning. Growth
hormone is an anabolic hormone that increases cellular amino acid uptake and
incorporation into protein (review; Porterﬁeld, 1996). Growth hormone is important for
galactopoiesis in certain species and is believed to facilitate the partitioning of glucose

and lipids to the mammary gland (Hart, 1983). Growth hormone is also a lipolytic

hormone, which promotes the mobilization of fats from adipose tissue. Growth hormone
favors the use of lipids as opposed to proteins as a source of energy for growth processes
(review; Porterﬁeld, 1996). Therefore, GH stimulates nitrogen retention and decreases
urea production (review; Porterﬁeld, 1996). Growth hormone can cause hyperglycemia
by decreasing cellular glucose uptake, and decrease insulin sensitivity (review; Cryer,
1981), and hypoglycemia stimulates GH secretion (review; Porterﬁeld, 1996). Glucose
concentrations were higher in gilts injected with growth hormone—releasing factor during
lactation (Farmer et al., 1992).

Administration of recombinant porcine somatotropin to sows depresses feed
intake, increases body weight loss and increases backfat loss (Cromwell et al., 1992).
Farmer et al. (1992) recorded similar responses when injecting gilts with growth
hormone-releasing factor during gestation, lactation or both; no effect on litter weight
gain was observed. Similar effects were seen by administering a human growth
hormone-releasing factor analog to lactating sows (Farmer et al., 1996).

In sows exposed to heat stress during lactation, GH concentration rises above
basal values (Barb et al., 1991). This is in agreement with Tucker and Wettemann (1976)
who reported that GH tended to increase in heifers exposed to heat stress. Conversely,
Collier et al. (1982) showed a decrease in GH and glucocorticoids in cattle exposed to
heat stress. Short-term heat exposure increased GH in cattle; however, long-term
exposure lowered GH (Johnson et al., 1976). In humans exposed for a short term to high
temperatures, GH concentration increases (Abdulaziz et al., 1996; Kukkonen et al.,
1988). The differential response may result ﬁ'om physiological adaptations during long-

term exposure to heat. In broilers exposed to heat stress, contradicting results have

10

reported either, a rise in GH concentration (Sinurat et al., 1987) or no effect on GH
concentration (Decuypere et al., 1993). An important point that must be considered when
drawing conclusions from previous studies is that the physiological state of a growing
animal differs from that of a lactating animal.

Some amino acids like arginine (I-Iertelendy et al., 1970; Davis, 1972; Takahashi '
et al., 1985), leucine (Davis, 1972), omithine (Handwerger et al., 1981), citrulline
(Handwerger et al., 1981), glycine (Pitwok et al., 1986), and aspartic acid (Sticker et al.,
2001) have been reported to stimulate GH secretion. Suckling stimuli of nursing pigs
increase GH in the lactating sow (Schams et al., 1994). However, both Vicini et al.
(1988) and Farmer et al. (1992) found no effect on milk production in sows with
increases in blood GH concentration.

Growth hormone acts via IGF-l , as it stimulates high IGF-l circulating levels
(Sejrsen et al., 1999). Concentrations of IGF-1 decrease in lactating sows exposed to
heat stress (Messias de Braganca and Prunier, 1998). In the catabolic sow, preprandial
levels of GH are higher while IGF-l is lower when compared to sows in a metabolic
balance (Einarsson and Rojkittikhurn, 1993; Quesnel and Prunier, 1995). This may
explain the increased GH concentration in sows under heat stress since they are always
catabolic.

2.3.3.4. Prolactin

Many functions have been attributed to prolactin depending on the species and
dose of administration. The main roles of prolactin on milk production are induction of
mammary gland development, and initiation and maintenance of lactation (Farmer,

2001). High levels of prolactin cause glucose intolerance and hyperinsulinemia (review;

11

Porterﬁeld, 1996). Massaging the udder stimulates prolactin secretion during lactation.
Sleep and dopamine antagonists also increase prolactin secretion. No effect on prolactin
has been recorded in the lactating sow exposed to hot environments, while in the cow and
the sheep, heat exposure is a major factor inﬂuencing prolactin. Exposure to high ambient
temperatures increases the concentration of prolactin in several species and this response
is independent of the physiological state of the animal. Stage of lactation affects the
concentration of prolactin. Prolactin concentrations peak before parturition and gradually
decrease as lactation progresses. This appears to be the result of a decrease in suckling
frequency (Varley and F oxcroft, 1990).

In the lactating sow, prolactin concentrations do not differ between non-heat
stressed and heat stressed sows (Barb et al., 1991) or ovariectomized gilts (Kraeling et al.,
1987). In male pigs (Klemcke et al., 1987), ewes (Hooley et al., 1979), cattle (Collier et
al., 1982) and heifers (Tucker and Wettemann, 1976; Ronchi et al., 2001), prolactin
concentrations rise under heat stress. In the non-pregnant, pregnant, and lactating ewe
and sheep, prolactin levels rose when these animals were exposed to high ambient
temperatures exceeding thermoneutrality (Hill and Alliston, 1981; Regnault et al., 1999;
Bell et al., 1989; Walker et al., 1990; Stephenson et al., 1980; Thompson et al., 1981).
Similar results have been observed in dairy cows (Thatcher, 1974) and buffaloes (Sharma
et al., 1999). In non-pregnant women, short—term exposure to high ambient temperatures
increases prolactin levels (Laatikainen et al., 1988). These studies suggest that under
high ambient temperatures, the rate of prolactin clearance may be decreased or the rate of
prolactin secretion may be increased. The question whether prolactin concentration is

decreased in the lactating sow exposed to heat stress deserves further investigation.

12

2.4. Sow and piglet behavior

Both the sow and nursing pig behavior inﬂuences the quantity of milk that nursing
pigs consume (Castrén et al., 1989b). Milk production from individual glands is a
function of suckling demand (Auldist et al., 1995). Whether inferior milk production in
sows exposed to heat stress is related to an alteration in suckling behavior has not been
fully investigated.

A paradox arises when comparing the behavioral aspects of sows and nursing pigs
at high ambient temperatures. As opposed to the sow, the nursing pigs thrive under high
ambient temperatures. The nursing pig’s behavior is thus less likely to inﬂuence lactation
behavior in a hot environment due to the fact that the nursing pig is within its
thermoneutral zone while the sow is above her critical limit, hence under heat stress.

The normal milking episode in the sow is divided in four distinct phases, beginning
with an initial massage, followed by a quiet suckling with slow mouth movements, a
rapid suckling with fast mouth movements, and a ﬁnal phase of udder massage (Fraser,
1973; Whittemore and Fraser, 1974). Rapid suckling is accompanied by milk ejection,
which is characterized by a rapid rise in intramammary pressure lasting approximately
from 8 to 40 seconds. Milk ejection is triggered by the release of oxytocin from the
posterior pituitary gland (Ellendorf et al., 1982), and is accompanied by rapid grunting by
the sow (Ellendorf et al., 1984). A milking interval is the period between milking
episodes were no milk is ejected. Milking interval in sows housed in a thermoneutral
environment is approximately 40 minutes (Prunier et al., 1997) and the duration of milk

ejection is approximately 14 seconds (Hartmann et al., 1997).

13

An altered nursing behavior may depress the frequency of milk ejection reﬂex and
consequently limit the transfer of milk from the sow to the litter (Hartmann et al., 1997).
Unsuccessful milkings occur in 20-30% of total milking attempts by the sow (Whatson
and Bertram, 1980; Jensen et al., 1991; Newberry and Wood-Gush, 1984) and are
characterized by the absence of milk ejection. There is scarce information in the
literature about suckling frequency or milk ejection in sows under heat stress. Although
unsuccessful milking episodes are considered to be a normal outcome of nursing behavior
(Castrén et al., 1989a), higher rates of unsuccessful milkings have been observed in sows
under heat stress (Fraser, 1970). An increase in the number of unsuccessful milkings
could reduce overall milk intake and decrease litter growth rate (N ewberry and Wood-
Gush, 1984; Castrén et al., 1989a).

Limited research has been conducted on the behavioral aspects of lactating sows under
heat stress. Hartrnann et al. (1997) proposed changes in sow’s suckling behavior under heat
stress where the piglets’ access to the gland may be impaired. It has been reported that the
sow uses postural adjustments to limit the access of nursing pigs to the mammary gland
(dePasillé and Robert, 1989). Recognizing that lactation is a highly energy demanding to
the sow, it is unknown whether the sow under heat stress attempts to decrease heat
production by altering nursing behavior via postural adjustments. Additionally, the stress
induced by coping with sub-optimal environment and the unavoidable increase in
metabolism required for each milking event may decrease milk production and ejection.
Consequently, the sow may be reluctant to assume lateral recumbence for each suckling

episode. Pigs tend to favor a ventral recumbence when exposed to a hot environment to

14

increase heat dissipation by conduction (Hicks et al., 1998), which in turn may affect

nursing behavior during lactation.

15

3. Role of arginine in nutrition and metabolism
3.1. Introduction
L-arginine is a dietary essential amino acid for lactation and maximal growth in
young mammals, but non-dietary essential during gestation (Easter, 1974) and in adult
mammals (review; Eremin, 1997).
3.2. Arginine metabolism
L-arginine is synthesized in the liver and kidney as a dibasic amino acid with four
nitrogen moieties in its molecule. Its primary catabolism occurs in the liver by arginase
to form urea and omithine in the last step of the urea cycle. L-arginine plays a vital role
in preventing ammonia intoxication in mammals (Roth, 1998).
L-arginine is widespread in tissues, mainly because of its action as a precursor in
the biosynthesis of the polyamines, creatinine, and pyrimidines (review; Eremin, 1997).
L-arginine is a secretatogue for various hormones, including those of the pancreas
(insulin, glucagon, pancreatic polypeptide, somatostatin), the pituitary gland (growth
hormone, prolactin), and the adrenal gland (catecholamines) (review; Eremin, 1997).
Furthermore, L-arginine is an important modulator of cellular (lymphocytes,
macrophages) and humoral immune responses in humans and animals (review; Eremin,
1997), and has been used in clinical situations to improve wound healing and immune
function in patients undergoing extensive protein catabolism (Daly et al., 1988; Barbul et
al.,1990).
L-arginine is the precursor of nitric oxide (N O), which is known as the
endothelium-derived relaxing factor due to its vasodilatory properties (Roth, 1998).

Nitric oxide is synthesized from L-arginine by the nitric oxide synthase (NOS) enzyme,

16

of which there are at least two general classes. The constitutive form is cat2 and is
calmodulin dependent (cNOS); it is present in neurons, platelets, mast cells, endothelial
cells, mesangial cells, and endocardial and myocardial cells; it exists primarily in a
membrane-bound particulate form; and produces a continuous low output of NO (review;
Eremin, 1997). The other type of NOS (iNOS) is induced by cytokines (e. g., interferon
and endotoxin); it is not calcium or calmodulin-dependent and it produces a twenty-fold
increase in NO, several magnitudes greater than cNOS (review; Eremin 1997). The
iNOS converts the terminal guanidine group of L-arginine to NO in a reaction requiring
NADP as a cofactor and O; to oxidize the L-arginine to NO and L-citrulline, the ﬁnal
reaction products (Roth, 1998).

The physiological target of NO in smooth muscle cells is guanylate cyclase,
which catalyzes the reaction of GTP to produce 3’, 5’-cyclic GMP (cGMP), an
intracellular second messenger. It resembles cAMP and induces smooth muscle
relaxation through stimulation of protein phosphorylase by a cGMP-dependent protein
kinase (Voet et al., 1999). In addition, the synthesis of NO can be inhibited by several L-
arginine analogues, which will ultimately increase vascular resistance (Lacasse et al.,
1996)

3.3. Arginine and mammary blood ﬂow

The rate of supply of amino acids is determined by mammary blood ﬂow, which
has been shown to increase in response to the local mammary epithelial secretion of the
ubiquitous vasodilator nitric oxide (NO) (Lacasse et al., 1996). L-arginine may regulate
NO bioavailability in the mammary vasculature (Chowienczyk, 1997). Oral arginine

administration was shown to increase NOZ/NO3 levels in humans (Battaglia et al., 1999).

17

Arginine in the feed or water alleviates pulmonary arterial pressure and pulmonary
vascular resistance in broilers with snared right pulmonary artery (Wideman Jr. et al.,
1996).

Although mammals are capable of synthesizing arginine in adequate amounts for
maintenance and productive functions via the urea cycle, the synthesis of arginine may
not be adequate for the synthesis of nitric oxide to maximize core and peripheral
vasodilation in times of elevated environmental temperatures. Under heat stress, arginine
administration restores NO synthesis (after inhibition by NO —nitro-L—arginine), which in
turn mediates the cutaneous capillary blood ﬂow responses to dissipate heat in the rat
(Zhang et al., 1994). Although the contribution of arginine to NO production has not
been quantiﬁed under heat stress, the arginine requirement in lactating sow diets under
heat stress may be higher than currently estimated.

In experiments involving denervation of the mammary gland, mammary blood
ﬂow variation still occurs, suggesting that locally produced or paracrine vasoactive
factors are involved in regulating mammary blood ﬂow (Lacasse et al., 1996). Such
factors could act on the arterioles, capillaries, or both to alter vascular resistance and
exchange rates in mammary tissue (Prosser et al., 1996).

Lacasse et al. (1996) measured the effects of NO on mammary blood ﬂow in
lactating goats. In their study, infusion of the nitric oxide donor diethylamine NONOate
increased local NO levels in mammary epithelial cells which in turn caused rapid and
sustained increase in mammary blood ﬂow in the mammary gland only, suggesting a
direct effect on the mammary vasculature. In contrast, infusion of an inhibitor of nitric

oxide synthase, Nm-nitro-arginine, decreased mammary blood ﬂow by up to 35%, and the

18

co-infusion of arginine, the precursor in the synthesis of nitric oxide, with Nm-nitro-
arginine signiﬁcantly diminished the latter’s ability to decrease mammary blood ﬂow.
Thus, the mammary gland produces and responds to nitric oxide, and through its
secretory epithelium, may control its own blood supply through nitric oxide secretion
(Lacasse et al., 1996).
3.4. Arginine and hormonal regulation

3.4.1. Amino acids

Several amino acids stimulate the secretion of key metabolic hormones. In
horses, increase in GH secretion by infusion of aspartic acid and glutamic acid has been
reported (Sticker etal., 2001). Arginine, lysine and glutamic acid stimulated the
secretion of prolactin but only arginine and lysine stimulated the secretion of insulin
(Sticker et al., 2001).

3.4.2. Insulin and glucose

Sener et al. (2000) suggested that arginine stimulates the secretion of insulin by
the carrier-mediated arginine transport in pancreas islet B-cells. Intravenous injection of
arginine at supraphysiological levels (132 g/kg body weight) to lactating cows increased
glucose and insulin concentrations, with basal values returning 45 minutes post injection
(Sano et al., 1999). Weaver et al. (1994) administered supraphysiological concentrations
of arginine (350 mg/ 100 g body weight) to growing male rats. At this level of arginine
administration, body weight was decreased, glucose clearance was decreased, as well as
the concentration of insulin in the pancreas.

Hertelendy et al. (1970) investigated the effect of arginine infusion on secretion of

insulin and glucose in the pig, sheep and cow. Infusion of arginine into sheep (0.5 g/kg)

l9

and cows (5 to 10 mg/kg) resulted in rises in insulin and glucose concentration. In
contrast, in the pig, arginine infused at 0.5 g/kg slightly increased insulin with no change
in plasma glucose. Arginine infusion also increased insulin concentration in the dairy
cow (Vicini et al., 1988). Chew et al. (1984) infused arginine to pregnant cows at a dose
of 0.1g /kg body weight which resulted in increasing insulin and urea nitrogen. Infusion
of arginine (0.5g/kg), phenylalanine (0.3 g/kg), or leucine (0.2g/kg) to non-lactating sheep
elevated insulin concentrations (Davis, 1972). Ponté et al. (1972) infused arginine to
human infants at a dose of 0.5g/kg. In this particular experiment, blood glucose was not
affected by arginine infusion. However, insulin and GH levels rose as arginine was
infused to the neonate. Atinmo et a1 (1978) showed that arginine infused to growing pigs
fed adequate diets for growth increased plasma insulin and glucose levels. The response
peaked at 45 minutes after the infusion and returned to basal levels at 2 hours after the
infusion. In this study arginine was inﬁrsed for 30 minutes at a dose of 0.5g/kg0'75 of
body weight.

3.4.3. Growth hormone

Infusion and oral administration of arginine stimulates GH secretion in several
species. However, the extent of this effect seems to be species speciﬁc and dose
dependent. Most of the scientiﬁc investigations performed during the 1960’s and 1970’s
focused on ruminant species were arginine was infused at doses that resulted in acute
responses (Vicini et al., 1988). Hertelendy et al. (1970) investigated the effect of arginine
infusion on secretion of GH in the pig, sheep and cow. Infusion of arginine to lactating
sheep (0.5 g/kg) and lactating cows (5 to 10 mg/kg) resulted in rises in GH. In contrast,

in the pig, arginine infused at 0.5 g/kg did not stimulate GH secretion. In the sheep,

20

hyperglycemia resulting from glucose inﬁrsion decreased the GH secretory response to
arginine inﬁrsion (Hertelendy et al., 1970). In growing pigs, plasma GH concentration

increased with arginine infused at a dose of 0.5 g/kg‘"75

of body weight (Atinmo et al.,
1978). In adult humans, arginine infusion (0.5g/kg) increased GH and PRL (Rakoff et
al., 1973). In the dairy cow, GH has been reported to increase with intravenous injection
of arginine at a dose of 0. 1 g/kg body weight with a peak response observed at 45 min
post infusion (Vicini etal., 1988). The rapid responses of GH to arginine infusion
indicate that these responses are most likely to be acute.

Although arginine supplementation has successﬁrlly stimulated the secretion of
GH in ruminants, improvements in milk production have been inconsistent. Chew et al.
(1984) infused arginine to pregnant cows at a dose of 0.1 g /kg body weight increasing
GH concentration. Milk production in this study tended to be higher in infused cows
compared to the controls for the ﬁrst 22 weeks of lactation. In lactating goats, arginine
infused at a rate of 25 g/d or 0.625 g/kg body weight failed to increase GH concentration
(Gow et al., 1979). Infusion of arginine (0.5g/kg) or leucine (0.2g/kg) to non-lactating
sheep consistently stimulated GH secretion (Davis, 1972). On the other hand,
phenylalanine (0.3 g/kg) did not cause an effect on GH concentration.

In humans, the stimulatory effect of arginine administration on GH has been
inconsistent. Ponté et al. (1972) infused arginine to human infants at a dose of 0.5g/kg.
In this particular experiment GH levels rose as arginine was infused to the neonate. In
adult humans, arginine infusion at the same dose (0.5g/kg) increased GH (Rakoff et al.,

1973). Isidori et al. (1981) showed that oral administration 1.2 g/d or 2.4 g/d of arginine

did not have any effect on human GH levels. However, a combination of arginine (1.2

21

g/d) and lysine (1.2 g/d) increased GH above basal levels. In contrast, older and younger
men administered with oral arginine and lysine (3g arg + 3g lys /day) had no effect on
GH concentration (Corpas et al., 1993). Marcell et al. (1999) administered 5 g arginine
to aged and young patients and reported no change in GH levels. Oral arginine
administration increased circulating IGF-l levels in humans (Battaglia et al., 1999).
Sows undergo a high rate of protein catabolism during lactation (Trottier, 1995).
It is unknown whether arginine plays any role in nitrogen balance during lactation.
Arginine inﬁrsion improves nitrogen retention in humans undergoing accelerated protein
degradation after trauma (Elsair et al., 1978; Daly et al., 1988). Arginine promotes
nitrogen retention in animals during sepsis, trauma, and severe burns (Saito et al., 1987;
Madden et al., 1988; Kirk and Barbul, 1990). It also improves the rate of wound healing
in rats (Seifter et al., 1978). The effect of arginine on nitrogen retention could possibly
occur via its stimulatory effect on GH (Seif’ter et al., 1978; Kelley et al., 1990; Gala,
1991). However, in growing calves infused with arginine at a dose of 0.5 g/kg of body
weight per day, increased average daily gain of calves before weaning occurred without
change in GH concentration however, followed by a decrease in GH concentration,
suggesting that arginine increased weight gain via an increase in milk arginine
concentration rather than increased GH (F ligger et al., 1997). Arginine concentration in
cow’s milk is below requirement for grth (Williams and Hewitt, 1979). Alternatively,
arginine may promote growth via an insulin-mediated mechanism, since arginine
promotes insulin secretion in several species (Fligger et al., 1997). Whether arginine
supplementation in lactating sow diets decreases the catabolic state or increases milk

arginine concentration is unknown.

22

3.4.4. Prolactin

Prolactin secretion by arginine supplementation has been documented in several
species. However, the effect of arginine on prolactin during lactation is not clear. The
inhibition of prolactin secretion by bromocriptine administration decreases milk
production (Farmer et al., 1998). However, exogenous prolactin administration increases
prolactin in plasma but does not increase milk yield (Farmer et al., 1999). In pregnant
cows infused with a dose of 0.1 g arginine per kg body weight, prolactin concentration
increased (Chew et al., 1984). Infusion of arginine (0.5g/kg), phenylalanine (0.3 g/kg), or
leucine (0.2g/kg) to non-lactating sheep elevated prolactin concentrations (Davis, 1972).
In adult humans, arginine infusion at the same dose (0.5g/kg) increased prolactin (Rakoff
et al., 1973). Kirchgessner (1991) has shown that dietary arginine supplementation to
lactating sows increases milk production by 10 %. Whether this occurred via an increase
in prolactin concentration has not been investigated.

3.5. Heat stress

Few studies have been performed investigating the effect of heat stress on
hormonal responses to arginine supplementation. Takahashi et al. ( 1985) reported altered
responses of insulin and glucagon to arginine supplementation in growing calves exposed
to heat stress. Insulin secretion was depressed while glucagon secretion was accelerated
when compared to the response observed in a thermoneutral environment. In non-
lactating dairy cows, the insulin response to arginine supplementation was depressed
under heat stress, while the glucagon response was enhanced (Itoh et al., 1998a). In
contrast, in the lactating dairy cow, heat exposure enhanced the secretory effect of

arginine on insulin and glucagon (Itoh et al., 1998b). These results suggest that the extent

23

to which heat exposure alters the stimulatory effect of arginine on insulin and glucagon
differs with physiological state, i.e., lactating vs. non-lactating. I am unaware of similar
studies performed in lactating sows.
3.6. Effects arginine on feed efﬁciency and performance

Weight gain and efﬁciency of feed utilization are enhanced with arginine
supplementation. Weight gains and feed conversion of broilers and turkeys were
improved when arginine was supplemented to increase the arginine to lysine dietary ratio
(Mendes et al., 1997; Waldroup et al., 1998). These responses were particularly
improved in a hot environment (Brake et al., 1998). Enhanced nitrogen retention by
dietary arginine supplementation is well documented in animals suffering from sepsis
(Saito et al., 1987; Madden et al., 1988; Kirk and Barbul, 1990). Arginine
supplementation minimizes post-traumatic weight loss and accelerates wound healing in
rats (Barbul et al., 1983). Nitrogen retention is improved in pigs, humans and rats,
especially after trauma (Sitren and Fisher, 1977; Elsair et al., 1978; Leibholz, 1982; Daly
et al., 1988). Wu et al. (1999) reported decrease feed intake and improved feed
efﬁciency when arginine is supplemented to rat diets compared to arginine-deficient
diets. Whether the improved nitrogen retention and feed efﬁciency observed with
arginine supplementation results from the secretory effect on anabolic hormones is
unclear.

This thesis focuses in the following research questions. In chapter 2, does heat
stress decrease lactation performance and does arginine supplementation improves sow
body condition and litter growth rate? In chapter 3, does heat stress modulate key

metabolic hormones and metabolites and does arginine supplementation ameliorates the

24

. effect of heat stress on these hormones? In chapter 4, does heat exposure decrease amino
acid concentrations in plasma and milk and does arginine supplementation increase nitric
oxide concentration? In chapter 5, is the sow’s nursing behavior altered during heat

stress?

25

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37

CHAPTER 2: EFFECT OF DIETARY L-ARGININE SUPPLEMENTATION AND
ENVIRONMENTAL TEMPERATURE ON SOW LACTATION PERFORMANCE.

Abstract

The objective of this study was to investigate whether supplemental dietary
arginine increases lactation performance in sows subjected to a hot environment. A total
of 66 multiparous sows were allotted in a 2 x 3 factorial arrangement, consisting of two
environments and three dietary treatments. Sows were maintained in a thermoneutral
environment of 20°C (TN) or heat-stressed in a hot environment of 294°C (HS), starting
on day 95 of gestation. From day 110 of gestation to weaning, sows were provided one
of three com-soybean meal based dietary treatments formulated to contain 0.96%
arginine (1:1 arginine:lysine), 1.34% arginine (1.4:1 arginine:lysine), and 1.73% arginine
(1.8:1 arginine:lysine), for control (C), medium (ME), and high (HI) dietary treatments,
respectively. Respiration rate and rectal temperature increased in HS sows compared to
TN sows (P < 0.01) and heart rate decreased in HS sows (P < 0.05) compared to TN
sows. Arginine supplementation did not affect heart rate and respiration rate; however, it
tended to decrease rectal temperature (P < 0.10) in sows fed the ME diet in the
thermoneutral environment. Litter weight gain (P < 0.05) and voluntary sow feed intake
(P < 0.01) decreased and sow weight loss increased (P < 0.01) in the hot environment.
Dietary arginine supplementation did not improve litter weight gain (P > 0.10) for either
HS or TN sows. Arginine supplementation reduced feed intake (P < 0.05) and tended to
reduce body weight loss (P = 0.06) during lactation. In conclusion, supplemental
arginine in lactating sow diets in a hot environment did not improve litter performance.
However, supplemental arginine in lactating sows subjected to a hot environment reduced

sow weight loss and increased sow feed efﬁciency.

38

Keywords: arginine, heat stress, lactation, sow, lysine

39

Introduction

Arginine is essential for growing mammals because the rate of endogenous
arginine synthesis cannot meet arginine requirements for optimal grth and health
(Visek, 1985). Although endogenous arginine synthesis rate is sufﬁcient to meet
requirements for maintenance and gestation in the sow, it is unknown whether it is
adequate to maximize milk synthesis during lactation in heat-stressed sows. High
ambient temperatures decrease voluntary feed intake by 20 to 55% (McGlone et al.,
1988; Messias de Braganca et al., 1998; Quiniou and Noblet, 1999; Johnston et al., 1999),
increase weight loss, and decrease milk production in lactating sows (Steinbach, 1973;
Black et al., 1993; Prunier et al., 1997; Johnston et al., 1999). A redirection of blood
ﬂow from peripheral organs to the skin occurs as a consequence of heat exposure (Black
et al., 1993) and a reduction in blood ﬂow to the mammary system may contribute to the
reduction in milk production in sows exposed to heat. Conversely, an increase in
mammary and peripheral blood ﬂow demand under a hot environment may increase the
metabolic requirement for arginine. Because arginine is the primary substrate for nitric
oxide (NO), the endothelium-derived relaxing factor (Ignarro et al., 1987), and nitric
oxide regulates vasodilation and blood ﬂow (V allance et al., 1989; Lacasse et al., 1996),
we hypothesized that high ambient temperature increases arginine metabolism and hence
its dietary requirement in the lactating sow. Thus, the objective of this study was to
determine whether increasing dietary arginine intake in heat stressed sows improved

lactational performance.

40

Materials and Methods
Environment

All-University committee on Animal Use and Care, Michigan State University
approved use of animals for this experiment. On day 95 of gestation, 66 multiparous (2
to 4 parity) sows (Landrace x Yorkshire) were randomly allocated to one of two
environments in 5 replicates: a thermoneutral environmental with a temperature
maintained at 20°C (TN), or a hot environment with a temperature maintained at 294°C
(HS). Sows allocated to the hot environment were acclimated to 294°C prior to
farrowing. Acclimation was achieved by gradually increasing the temperature on a
weekly basis (3.3°C increments). Temperature was decreased to 24°C from 20. 00 to 8.
00 hours to simulate a natural summer setting. Photoperiod was controlled with 12h of
light and 12h of darkness daily. Sows were individually housed in farrowing crates
equipped with plastic ﬂooring at the Michigan State University Swine Research and
Teaching Facility.
Diet and feeding

All sows were fed a gestation diet meeting nutrient requirements for maintenance
and gestation (NRC, 1998) until day 110 of gestation. On day 110 of gestation, sows
were allocated randomly to three dietary treatments consisting of com-soybean meal
based diets with varying concentrations of arginine. Diets were as follow: 0.96%
arginine (control = C), 1.34% arginine (medium = ME), and 1.73% arginine (high = HI).
Ingredients and nutrient composition of the experimental diets are shown in Table 2.1 and

2.2, respectively. Sows were fed 2kg per day from day 110 until farrowing. Sows were

41

fed in a stair-step fashion from day one post-farrowing to day ﬁve (1 kg increment per
day), and provided ad libitum access to feed thereafter. Sows had free access to water.
Data collection

Backfat depth and loin eye area were measured 5cm lateral from the spine and
centered over the 10th rib (Ultrasound Scanner 200 Vet with ASP-18 linear probe, Pie
medical Equipment B.V., Maastricht, The Netherlands) on day 112 of gestation and on
day 22 of lactation. Feed intake was recorded and orts collected and weighed daily.
Sows were weighed before the morning meal on day 1 post farrowing and weekly
thereafter. Within 24 hours after birth, piglets were weighed, tails were docked, needle
teeth clipped, iron dextran administered, and males castrated. Litters were weighed on
day l of lactation and every three days until weaning on day 22 of lactation. Litters were
equalized to 10 or 11 piglets depending on piglet availability, by cross-fostering within
36 hours post-farrowing. To monitor the effect of ambient temperature on physiological
indicators of heat stress, respiration rate, heart rate, and rectal temperature were recorded
on day 1 post farrowing and weekly thereafter.

The effects of environment, diet, and the environment by diet interaction were
determined by analysis of variance using the MIXED procedure of SAS (1998) with
repeated measures analysis using sow within diet and environment as the error term.
Results

Lactational performance results are presented in Table 2.3.1 and 2.3.2. Overall,
heart rate decreased in HS sows compared to TN sows (102 beats per minute vs. 106
beats per minute, respectively; P < 0.05). No diet or environment by diet interaction

effects on heart rate were found (P > 0.10 and P > 0.10, respectively). Respiration rate

42

increased in HS sows compared to TN sows (89 breaths per minute vs. 45 breaths per
minute respectively; P < 0.001). No diet and diet by environment interaction effects
were found (P > 0.10 and P > 0.10, respectively). Rectal temperature was higher for HS
sows compared to TN sows (39.9 °C vs. 39.1 °C, respectively; P < 0.001). Arginine
supplementation tended to decrease rectal temperature of TN sows fed the ME diet (38.9
°C) compared to TN sows fed the C or HI diets (39.3 °C and 39.2 °C, respectively; P <
0.10). No diet by environment interaction effects on rectal temperature were found (P >
0.10). Voluntary feed intake in HS sows declined by 20% compared to TN sows (5.12 kg
per day vs. 6.43 kg per day, respectively; P < 0.001). In the hot environment, feed intake
decreased in sows fed the HI diet compared to the sows fed the C diet (5.01 kg per day
vs. 5.26 kg per day, respectively; P < 0.05), but in the thermoneutral environment, feed
intake decreased in sows fed the ME diet (6.29 kg per day) compared to the C (6.48 kg
per day) and HI (6.52 kg per day) diets (P < 0.05).

Sow weight change during lactation was adversely affected by heat stress (Table
2.3.1 and 2.3.2 and Figure 2.1). Heat-stressed sows lost more weight during the 21-d
lactation period compared to TN sows (11.33 kg vs. 2.99 kg, respectively; P < 0.001 ).
Under the hot environmental treatment, sows fed the C diet had a higher weight loss
(11.24 kg) during lactation compared to sows fed either the ME diet (5.50 kg; P < 0.05)
or the HI diet (4.74 kg; P < 0.05). Sows under the thermoneutral environment exhibited
no difference between diets.

Average daily gain (ADG) per piglet decreased under the hot compared to
thermoneutral environment (P < 0.05). Piglets nursing TN sows had an ADG of 21 1.67 :L-

10.62 g per day compared to 196.79 :1: 11.73 g per day for piglets nursing HS sows.

43

Dietary treatments and diet by environment interaction did not affect piglet average daily
gain (P > 0.10 and P > 0.10, respectively). Diets, environments, and the interactive effect
of enviromnent by diet were not signiﬁcant (P > 0.10) for changes in backfat thickness
and loin-eye area.
Discussion

The purpose of this study was to test whether dietary arginine supplementation
improved lactational performance of sows exposed to hot environmental temperature.
Current estimates of arginine requirement for the lactating sow are derived from
estimates of lysine requirement and the relative proportion of arginine to lysine in porcine
milk (NRC, 1998). However, the amount of arginine uptake by the porcine mammary
gland is higher compared to the amount secreted in milk (Trottier et al., 1997), suggesting
the total requirement for arginine may be higher than the requirement estimated from
amino acid proﬁle of milk. A higher mammary uptake of arginine compared to arginine
output into milk has also been observed in bovine and rabbit mammary glands (Clark et
al., 1975). This net positive balance for arginine across the mammary gland may be
attributed to its use as an essential substrate for the synthesis of numerous biological
compounds. Nitric oxide is a free radical synthesized from L-arginine by NO synthase
(NOS) in all mammalian cells (Morris, 1998). Dietary arginine deﬁciency in young rats
decreases plasma concentrations of arginine and impairs NO synthesis (Wu et al., 1999).
Nitric oxide plays a critical role in vasodilation (Vallance et al., 1989), and evidence
indicates that NO regulates blood ﬂow to the mammary gland. For instance, Lacasse et

a1. (1996) found a rapid increase in mammary blood ﬂow in lactating goats in response to

44

intramammary infusion of 1,1—diethyl-2-nitroso-hydrazine (diethylamine NONOate), a
NO generator M.

Mechanisms by which high ambient temperature decreases lactation performance
are unclear. In lactating rabbits exposed to high temperatures, blood ﬂow to the
mammary gland was reduced (Lublin and Wolferson, 1996). The reduction of blood
ﬂow to the mammary system may consequently decrease milk synthesis and production
in lactating animals under hot environmental conditions. This study was designed to
induce heat-stress in lactating sows and to examine whether dietary arginine
supplementation improves lactational performances. While milk production and blood
ﬂow were not measured, litter growth was used as an indicator of milk production. The
feed intake, respiration rate, heart rate and rectal temperature responses in sows exposed
to a hot environmental temperature in this study indicated that the sows were heat
stressed. Sows reduced their voluntary feed intake by 20%, increased their respiration
rate and rectal temperature when compared to sows housed under a thermoneutral
environment. This is in agreement with other studies, where respiration rate and rectal
temperature were increased in sows subjected to a hot environmental temperature (Barb
et al., 1991; Prunier et al., 1997; Johnston et al., 1999). In contrast to these studies, but in
agreement with Fraser (197 0), heart rate decreased in sows under the hot environment.

In the present study, litter weight gain decreased with an environmental
temperature maintained at 294°C compared to 20°C. Prunier et al. (1997), Messias de
Braganca et al. (1998), and Quiniou and Noblet (1999) found a decrease in litter weight
gain with an increase in ambient temperature above 27 °C. However, there was no

indication in this study that dietary arginine supplementation at the highest concentration

45

inﬂuenced milk production as measured by litter weight gain in sows exposed to a hot
environment. While sows in our study demonstrated physiological signs of heat stress, it
is possible that the temperature selected was not sufﬁciently high to increase arginine
requirement. Indeed, hot environment reduced litter weight gain in sows fed the ME diet.
However, it is possible that the response observed is not a result of supplementing
arginine as this was not observed with the HI arginine diet. In studies conducted by
Biensen et al. (1996) and McGlone et al. (1988), high environmental temperatures did not
depress litter weight gain. Our results indicate that sufﬁcient arginine is present in com-
soybean meal based diets for lactating sows housed under either a thermoneutral or hot
environment of up to 294°C.

Interestingly, while feed intake decreased in response to increasing dietary
arginine concentrations, weight loss also decreased. This was observed in sows under the
hot environment. Similarly, Wu et al. (1999) found that rats fed a 1.0% arginine diet
consumed less feed and lost less weight than those fed isocaloric but 0.0% and 0.3%
arginine-deﬁcient diets. Increasing arginine to lysine ratios in chickens exposed to high
ambient temperatures (Brake et al., 1998) and in turkeys under thermoneutral
environment (Mendes et al., 1997) improved feed utilization. In addition, supplemental
dietary arginine increased nitrogen retention after trauma in rats (Sitren and Fisher,

1977). The results of our study and those of the studies mentioned above indicate that
arginine given at dietary concentrations above requirements for growth and lactation
improves nutrient utilization, particularly under hot environmental temperatures. The
mechanisms by which arginine improves nutrient utilization are not known, but may

involve key metabolic hormones. The secretagogue effect of arginine on insulin,

46

prolactin, grth hormone, and glucagon has been well documented in humans (Sigal et
al., 1992), cows and growing calves (Sano et al., 1999), and growing pigs (Atinmo et al.,
1978). Whether arginine promotes the synthesis and (or) secretion of these hormones in
the lactating sow is unknown, and warrants further research.
Implications

The results of this study indicate that arginine given at dietary concentrations
above requirements for growth and lactation improves nutrient utilization, particularly
under hot environmental temperatures, but does not improve milk production as
measured by litter growth.
Acknowledgments
The authors wish to thank Nutri-Quest, Inc. for partial funding and Dr. Gretchen Hill for

reviewing the manuscript.

47

 

Table 2.1 Ingredient composition of experimental diets on as fed basis (%)

 

 

Diets
Ingredients LOW MED HIGH
Corn, dent yellow 62.18 62.18 62.18
Soybean meal, solv 22.60 22.60 22.60
Corn oil 3.72 3.72 3.72
Dicalcium phosphate 1.97 1.97 1.97
Limestone 0.87 0.87 0.87
Trace mineral premix"1 0.50 0.50 0.50
Vitamin premixb 0.60 0.60 0.60
Salt, NaCl 0.30 0.30 0.30
Sowpacc 0.30 0.30 0.30
Sucrose 3.96 4.87 5.84
Glutarnic acid 2.65 1.35 0.00
Amino Acid Mixd 0.35 0.35 0.35
Arginine 0.00 0.39 0.77

 

'Provided the following per kilogram of diet: 335 g Ca, 5 g Fe, 5 g Zn, 5 mg Cu, 150 ug
Se, and 75 pg 1.

bProvided the following per kilogram of diet: 4,583 IU vitamin A, 458 IU vitamin D3, 55
IU vitamin E, 11 mg vitamin K, 3.66 mg menadione, 0.0275 mg vitamin B12, 3.66 mg
riboﬂavin, 14.67 mg D-pantothenic acid, 22 mg niacin, 0.913 mg thiamine, 0.825 mg
pyridoxine.

° Provided the following per kg premix: 918583 IU vitamin A, 73487 mcg biotin, 128602
mg choline, 551 mg folic acid. ,

° Amino Acid mix: 0.15774% lysine-HCl + 0.05092% threonine + 0.0053% tryptophan +
0.1149% valine + 0.02452% phenylalanine

48

 

Table 2.2 Nutrient composition of experimental diets on as fed basis (%)

 

 

Diets
Calculated Analysis LOW MED HIGH
CP, % 16.97 16.97 16.97
DE, kcal/kg 3456 3491 3527
ME, kcal/kg 3301 3335 3370
Ca, % 0.91 0.91 0.91
P, % 0.69 0.69 0.69
Ca:P 1.33 1.33 1.33
NDF, % 8.98 8.98 8.98
Arginine, % in diet 0.96 1.34 1.73

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49

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51

Figure 2.1 Effect of environmental temperature and dietary arginine supplementation on sow
weight change during a 21-d lactation period. Gray shaded bars represent control group
(0.96% arginine); Black bars represent medium group (1.34% arginine); Dark gray bars
represent high group (1 .73% arginine). a: = different at a P < 0.05 compared to control group.
NS = non-signiﬁcant compared to control group. The main effect of environmental
temperature is represented by different letters.

52

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53

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Atinmo, T., Baldijao, C., Houpt, K. A., Pond, W. G. and Barnes, R. H. 1978. Plasma levels
of growth hormone and insulin in protein malnourished vs. normal growing pigs in response
to arginine or glucose infusion. J. Anim. Sci. 46: 409-416.

Barb, C. R., Estienne, M. J ., Krailing, R. R., Marple, D. N., Rarnpacek, G. B., Rahe, C.
H., Sartin, J. L. 1991. Endocrine changes in sows exposed to elevated ambient
temperature during lactation. Domest. Anim. Endocrinol. 8: 117-127.

Biensen, N. J ., von Borell, E. H. and Ford, S. P. 1996. Effects of space allocation and
temperature on periparturient maternal behaviors, steroid concentrations, and piglet grth
rates. J. Anim. Sci. 74: 2641-2648.

Black, J. L., Mullan, B. P., Lorschy, M. L. and Giles, L. R. 1993. Lactation in the sow
during heat stress. Livest. Prod. Sci. 35: 153-170.

Brake, J ., Balnave, D. and Dibner, J. J. 1998. Optimum dietary arginine:lysine ratio for
broiler chickens is altered during heat stress in association with changes in intestinal
uptake and dietary sodium chloride. Br. Poult. Sci. 39: 639-647.

Clark, J. H., Derrig, R. G., Davis, C. L. and Spires, H. R. 1975. Metabolism of arginine
and omithine in the cow and rabbit mammary tissue. J. Dairy Sci. 58: 1808-1813.

Fraser, A. F. 1970. Studies on heat stress in pigs in a tropical environment. Trop. Anim.
Health Prod. 2: 76-86.

Ignarro, L. J ., Buga, G. M., Wood, K. S. and Byms, R. E. 1987. Endothelium-derived
relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl.
Acad. Sci. 84: 9265-9269.

Johnston, L. J ., Ellis, M., Libal, G. W., Mayrose, V. B. and Weldon, W. C. 1999. Effect
of room temperature and dietary amino acid concentration on performance of lactating
sows. J. Anim. Sci. 77: 1638-1644.

Lacasse, P., Farr, V. C., Davis, S. R. and Prosser, C.,G. 1996. Local secretion of nitric
oxide and the control of mammary blood flow. J. Dairy Sci. 79: 1369-1374.

Lublin, A. and Wolfenson, D. 1996. Lactation and pregnancy effects on blood ﬂow to
mammary and reproductive systems in heat-stressed rabbits. Comp. Biochem. Physiol.
115A: 277-285.

McGlone, J. J ., Stansbury, W. F., Tribble, L. F. and Morrow, J. L. 1988. Photoperiod and

heat stress inﬂuence on lactating sow performance and photo period effects on nursery
pig performance. J. Anim. Sci. 66: 1915-1919.

54

Mendes, A. A., Watkins, S. E., England, J. A., Saleh, E. A., Waldroup, A. L. and
Waldroup, P. W. 1997. Inﬂuence of dietary lysine levels and arginine:lysine ratios on
performance of broilers exposed to heat or cold stress during the period of three to six
weeks of age. Poult. Sci. 76: 472-481.

Messias de Braganca, M., Mounier, A. M. and Prunier, A. 1998. Does feed restriction
mimic the effects of increased ambient temperature in lactating sows? J. Anim. Sci. 76:
2017-2024.

Morris, S. M., Jr. 1998. Arginine synthesis, metabolism, and transport: regulators of nitric
oxide synthesis. In: Laskin, J. and Laskin, D. (Eds). Cellular and molecular biology of
nitric oxide. Marcel Dekker, New York, NY. pp. 57-85.

NRC. 1998. Nutrient requirements of swine (10th Rev. Ed.). National Academy Press,
Washington, DC.

Prunier, A., Messias de Braganca, M. and Le Dividich, J. 1997. Inﬂuence of high ambient
temperature on performance of reproductive sows. Livest. Prod. Sci. 52: 123-133.

Quiniou, N. and Noblet, J. 1999. Inﬂuence of high ambient temperatures on performance
of multiparous lactating sows. J. Anim. Sci. 77 : 2124-2134.

Sano, H., Arai, I-I., Takahashi, A., Takahashi, H. and Terashima, Y. 1999. Insulin and
glucagon responses to intravenous injections of glucose, arginine and propionate in
lactating cows and growing calves. Can. J. Anim. Sci. 79: 309-314.

SAS, 1998. SAS/STAT User’s Guide (Release 7.1) SAS Inst. Inc., Cary, NC.

Sigal, R. K., Shou, J. and Daly, J. M. 1992. Parenteral arginine infusion in humans:
nutrient substrate or pharmacoligic agent? J. Parent. Enteral. Nutr. 16: 423-428.

Sitren, H. S. and Fisher, H. 1977. Nitrogen retention in rats fed on diets enriched with
arginine and glycine. Improved N retention after trauma. Br. J. Nutr. 37: 195-208.

Steinbach, J. 1973. Bioclimatic inﬂuences on the reproductive processes in swine in a
humid tropical environment. Int. J. Biometeor. 17: 141-145.

Trottier, N. L., Shipley, C. F. and Easter, R. A. 1997. Plasma amino acid uptake by the
mammary gland of the lactating sow. J. Anim. Sci. 75: 1266-1278.

Valance, P., Collier, J. and Moncada, S. 1989. Nitric oxide synthesized from L-arginine
mediates endothelium dependent dilatation in human veins in vivo. Cardiovasc. Res. 23:
1053-1057.

Visek, W. J. 1985. Arginine and disease states. J. Nutr. 115: 532-541.

55

Wu, G., Flynn, N. E., Flynn, S. P., Jolly, C. A. and Davis, P. K. 1999. Dietary protein or
arginine deﬁciency impairs constitutive and inducible nitric oxide synthesis by young
rats. J. Nutr. 129: 1347-1354.

56

CHAPTER 3: INSULIN, GLUCOSE, CORTISOL, GROWTH HORMONE AND
PROLACTIN RESPONSES TO ORAL L-ARGININE SUPPLEMENTATION TO
LACTATING SOWS UNDER HEAT STRESS.
Abstract

The objective of this study was to determine whether dietary arginine decreases
weight loss of lactating sows via alteration of key metabolic hormone concentrations.
Sows were exposed to a thermoneutral (TN=20°C) or hot (HT=29.4°C) environment and
fed one of three dietary treatments in a 2 x 3 factorial design. Dietary treatments were
com-soybean meal based diets formulated to contain 0.96%, 1.34%, and 1.73% arginine
for control (C), medium (ME), and high (HI) dietary treatments, respectively. Blood
samples were obtained 2 h post-meal on day 7, 14, and 21 of lactation. Plasma insulin
and prolactin concentrations did not differ (P>0.10) in HT sows when compared to TN
sows. Plasma growth hormone (P<0.05) and glucose (P<0.01) concentrations were
higher and salivary cortisol not different (P>0.05) in HT sows compared to TN sows.
Arginine supplementation increased plasma insulin concentration (P<0.05) in sows fed
the HI and ME diet compared to C in both environments. Growth hormone concentration
in plasma decreased in sows fed the ME and HI diets compared to C diet in both
environments (P<0.05). Plasma prolactin and glucose, and salivary cortisol did not differ
(P>0.10) between C, ME, and HI diets. Arginine may mediate improvement in nutrient
utilization by regulating both insulin and GH metabolism.
Keywords:

Arginine; heat stress; sow, lactating; sow, performance; hormone

57

Introduction

Impaired lactation performance such as increased body weight loss and decreased
milk production has been well documented in sows exposed to high ambient temperatures
(Barb et al., 1991; Black et al., 1993; Messias de Braganca et al., 1998; Johnston et al.,
1999; Quiniou and Noblet, 1999). Excessive loss of body weight during lactation results
in extended weaning to estrous interval and potentially impairs subsequent reproductive
performance (Prunier et al., 1997 ; Messias de Braganca et al., 1998; Johnston et al.,
1999). It is unclear how heat stress reduces lactation performance. Few studies
conducted have shown impaired endocrine functions in heat-stressed sows (Barb et al.
1991; Prunier et al., 1997; Messias de Braganca et al., 1998; Messias de Braganca and
Prunier, 1999).

Recently, we have found that in lactating sows exposed to high environmental
temperature, supplemental dietary arginine decreases body weight loss (Chapter 1).
Other studies have shown that weight gain and feed conversion of broilers is improved
when dietary arginine is supplemented to increase the dietary arginine to lysine ratio in
hot environments (Brake et al., 1998). During post-surgical recovery, dietary arginine
supplementation increases feed utilization and increases nitrogen retention in rats (Kari et
al., 1981; Mulloy et al., 1982; Sitren and Fisher, 1977). The apparent alleviating effect of
arginine on catabolic states is poorly understood. It is well documented that arginine
stimulates the secretion of key anabolic hormones such as insulin, growth hormone, and
prolactin (Visek, 1986; Wu et al., 1998). Taken together, these notions led us to
hypothesize that arginine supplementation decreases weight loss of heat-stressed sows via

regulation of key metabolic hormones. We tested whether decreased lactation

58

performance in sows exposed to heat is related to an impaired glucose metabolism and to
a decrease in plasma GH and prolactin concentration. Second, we tested whether weight
loss reduction in heat stressed sows fed supplemental arginine is associated with an
increase in plasma insulin and GH concentration. The objectives of this study were two
fold. First, to determine whether exposure to heat modiﬁes postprandial plasma GH,
insulin, prolactin, glucose and cortisol concentration responses. Second, to determine
whether chronic dietary arginine supplementation modiﬁes the concentration response of
GH, insulin, prolactin, and glucose.
Materials and methods
Animal management, experimental design, and diets

All-University committee on Animal Use and Care, Michigan State University
approved use of animals for this experiment. Experimental procedures on animal
management, experimental design, and diets have been previously described in details
elsewhere (Chapter 1). Brieﬂy, sixty-six crossbred (Landrace x Yorkshire) multiparous
sows were used and housed in standard farrowing crates with plastic ﬂooring throughout
the duration of the experiment. Sows were weighed on d 1 post farrowing and weekly
thereafter before the morning meal. Litters were equalized to 10 or 11 pigs by cross
fostering within the ﬁrst 36 h after farrowing.

Sows were allotted to a 2 x 3 factorial design. Three weeks prior to farrowing,
sows were allocated to either a thermoneutral environment (TN: 20°C) or a hot
environment (HT: 294°C). Sows allotted to the HT environment were acclimated by

gradually increasing the room temperature in 33°C weekly increments until a maximum

temperature of 294°C was reached. On day 110 of gestation, sows were randomly

59

allocated to three dietary treatments consisting of corn-soybean meal based diets with
varying concentrations of arginine, i.e., control (C: 0.96% arg), medium (MB: 1.34%
arg), and high (HI: 1.73% arg). All diets contained 17% crude protein and 3,400 kcal/kg,
and formulated to meet or exceed NRC (1998) requirements for all other nutrients. The
M and H diets contained crystalline L-arginine. Diets were formulated to be
isonitrogenous by addition of L-glutamic acid to the C and M diets. Sows were fed 2
kg/day of their respective diet from day 110 of gestation until farrowing. Sows were fed
in a stair-step manner from day 1 to day 5 of lactation, and provided ad libitum access to
feed until weaning on d 22. Feed refusals were collected and feed intake recorded daily.
Sows had free access to water throughout the duration of the experiment. A total of six
sows were removed from the study due to a variety of reasons such as small litter size,
abortion, and (or) aggressiveness.
Sample collection and analysis
Blood

Feed was removed at 2000 to ensure a 12-hr post-absorptive status on the
following moming. Sows were fed a 2-kg meal at 0800 and blood was collected at 1000.
Blood samples were collected on d 7, 14 and 21 of lactation by jugular or ear
venipuncture using 7.0 mL BD VacutainerTM glass whole blood tubes containing EDTA
(2.1 % EDTA/mL blood) (Becton, Dickinson and Company, USA). Blood samples were
immediately put on ice and centrifuged within 30 min of collection. Sows were provided
ad libitum access to feed following blood sample collection. Plasma was aliquoted into
2-mL vials and stored at -20°C until analysis. Plasma samples were analyzed for insulin,

glucose, growth hormone, and prolactin concentration. Insulin concentrations were

60

determined in duplicate samples using a validated porcine insulin radioimmunoassay kit
(Linco, St. Charles, MO, USA). The intra assay CV was 14 %. Glucose concentrations
were enzymatically determined in duplicate samples using a commercial colorimetric
assay kit (Sigma Diagnostics, Cat. No. 315, St. Louis, MO, USA). Any duplicate sample
exceeding a CV of 10% were not included in the data set. Concentrations of growth
hormone were determined according to a previously described radioimmunoassay
(Dubreuil et al. 1990) which was validated in the laboratory of Dr. Farmer. The intra
assay CV was 4.56 % and the inter assay CV was 3.36 %. Prolactin concentrations were
determined according to previously described radioimmunoassay (Robert et al., 1989).
The cold prolactin was donated by Dr. A.F. Parlow (US National Hormone and Pituitary
Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance,
CA). Parallelism of a pool from gestating gilts was demonstrated and average recovery,
which was calculated by addition of various doses of unlabelled hormone to a pooled
sample, was 109.1%. Six samples of a representative pool of plasma or serum were
can'ied in duplicates in all assays in order to calculate coefﬁcient of variation (CV). The
intraassay CV was 4.5% and the interassay CV was 4.3%. Assay sensitivity (per tube)
was 0.1 ng.
Saliva

Saliva samples were collected from each sow at 0700 before the morning meal on
day 8, 15 and 22 of lactation. Samples were collected following the procedure described
by Gemus (1998). Samples were collected in 14-mL polypropylene vials and stored at
-20°C for later analysis of cortisol. For analysis, samples were thawed and centrifuged.

Saliva cortisol concentration was determined in duplicate samples using a commercially

61

available radioimmunoassay kit (Diagnostic Products Corp., Cat. No. TKCOZ, Los
Angeles, CA). Any duplicate sample exceeding a CV of 10 % were not included in the
data set.
Statistical analysis

Differences in plasma growth hormone, prolactin, insulin, glucose, and salivary
cortisol were determined by analysis of variance. Sources of variation in the ﬁll] model
included environment, diet, day of lactation, and trial as classiﬁcation variables and their
corresponding two-way and three-way interactions. The reduced model included
environment, diet, environment x diet interaction, and day of lactation. Sow nested
within environment x diet was included as a random effect. Statistical analysis was
performed using the MIXED procedure of SAS (1998). Repeated measures analysis was
used for repeated measures over days of lactation based on different covariance structures
(Litell et al., 1998). The best ﬁtting repeated measures covariance structure was
determined using the Akaike information criterion (AIC). Separation of means was
performed by the least squares estimates for unbalanced design. Least square means
were considered different at P<0.05. Tendency for differences between treatments was
considered at P<0.10. Data for growth hormone was log transformed to satisfy
assumptions of normal distribution. Back transformed data are presented. To test
whether the dietary treatment effect on sow body weight loss was independent of its
effect on litter growth rate, multivariate analysis of variance was performed using the
MIXED procedure of SAS (1998). Sources of variation in the full model included
environment, diet, and trial as classiﬁcation variables and all corresponding two-way and

three-way interactions. The reduced model included environment, diet, environment x

62

. diet interaction, and trial. The residual correlation (r = -0. 14) was not different from zero
(P = 0.29), indicating that treatment effect on body weight loss was independent of litter
grth rate.
Results and Discussion
Heat stress

Insufﬁcient voluntary feed intake coupled with high nutrient demand for milk
production is the primary cause of body weight loss during lactation in the sow. Under
conditions exceeding thermoneutrality, reduction in feed intake, increased weight loss,
and decreased milk production is often observed (Black et al., 1993; Messias de Braganca
et al., 1998; Quiniou and Noblet, 1999; Johnston et al., 1999). In this study, sows
exposed to 294°C had higher weight loss (P<0.001), (Fig. 3.1) and lower litter growth
rate (P<0.05), (Fig. 3.2) compared to sows exposed to 20°C. The magnitude of body
reserves mobilization is regulated by alterations in both anabolic and catabolic hormones
(Bauman and Currie, 1980). In this study, high environmental temperatures did not
decrease (P>0.10) insulin concentrations (Fig. 3.3), agreeing with previous studies in
which post-prandial insulin concentration was not affected by heat exposure in calves
(Takahashi et al., 1985) and in broiler chickens (Geraert et al., 1996). In contrast, others
have shown that the post-absorptive insulin concentrations decreased in animals exposed
to heat, such as lactating sows (Messias de Braganca and Prunier, 1999), non-lactating
dairy cows (Itoh et al., 1998a), rabbits (Trammell et al., 1988), and rats (Souza et al.,
1993). Conversely, post-absorptive insulin concentration was increased in lactating dairy
cows (Itoh et al., 1998b), growing calves (Takahashi et al., 1986), and sheep (Terashima

et al., 1996) under heat stress. Our samples were obtained 2 h post-feeding, thus

63

explaining in part the differences between the insulin response in this study and Messias
de Braganca and Prunier (1999). Plasma glucose concentration was higher (P<0.05) in
HT sows compared to TN sows (Fig. 3.4). Prunier et al. (1997) also reported higher post-
prandial glucose concentration in lactating sows exposed to heat stress. Heat stress may
reduce glucose clearance rate. We are unaware whether heat exposure causes insulin
insensitivity. In dairy cows exposed to heat stress, tendencies to lower basal glucose and
higher basal insulin concentrations have been reported (Itoh et al., 1998b). Similarly, in
non-lactating sheep subjected to heat stress, insulin concentration and glucose clearance
rate increased following a glucose challenge (Achmadi etal., 1993). Ruminant species
rely on gluconeogenesis as opposed to dietary glucose to maintain normal blood glucose
levels. For this reason, the glucose response to heat stress may differ between ruminant
and non-ruminant species.

Thus, a higher tissue mobilization for glucose synthesis combined with a reduced
utilization may in part explain the increased weight loss in sows under heat stress. We
did not measure glucagon, but heat may reduce the insulin to glucagon ratio, leading to a
higher rate of gluconeogenesis. Heat exposure increases glucagon in growing calves
(Takahashi et al., 1986). The role of cortisol, a potent proteolytic and gluconeogenic
hormone (review; Porterﬁeld, 1996), in contributing to higher blood glucose
concentration in heat stressed sows in both this study and Prunier et al. (1997) is minor.
Salivary cortisol from sows in the HT group did not differ from those of sows in the TN
group (Fig. 3.5). Furthermore, others have reported lower plasma cortisol concentrations
in sows exposed to 30°C during lactation (Barb et al., 1991; Messias de Braganca et al.,

1998), and in calves and heifers (Takahashi et al., 1986; Ronchi et al., 2001). It has been

64

suggested that lower cortisol concentration results ﬁom an adaptive decrease in
metabolism in order to reduce heat production (Barb et al., 1991; Messias de Braganca et
al., 1998). Because cortisol modulates nutrient partitioning toward milk production
(Motil et al., 1994) lower blood cortisol may contribute to decreased milk production in
hot environments (Messias de Braganca et al., 1998). Alternatively, glucose uptake by
the mammary gland may be decreased in sows exposed to heat stress. This later may
explain the decrease in litter weight gain reported in this study and by others (Black et al.,
1993; Messias de Braganca et al., 1998; Quiniou and Noblet, 1999; Johnston et al., 1999).

High ambient temperatures increased growth hormone (GH) concentration
(P<0.05). Growth hormone can cause hyperglycemia and decrease insulin sensitivity
(review; Cryer, 1981), which might explain the higher blood glucose concentration in HT
sows. A decrease in glucose utilization by peripheral tissue would ultimately increase
body nutrient utilization and increase weight loss. High GH concentration and higher
weight loss have been reported in the heat-stressed sow (Barb et al., 1991). F urtherrnore,
in the lactating sow, porcine GB or grth hormone-releasing factor (GRF)
administration increases sow weight loss (Farmer et al., 1992; Farmer et al., 1996). It is
unclear why GH concentration is higher in heat stressed lactating sows. The fact that GH
seems to decrease nutrient utilization in the lactating sow is puzzling as high GH
concentration in the growing pig increases weight gain (Etherton et al., 1987; Evock et
al., 1988; Wester et al., 1998). Feed restriction increases GH concentrations, but
decreases IGF-l concentration in pigs (Buonomo and Baile, 1991), heifers (Yambayama
et al., 1996; Choi et al., 1997), and dairy cows (Lapierre et al., 1995) housed in

thermoneutral environments. In our study, sows exposed to heat stress reduced their

65

voluntary feed intake (Chapter 1). In lactating sows exposed to heat stress, Messias de
Braganca and Prunier (1999) showed that feed intake was decreased, and although they
did not measure GH, IGF-l was decreased. Thus, the increase GH concentration may
have resulted from a depressed feed intake. Therefore, the increase loss of body weight
may have been mediated via a decrease in IGF-l.

Environmental temperature did not decrease prolactin concentration in plasma
(Fig. 3.7). In the lactating sow, prolactin is the main lactogenic hormone (Forsyth, 1986)
and its absence inhibits milk secretion (Farmer et al., 1998). Other studies conducted in
the lactating sow (Barb et al., 1991; Messias de Braganca etal., 1998) and
ovariectomized gilt (Kraeling et al., 1987) has found no effect of heat stress on prolactin
concentrations. The response in ruminant species differs from the lactating sow. In the
absence of prolactin, lactating ruminants species do not experience suppression of milk
secretion (Forsyth, 1986). In the lactating ewe exposed to heat stress, prolactin
concentration increased while milk yield remain unchanged (Stephenson et al., 1980). In
the lactating goat, prolactin concentration increased while milk yield decreased (Sano et
al., 1985). In heat stressed heifers (Tucker and Wettemann, 1976; Ronchi et al., 2001)
and ewes (Hooley et al., 1979), higher prolactin concentrations have been reported. Our
results suggest that the adverse effects of high temperatures on milk production do not
occur via a decrease in plasma prolactin concentration in the sow.
Arginine supplementation

Dietary arginine supplementation decreased weight loss in HT sows (Chapter 1).
Intravenous arginine supplementation at supraphysiological levels has been reported to

have anti-catabolic effects (Barbul etal., 1984). In this study, sows were not fed

66

supraphysiological levels of arginine, and similar responses have been observed in other
studies with dietary arginine administration. In young pigs, arginine supplemented to
diets increased weight gain and nitrogen retention (Leibholz, 1982). In humans and rats,
dietary arginine supplementation improved nitrogen balance post-trauma (Daly et al.,
1988; Elsair et al., 1978; Sitren and Fisher, 1977). The anti-catabolic properties of
arginine are not well understood. Accelerated protein catabolism occurring during
lactation, growth and post-trauma may increase arginine requirements via an increasing
demand for tissue repair and formation. Arginine can enhance reparative collagen
synthesis in humans (Barbul et al., 1990). Arginine is a precursor for omithine required
in polyamine synthesis (V isek, 1986; Jackson et al., 1986), which play an important role
in cell division, tissue growth, and differentiation (Pegg and McCann, 1982; Auvinen,
1997). Immunosupression during recovery from trauma may increase arginine
requirement via a higher demand for nitric oxide synthesis, which in turn plays an
important role in the immune response (Moncada and Hi ggs, 1995). Arginine
requirements may also increase with a higher demand on the urea cycle needed to
detoxify high ammonia levels arising from high catabolism rate (Sitren and Fisher, 1977 ;
Visek, 1986).

The anti-catabolic property of arginine may be mediated via its secretogogue
effect on speciﬁc anabolic hormones. In this study, dietary arginine supplementation
increased insulin concentration (P<0.05) (Table 3.1, Fig. 3.3). Arginine supplementation
either by oral or intravenous administration increased insulin concentration in the human
neonate (Ponté et al. 1972), growing pigs (Hertelendy et al., 1970; Atinmo et al. 1978),

dairy cows (Hertelendy et al., 1970; Chew et al., 1984; Vicini et al. 1988), sheep

67

(Hertelendy et al., 1970; Davis, 1972), lactating cows and growing calves (Takahashi et
al., 1985; Sano et al., 1999). Arginine supplementation increased plasma glucose
concentration (P<0.05) in TN sows (Table 3.1, Fig. 3.4), but did not further increase
plasma glucose concentration in sows in the HT environment relative to the C sows.
Others have shown that arginine administered orally or intravenously stimulates both
insulin and glucose secretion rapidly after administration with values retuming to basal
concentrations within 45-60 minutes (Vicini et al., 1988; Ponté et a1. 1972). A positive
association between insulin and nitrogen balance has been reported in the lactating
woman (Motil et al., 1994). Thus, the decrease in body weight loss upon arginine
supplementation may have been mediated via an increase in insulin.

Growth hormone concentration was not enhanced by arginine supplementation in
this study. The role of GH as an anabolic hormone is well documented in growing
animals. Exogenous porcine GH administration to neonatal pigs and adult pigs increases
weight gain and nutrient utilization for protein accretion (Etherton et al., 1987; Evock et
al., 1988; Wester et al., 1998). However, the role of GH in the lactating sow is less
understood. In this study, GH was lower (P<0.01) in sows fed supplemental arginine in
both environments compared to the C sows. Kirchgessner et a1. (1991) fed supplemental
arginine to lactating sows and reported no change in plasma GH concentration. In calves,
dietary arginine decreased GH via depression of GH pulse amplitude (Fligger et al,
1997). In contrast, other studies conducted with human neonates (Ponté 197 2), adult
humans (Rakoff et al., 1973; Isidori et al., 1981; Corpas et al., 1993), dairy cows

(Hertelendy et al., 1970; Chew et al., 1984; Vicini, 1988), sheep (Hertelendy et al., 1970;

68

Davis, 1972), and growing pigs (Atimno et al., 1978), have shown that arginine
administration increases GH concentration.

While arginine supplementation decreased weight loss in heat-stressed sows it did
not improve their litter grth rate. Litter growth rate was lower in the ME group, which
also corresponded to lower plasma prolactin concentration (Fig. 3.7). Prolactin secretion
is stimulated by intravenous arginine administration in adult humans (Rakoff et al.,
1973), dairy cows (Chew et al., 1984), sheep (Davis, 1972), and growing pigs (Atinmo et
al., 1978). In this study, arginine was not administered intravenously, and the dietary
dose corresponded to 0.05 to 0.2 g/kg body weight. The stimulatory effect of arginine on
prolactin seen in other studies occurs with doses well exceeding physiological levels.

The fact that sows in the ME diet also had higher insulin concentration may offer another
explanation for the reduction in litter growth rate. Exogenous insulin administration to
lactating sows decrease weight loss but decrease litter growth rate (Reynolds and Rook,
1977)

The results of this study indicate that arginine given at dietary concentrations
above NRC (1998) requirements for maintenance and lactation under hot environmental
temperatures reduces weight loss, but does not improve litter grth rate.

Implications

Alterations in GH metabolism and reduction in glucose utilization seem to play an
important role in the depressed performances observed in sows exposed to heat stress.
The reduction in sow body weight loss with dietary arginine supplementation may be
mediated via an increase in insulin concentration and peripheral glucose utilization.

Dietary supplementation with crystalline L-arginine is beneﬁcial in decreasing body

69

weight loss. However, with the notion that arginine does not improve litter grth rate,
the use of L-arginine in lactating sow diets may not be recommendable, nor practical.
Acknowledgements

The authors wish to thank Nutri-Quest, Inc. for partial funding of this experiment.
We also wish to acknowledge Dr. Pierre Lacasse for his input and ideas concerning the
metabolism of the hormones studies and Alan Snedegar and the staff at the Michigan

State University Swine Farm for their help in this project with animal care and assistance.

70

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Table 3.2 Effect of L-arginine supplementation on metabolic hormones glucose

 

 

Item Overall
Cb + Argc P-valuec

n = 43

Insulin, uU/mL 13.5 :t 2.1 19.9 :t 2.1 <0.01
Glucose, mg/dL 93.9 d: 1.3 97.4 i 1.3 0.04
Cortisol, ug/dL 0.35 d: 0.04 0.43 :t 0.05 0.11 '
Growth hormone, ng/mLf 5.2 :t 0.3 4.1 i 0.3 <0.0l
Prolactin, ng/ml 31.0 at 2.8 28.3 i 2.8 0.40

 

‘ Data are least square means i SEM.

b Control diet (0.96% arginine).

° Supplemental arginine (1 .34% and 1.74% arginine).

d Main effect of supplemental arginine within environmental temperature.
° Main effect of dietary arginine supplementation.

f Backstransformed data represents medians and 95% conﬁdence intervals.

72

Fig. 3.1 Effect of environmental temperature on sow body weight loss during a 21-day
lactation period. * indicates difference at P<0.05 between HT and TN. No difference at
P>0.05 is indicated by ns.

73

 

 

 

 

 

 

0.7 -

0.6 -

0.5 r

0.4 -

29.

0.3 —

0.2 -

0.1 -

0.0

74

Fig. 3.2 Effect of environmental temperature on litter average daily gain during 21 day
lactation period. * indicates difference at P<0.05 between HT and TN. No difference at
P>0.05 is indicated by ns.

75

g/d

240 -

220 4

200 —

180

 

 

 

76

Fig. 3.3 Effect of environmental temperature and arginine supplementation on plasma
insulin concentration. * indicates differences at P<0.05, comparing the control diet (C) to
the medium (M) and high (H) diets within each environment. Different letters represent
difference at P<0.05 between TN and HT sows fed C diet. No difference at P>0.05 is
indicated by ns.

77

 

 

 

 

  

 

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.. t.. y- it...»
. , .‘X‘ (a?

    

 

 

 

30-

 

.53...

78

Fig. 3.4 Effect of environmental temperature and arginine supplementation on plasma
glucose concentration. * indicates differences at P<0.05, comparing the control diet (C)
to the medium (M) and high (H) diets within each environment. Different letters
represent difference at P<0.05 between TN and HT sows fed C diet. No difference at
P>0.05 is indicated by ns.

79

120 '

110 '

 

 

 

 

//
//

 

80

Fig. 3.5 Effect of enviromnental temperature and arginine supplementation on salivary
cortisol concentration. "' indicates differences at P<0.05, comparing the control diet (C)
to the medium (M) and high (H) diets within each environment. Different letters
represent difference at P<0.05 between TN and HT sows fed C diet. No difference at
P>0.05 is indicated by ns.

81

3

 

.9. ,
n. N
Mg...”

’5

.(vv

\.

 

 

 

 

 

20-

18-

16-

u
4
1

u
2
1

u
o
1

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82

Fig. 3.6 Effect of environmental temperature and arginine supplementation on plasma
GH concentration. Back transformed data is presented. * indicates differences at
P<0.05, comparing the control diet (C) to the medium (M) and high (H) diets within each
environment. Different letters represent difference at P<0.05 between TN and HT sows
fed C diet. No difference at P>0.05 is indicated by ns.

83

 

  
 

 

 

 

 

 

   

.

I an.

    

«1‘

BBL. c

 

 

n

 

LDA
Q.<.w

 

    
 

       

   

 

 

84

Fig. 3.7 Effect of environmental temperature andarginine supplementation on plasma
prolactin concentration. * indicates differences at P<0.05, comparing the control diet (C)
to the medium (M) and high (H) diets within each environment. Different letters
represent difference at P<0.05 between TN and HT sows fed C diet. No difference at
P>0.05 is indicated by ns.

85

 

 

 

 

 

 

30-

:59.

10-

 

86

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92

CHAPTER 4: SUPPLEMENTAL L-ARGININE IN DIETS OF LACTATING SOWS
UNDER HEAT STRESS: EFFECT ON PLASMA NITRIC OXIDE AND MILK
AMINO ACID CONCENTRATION
Abstract

The objective of this study was to determine whether heat stress decreases plasma
and milk arginine concentration and increases nitric oxide production, and whether
arginine supplementation increases plasma and milk arginine concentration. Sows were
exposed to a thermoneutral (TN=20°C) or hot (HT=29.4°C) environment and fed one of
three dietary treatments in a 2 x 3 factorial design. Dietary treatments were com-soybean
meal based diets formulated to contain 0.96%, 1.34%, and 1.73% arginine for control
(C), medium (M), and high (H) dietary treatments, respectively. Heat exposure decreased
plasma amino acid concentration of isoleucine, phenylalanine, and threonine (P<0.05)
and decreased milk concentrations of arginine, leucine, lysine, phenylalanine and valine
(P<0.05) compared to TN sows. Arginine supplementation increased arginine and
threonine concentrations in plasma (P<0.05) and increased proline concentration in milk

(P<0.05). Nitric oxide production was not increased by either heat exposure or arginine

supplementation.

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Introduction

Exposure to high ambient temperatures reduces milk production in sows as
estimated by decreased litter weight gain (Black et al., 1993; Prunier et al., 1997; Johnston et
al., 1999). Heat stress has been shown to decrease milk protein percentage in dairy cows
(Knapp and Grummer, 1991). Plasma amino acids are the precursors for milk protein
synthesis and the rate of amino acid uptake by the mammary gland depends on plasma
amino acid concentration, blood ﬂow rate through the mammary gland, and mammary
amino acid transport system regulation (Trottier et al., 1997). The consequence of heat
exposure in lactating sows on plasma and milk amino acid concentration has not been
investigated. Heat stress increases blood ﬂow to cutaneous tissue to facilitate heat
dissipation (Zhang et al., 1994). This may result in the following: 1) decrease blood ﬂow to
the mammary gland, decreasing nutrient availability for milk protein synthesis (Black et al.,
1993); 2) increase arginine metabolism since arginine is the precursor for nitric oxide.
Under normal physiological conditions, uptake of arginine (Arg) by the porcine mammary
gland exceeds the amounts secreted in milk (Trottier, 1997) and similar results have been
reported in the bovine and rabbit mammary gland (Clark et al., 1975). Higher retention of
arginine by the mammary gland suggests that arginine may be required for processes other
than milk protein synthesis, thus increasing the current requirement estimates published by
NRC (1998). Empirical arginine requirement estimates for the lactating sow have not been
determined. Current requirement estimates have been derived factorially from arginine
output in milk (NRC, 1998). this does not account for arginine required for optimal
mammary growth as demonstrated in the rat (Pau and Milner, 1982) and for the synthesis of

proline (Pro), omithine (Om), citrulline (Cit), glutamate (Glu), creatine, and nitric oxide

94

(N O). Arginine deﬁciency decreases plasma concentrations of arginine in animals, and
impairs NO synthesis in young rats (Wu et al., 1999). Arginine is reported to be deﬁcient in
milk of humans (Davis etal., 1994) and sows (Wu and Knabe, 1994) to meet the offspring’s
arginine requirement for grth (Wu et al., 1997). The sow’s milk provides approximately
40 % or less of the daily arginine required by 7-day old nursing pigs, based on arginine
deposition and catabolism (Wu et al., 2000).

The objectives of this study were ﬁrst, to determine whether exposure to heat
stress decreases plasma and milk arginine concentration. Second, to determine if arginine
supplementation increases NO production and milk arginine concentration.

Materials and methods
Animal management and Housing

Use of animals for this experiment was approved by All-University Committee on
Animal Use and Care, Michigan State University. Experimental procedures have been
previously described in details elsewhere (Chapter 1). Brieﬂy, forty-two crossbred
(Landrace x Yorkshire) multiparous sows were used in three replicates (rep. 1, n=12; rep.
2, n=12; rep. 3, n=18) and housed in standard farrowing crates with plastic ﬂooring
throughout the duration of the experiment. Sows were weighed on day 1 post farrowing
and weekly thereafter before the morning meal. Litters were equalized to 10 or 11 pigs
by cross fostering within the ﬁrst 36 hours after farrowing.

Design and diets
Sows were allotted to a 2 x 3 factorial design. Three weeks prior to farrowing,

sows were allocated to either a thermoneutral environment (TN: 20°C) or a hot

environment (HT: 29.4°C). Sows allotted to the HT environment were acclimated by

95

gradually increasing the room temperature in 33°C weekly increments until a maximum
temperature of 294°C was reached. On day 110 of gestation, sows were randomly
allocated to three dietary treatments consisting of com-soybean meal based diets with
varying concentrations of Arg, i.e., control (C: 0.96% arg), medium (M: 1.34% arg), high
(H: 1.73% arg). A detailed description on the dietary ingredient and nutrient composition
has been described previously (Chapter 1). Brieﬂy, all diets contained 17% crude protein
and 3,400 kcal/kg and formulated to meet or exceed NRC (1998) requirements for all
nutrients. The M and H diets contained crystalline L-arginine. Diets were formulated to
be isonitrogenous by addition of L-glutamic acid to the C and M diets. Sows were fed 2
kg/day of their respective diet from day 110 of gestation until farrowing. Sows were fed
in a stair-step manner from day 1 to day 5 of lactation, and provided ad libitum access to
feed until weaning on d 22. Feed refusals were collected and feed intake recorded daily.
Sows had free access to water throughout the duration of the experiment.
Sample collection and analysis
Blood

Feed was removed at 2000 to ensure a 12-hr post-absorptive status on the
following morning. Sows were fed a 2-kg meal at 0800 and blood was collected at 1000.
Blood samples were collected on day 21 of lactation by jugular or ear venipuncture using
7.0 mL BD VacutainerTM glass whole blood tubes containing EDTA (2.1 % EDTA/mL
blood) (Becton, Dickinson and Company, USA) and immediately put on ice and
centriﬁrged. Sows were provided ad libitum access to feed following blood sample
collection. Plasma was aliquoted into 2-mL vials and stored at -20°C until analysis.

Plasma samples were analyzed for amino acids (rep. 3 only) and nitric oxide

96

concentration (rep. 1, 2 and 3). Plasma amino acids were analyzed by reverse phase-high
pressure liquid chromatography (Pico-Tag, Waters USA) following the procedure
described by Guan (2000). Concentrations of nitric oxide were determined based on
nitrite in duplicate samples using a validated colorimetric assay (Roche Diagnostics, Cat.
No. 1 756 281, Indianapolis, IN, USA). Samples were initially ﬁltered by centrifuging
one part plasma with one part sodium phosphate buffer using Amicon Centriﬁ'ee 30,000
M. Wt. cut off centrifugal ﬁlters (Millipore Corporation, Cat. No. 4104, Bedford, MA,
USA). Centriﬁrgation was performed at 1250 x g for 45 minutes at 20°C. Any duplicate
sample exceeding a CV of 10% were not included in the data set.
Milk

On (1 7, 14, and 21 of lactation milk samples were obtained from sows in rep. 3
(n=18). Milk was removed manually from the ﬁrst two anterior mammary glands
following an I.V. injection of 10 I.U. oxytocin. Milk samples on day 21 were collected
immediately following blood collection. Amino acid concentration in milk was
determined following acid hydrolysis by reverse phase-high pressure liquid
chromatography (Pico-Tag, Waters USA) following the procedure described by Guan
(2000)
Statistical analysis

Differences in plasma amino acid, plasma nitric oxide, and milk amino acid
concentrations were determined by analysis of variance. For plasma amino acid and
nitric oxide concentrations, sources of variation in the model included environment and
diet as classiﬁcation variables and their corresponding two-way interaction. For milk

amino acid concentration, day of lactation was included in the model with repeated

97

measures statement. Repeated measures analysis was used for repeated measures over
days of lactation based on different covariance structures (Littell et al., 1998). Sow
nested within environment x diet was included as a random effect. Statistical analysis
was performed using the MIXED procedure of SAS (1998). Separation of means was
performed by the least squares estimated for unbalanced design. Least square means
were considered different at P<0.05. A tendency to differ was considered at P<0.10.
Results and Discussion
Eﬂect of heat stress

Sows in both environments were fasted overnight (12 hrs) and fed a 2-kg morning
meal. This ensured that all sows had consumed 2 kg of their respective diets at the time of
bleeding. Differences in plasma amino acid concentration should thus reﬂect differences in
metabolism of these amino acids and not differences in feed intake. Heat exposure did not
decrease plasma arginine concentration (P>0.10) but decreased plasma concentrations
(P<0.05) of isoleucine (Ile), phenylalanine (Phe), and threonine (Thr) compared to TN sows
(Table 4.1). Leucine (Leu) and valine (Val) tended to be lower (P=0.07) in HT sows
compared to TN sows. Of the dispensable amino acids, Pro and tyrosine (Tyr) were lower
(P<0.05), while asparagine (Asn) tended to be lower (P=0.08) in HT sows compared to TN
sows (Table 4.2). The synthesis of Pro and arginine may have been reduced in HT sows as
a mean to conserve arginine for synthesis of NO. A reduction in plasma amino acid
concentration in lactating sows exposed to heat stress has not been reported before. Indeed,
there is a dearth of information on heat stress and amino acid metabolism in the non-
rtnninant species. A deﬁciency of a single amino acid relative to the mammary gland’s

requirement can decrease protein synthesis by the mammary gland (V andeHaar, 1999). The

98

decrease in branched-chain amino acids in plasma may be indicative of higher uptake by
muscle and a higher rate of degradation. High rate of degradation for the branched-chain
amino acids has been reported in muscle during low protein intake (Reeds and James, 1983).
This is unlikely since Ala concentration did not change with heat exposure in this study and
the branched-chain aminotransferase enzyme activity in muscle is low during lactation
(DeSantiago et al., 1998). DeSantiago et a1. (1998) reported that the capacity of the
mammary gland to catabolize branched-chain amino acids is increased during lactation,
however this is unlikely to be increased under heat stress. Thermal stress can cause
respiratory alkalosis (review; Hill and Wyse, 1989), but we are unaware as to whether
alkalosis increases hepatic amino acid uptake and metabolism. Acute acidosis inhibits
amino acid uptake by the liver (Boon et al., 1994).

Catecholarnines are synthesized from Phe and Tyr (Rios et al., 1999) and we are
unaware of whether thermal stress stimulates the synthesis and/or secretion of
catecholamines. With this notion in mind, the lower plasma Phe and Tyr concentration in
HT sows may have resulted ﬁom increased synthesis of catecholamines. We have no
reason to postulate that impaired gut absorption or amino acid catabolism at the gut level
may have occurred in HT sows. Of the eight amino acids found to decrease, seven can be
glucogenic. It is possible that HT sows had a higher rate of utilization of these amino acids
for fuel purposes. We have previously reported that sows exposed to heat stress have higher
plasma glucose concentrations than sows housed in a thermoneutral environment (Chapter
2).

Since milk samples were collected approximately 5 minutes after blood samples

were obtained, we expect the response in milk amino acids to result from differences in

99

metabolism of these amino acids and not from differences in feed intake. Milk from
sows in the HT environment had lower concentrations (P<0.05) of Arg, Leu, lysine
(Lys), Phe, and Val, while tended to have lower concentrations (P=0.07) of histidine
(His). Heat exposure also decreased (P<0.05) the concentration of the dispensable amino
acids Asn and Pro, and tended to decrease Ala (P=0.06), Glu (P=0.09), and glycine
(P=0.07) in milk.

Black et al. (1993) proposed that heat stress decreases blood ﬂow to the mammary
gland, which in turn may decrease the availability of plasma amino acids for milk
synthesis. Under this condition, NO production would be expected to increase in the
peripheral circulation. For instance, increased NO production stimulates increased
capillary blood ﬂow to the rat tail in response to heat exposure for heat dissipation
(Zhang et al., 1994). However, in this study, heat stress did not increase nitric oxide
production (P>0.10; Fig 4.1). This could suggest that blood ﬂow is not the limiting
factor in nutrient availability to the mammary gland under heat stress. An impaired
uptake of amino acids by the mammary gland in animals exposed to heat stress has not
been reported. In turn, the decreased availability in plasma of Leu, Phe, Pro, and Val
may possibly decrease the concentration of these amino acids in milk. For instance,
decreased Lys concentration in milk may suggest that the uptake of lysine by the gland
was depressed since Lys concentration in plasma was unchanged by heat exposure. In
vitro, high concentrations of insulin, cortisol, and prolactin increased arginine uptake by
the rat mammary gland (Shanna and Kansal, 2000). Insulin and prolactin are positively
correlated in vivo with amino acid uptake by the porcine mammary gland (Guan et al.,

2000). However, we have previously reported that insulin, prolactin, and cortisol are not

100

affected in HT sows (Chapter 2), suggesting that other factors are responsible for
decreasing milk amino acid concentration in HT sows.
Arginine supplementation

Plasma amino acid concentrations as affected by arginine supplementation are
shown in Table 4.1 and 4.2. Arginine supplementation increased plasma concentrations
of Arg and Thr (P<0.05) and tended to increase Ile (P<0.10). Sows fed the ME diet had
higher concentrations (P<0.05) of these amino acids than sows fed the HI diet.
Concentrations of Glu and Ala tended to be lower (P<0.10) in sows fed the ME and'HI
diet compared to C (Table 4.2). This could in part be explained by the substitution of
crystalline L-arginine by L-glutamic acid as a source of nitrogen to formulate
isonitrogenous diets. Arginine infusion increased Arg, Orn, and urea in plasma of cows
(V icini et al., 1988), and increased plasma Orn in the pig (Southern and Baker, 1982). In
our study, Om seemingly increased without reaching statistical signiﬁcance (P=0.16;
Table 4.1). Because citrulline concentrations did not increase, and we did not measure
plasma urea nitrogen, we cannot conclude whether arginine degradation via the arginase
pathway was increased. Although arginine and lysine share the same transport system
across cells (Wu and Morris, 1998), lysine concentration in the plasma were not affected
by arginine supplementation, suggesting that absorption of Lys at the gastrointestinal
tract was not impaired by high Arg supplementation. Similar results have been reported
in growing pigs were increasing arginine to lysine ratio did not affect lysine absorption
(Anderson et al., 1984). Uptake of lysine by the lactating porcine mammary tissue was
strongly inhibited by arginine (Hurley et al., 2000) but this competitive inhibition

occurred at concentrations exceeding physiological levels.

101

Arginine supplementation increased (P>0.05) milk proline concentration in HT
sows, but did not increase milk arginine concentration in either TN or HT sows (Table
4.3). This suggests that Arg was being converted to Pro within the mammary gland. Yip
and Knox (1972) reported the presence of arginase in the rat mammary gland, and the
absence of carbamoyl synthetase I and omithine carbamoyl transferase. Thus, high
arginase activity in the intact gland corroborates Pro synthesis from Arg (Yip and Knox,
1972). Clark et al. (1975) reported that the metabolic fate of arginine in the bovine and
rabbit mammary gland was for the synthesis of Pro and Glu. It has been reported that
sows milk is deﬁcient in arginine (Wu et al., 2000), and that proline can ameliorate
arginine deﬁciency in young pigs (Brunton et al., 1999). Ball et a1. (1986) has shown that
proline is a dietary essential amino acid in the nursing pig. However, albeit arginine
dietary supplementation was shown to increase milk proline concentration in this study,
there was no improvement in litter weight gain (Chapter 2).

Mammary blood ﬂow is rapidly increased by intramammary inﬁrsion of
diethylamine NONOate, a NO donor (Lacasse et al., 1996). Nitric oxide synthesis, as
well as the presence of NOS, was detected in the mammary gland of the goat suggesting
that the epithelial cells can control their own supply of blood and nutrients (Lacasse et al.,
1996). Furthermore, in the early lactating rabbit, heat stress diverts mammary blood ﬂow
from the mammary gland (Lublin and Wolfenson, 1996). If heat stress increases demand
for blood ﬂow to the mammary gland, a higher demand for NO production may increase
arginine requirement to the mammary gland. Arginine supplementation failed to increase
NO concentration in both TN and HT sows (P>0.10). Therefore, it is unlikely that heat

exposure increases the arginine requirement for NO production in lactating sows.

102

Heat exposure to lactating sows decreased the concentration of an important
number of amino acids in both plasma and milk but did not increase peripheral NO
concentration. Dietary arginine supplementation increased arginine and threonine in
plasma and increased proline concentration in milk, but did not increase NO production
in heat stressed sows compared to thermoneutral sows.

Implications

Alterations in the milk amino acid proﬁle of sows exposed to high ambient
temperatures may affect grth rates of the nursing pigs by offering a less than optimal
milk amino proﬁle. This could explain the decreased litter weight gain observed in sows
exposed to high ambient temperatures. Our results suggest that arginine requirement for
milk production is not increased by heat exposure. This implication is based on two
facts: 1) NO production was not increased in sows exposed to heat stress and (or)
receiving dietary arginine supplementation; 2) heat exposure did not decrease arginine
concentration in plasma. Future investigation on amino acid metabolism by the
mammary gland in sows exposed to heat stress may provide useful information for
implementation of new nutritional strategies in sows suffering from heat stress.
Acknowledgements

The authors wish to acknowledge Nutri Quest, Inc. for partial funding of this
project. We wish to acknowledge Alan Snedegar and staff at the Michigan State
University Swine Farm for their help in this project with animal care and assistance. We

would also like to acknowledge Dr. Xinfu Guan for his input in amino acid metabolism.

103

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107

Fig. 4.1 Effect of environmental temperature and arginine supplementation on plasma
NO concentration. * indicates differences at P<0.05, comparing the control diet (C) to
the medium (M) and high (H) diets within each environment. Different letters represent
difference at P<0.05 between TN and HT sows fed C diet. No difference at P>0.05 is
indicated by ns.

108

 

 

 

 

 

 

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109

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112

CHAPTER 5: EFFECT OF HEAT STRESS ON SOW NURSING BEHAVIOR AND
CONSEQUENT LITTER PERFORMANCE.

Abstract

The objective of this study was to investigate whether high ambient temperatures
impair milk transfer by altering the sow’s nursing behavior. A total of 24 multiparous
sows were allotted in a randomized block design in two replicates. Sows were
maintained in a thermoneutral environment of 20°C (TN) or heat-stressed in a hot
environment of 294°C (HT), starting on day 95 of gestation. Sows were videotaped with
their litters on day 3, 6, 12, 15, and 21 of lactation for a period of 8 hrs. Behaviors
observed were nursing attempts, ratio of successful vs. unsuccessful nursings, interval
between successful nursings, behavior of the piglets before a nursing event, behavior of
the sow to terminate a nursing event. The number of nursing attempts, the interval
between successful sucklings, and the frequency of successful suckling events was not
affected by environmental treatment (P>0.10). In TN sows, the odds of a successful
suckling were higher in 21 day old pigs than younger pigs (P<0.05). Interestingly, at 3
days of age the interval between successful sucklings was longer and the odds of a
successful suckling were lower in the HT environment (P<0.05). Heat stress decreased
the ﬁ'equency of the sow terminating a nursing event by sitting up (P>0.001). Piglets’ in
the HT environment fell at sleep at udder more often than pigs in TN environment
(P<0.001 ).

Keywords: suckling behavior, lactation, heat stress, sow

113

Introduction

Sows exposed to temperatures exceeding 12-22°C, their thermoneutral zone,
(Black et al., 1993) show decreased milk production characterized by a 10 to 35 %
decrease in average daily gain of nursing pigs (Schoenherr et al., 1989; Vidal et al.,
1991). Physiological and endocrinological adaptations to heat stress cannot fully explain
the decrease in milk production in heat-stressed sows (Chapter 2 and 3). In mammals,
milk transfer to the offspring during lactation is a major maternal investment (Pond,
1977; Lee, 1987). The impact of environmental temperature on maternal investment has
not been investigated in the sow. The difﬁculty in measuring milk transferred to the
offspring has led investigators to rely on behavioral measures of suckling to estimate
milk output (Cameron, 1998). Furthermore, methods of measuring milk intake, such as
weigh-suckle-weigh, interfere with normal suckling patterns (Pettigrew, 1985), and could
potentially alter normal milk output. Despite a high motivation to nurse from the piglets,
not all sucklings result in milk transfer (N ewberry and Wood-Gush, 1985; Rushen and
Fraser, 1989; Bee and Jensen, 1995), with 20 to 30 % of nursing attempts being
unsuccessful (Whatson and Bertram, 1980; Newberry and Wood-Gush, 1985; Jensen et
a1, 1991). Milk ejection is identiﬁed by the presence of the nursing phase during which
piglets assume stance position with characteristic rapid sucking (Castrén et al., 1989b).
An unsuccessful suckling (US) or a non-nutritive suckling event is deﬁned as a nursing
attempt where milk ejection is absent, this is characterized by no increase in intra-
mammary pressure and no oxytocin release (Ellendorff and Poulain, 1984).

Behavioral indicators of US may be restlessness of nursing pigs, intense

stimulation of the udder and frequent attempts to suckle, while the sow may be restless

114

and change positions repeatedly (Ellendorff and Poulain, 1984). Castrén et al. (1989a)
characterized an US as a nursing event where the grunting of the sow was not increased
and rapid sucking movements by the pigs were not observed. There is some evidence
that US could be caused by premature resumption of nursing (Fraser, 1977; Whatson and
Bertram, 1980; Ellendorff et al., 1982). In a thermoneutral environment, success of milk
ejection varies between 69 and 77% of all nursing attempts of sows (Jensen et al., 1991).
Higher rates of US have been suggested to occur in sows under heat stress (Fraser, 1970).
An altered suckling behavior may depress the frequency of milk ejection reﬂex and
consequently limit transfer of milk from the sow to the litter (Hartmann et al., 1997).
Studies looking at sow and piglet behavior before an US have reported no
changes in the behavior of the sow that could predict the failure of the nursing event
(Ellendorff et al., 1982). Previous research has demonstrated that, pigs are more active
during the time previous to an US compared to a successful one (Fraser, 1977; Ellendorff
et al., 1982; Castrén et al., 1989a). Castrén et al. (1989) speculated that the increased
activity of the piglets before an US may be a contributory cause to the outcome of the
event. If the piglets are active it may be more difﬁcult for them to synchronize the tactile
stimuli to the udder. Milk ejection occurs only when most of the litter simultaneously
massages the udder (Ellendorff et al., 1982; Algers et al., 1990). Furthermore, Ellendorff
et al. (1982) reported that milk ejection was never observed when the piglets do not
synchronize their efforts. A high suckling stability within the litter helps to ensure that
piglets suckle frequently with less competition (DePasillé et al., 1989). This could
potentially explain an impaired milk production, hence a depressed litter weight gain

observed in heat stressed sows.

115

An alternative explanation is that the sow may be restricting the piglets access to
the gland by postural changes and therefore decrease the frequency of suckling events as
suggested by Hartmann et al. (1997). Postural changes, such as increase time spent
standing, sitting, and in stemal recumbence may limit the pigs access to the udder
(dePasillé and Robert, 1989; Gotz, 1991), which can translate in a decrease willingness to
nurse by the sow. In early lactation, half of the suckling events are terminated by the pigs
moving away from the udder (Whatson and Bertram, 1980). As lactation progresses, the
sow terminates most of the suckling events by standing, sitting or restricting access to the
gland (Whatson and Bertram, 1980). The behavior of sows under heat stress could
impair the pigs’ access to milk under hot environments. Growing pigs exposed to high
ambient temperatures exhibit more lying behavior (Hicks et al., 1998), probably for heat
dissipation as well as to reduce physical activity, hence heat production. Therefore, an
increased time spent in stemal recumbency may help the sow dissipate heat and may, as a
consequence, limit the piglets’ access to the mammary gland. Previous work has failed
to identify alterations of the lactating sow’s physical activities, i.e., standing and sitting,
when kept under high ambient temperatures (Kelley and Curtis, 197 8; Biensen et al.,
1996). The possible reasons for the lack of association are a small sample size (n = 10) in
one study (Kelley and Curtis, 1978), while in the other study environmental temperature
was not controlled (18.3 to 35.5 °C) and for the lactating sow 18.3° C is in the
thermoneutral zone (Biensen et al., 1996).

The aims of this study were ﬁrst, to determine if sows exposed to heat stress
diminish their maternal investment measured by the ratio of US to successful sucklings

(SS). Second, to determine if the decrease in milk production observed under heat stress

116

is the result of alterations in nursing behavior. We hypothesized that a decreased
frequency of SS and an increased interval between SS occur during periods of heat stress.
Materials and methods
Experimental Design

Twenty-four crossbred (Landrace x Yorkshire) multiparous lactating sows and
their litters were used in a randomized block design in two replicates. Litter size was
adjusted to 10 or 11 piglets by cross-fostering within 36 hrs aﬁer birth. Sows and their
litters were housed in standard farrowing crates (24” x 7’) throughout the lactation period
at the Michigan State University Swine Research and Teaching Facility. Heat lamps
were provided to the piglets for the ﬁrst 3 days of life. Sows were allocated to one of two
environmental temperatures on day 95 of gestation. Environmental temperatures were
adjusted in a control room at 20°C (TN: thermoneutral) or a hot room at 294°C (HT: heat
stress). Photoperiod was controlled to 12 hrs of light and 12hrs of darkness. Sows were
provided ad libitum access to feed and water throughout the study. No creep feed was
offered to the litters. Ventilation in the farrowing rooms was provided by mechanical
forced air.
Behavioral observations

Sows were videotaped with their litters on day 3, 6, 12, 15, and 21 of lactation.
Behavioral observations were carried out for 8 hrs from 0800, after the morning meal, to
1600, before the evening meal. Sows were allowed to nurse their piglets without any
human intervention for the data collection period. The equipment used were CCTV
cameras (Panasonic, Model No. WV-BP330) mounted with 4.5 mm aspherical TV lenses

(Panasonic, Model No., WV-LA408C3). Cameras were attached to a multiplexer video

117

monitor (Panasonic, Model No., WV-CM146) and data recorded into a time lapse VCR
(Panasonic, Model No., AG-6740. A trained observer decoded tapes using check sheets.
Behavioral sampling and continuous recording were the techniques used (Martin and
Bateson, 1986). The occurrence of each nursing event was recorded. The number of
nursing attempts, success of milk ejection by presence of characteristic suckling phase
and stance of pigs, and the interval between SS were noted. To assess if the piglet’s
activity inﬂuence the outcome of a nursing event, i.e., SS or US, the activity of the
nursing pigs 5 minutes before a nursing attempt was characterized as sleeping or active.
The pigs’ activity 5 minutes after a nursing attempt was characterized as sleeping at
udder, or massaging (and suckling) the udder in order to assess if the piglets’ behavior is
altered by the outcome of the nursing event. Nursing events were terminated by the sow
through postural changes, i.e., stands, sits, or restricts access to udder. The piglets’
behavior after a suckling event and the sows’ behavior in terminating a suckling event
were considered mutually exclusive variables. That is, if the sow terminates a nursing by
sitting, standing, or restricting access to the udder, the piglets cannot sleep at the udder or
continue massaging the udder. An activity was recorded only when 80 % or more of the
piglets were engaged in it.
Statistical Analysis

Differences in the number of nursing attempts and the interval between SS were
determined by analysis of variance. Sources of variation in the full model included
environmental temperature, day of lactation, and their two-way interaction. Sow nested
within environment was included as a random effect. Statistical analysis was performed

using the MIXED procedure of SAS (1998). Repeated measures analysis was used for

118

repeated measures over day of lactation based on different covariance structures (Litell et
al., 1998). The best ﬁtting repeated measures covariance structure was determined using
the Akaike information criterion (AIC). Separation of means was performed by the least
square estimates for unbalanced design. Least square estimates were considered different
at P<0.05.

The difference in the ratio of SS to (US + SS) between environments was
determined by logistic regression analysis using GENMOD procedure of SAS (1998).
Sources of variation in the full model included environmental temperature, day of
lactation, and the two-way interaction.

The effect of heat stress and success of suckling on the activity of nursing pigs
before and aﬁer a nursing event was analyzed using the Fisher’s exact test. The
association between success of suckling and the behavior of the piglets and sows after a
nursing between environments was analyzed using the McNemar’s exact test.

Results

Overall, there was no difference in the interval between SS of TN and HT sows;
60.0 min and 60.4 min, respectively (P>O. 10). Over the entire lactation period, the odds
ratio of a SS event was 1.1 for TN sows versus HT sows (P>0.10; Fig. 5.1). The number
of nursing attempts was not different between TN and HT sows; 1.3 nursings/hr and 1.4
nursings/hr, respectively (P>0.10).

In the TN environment, age of nursing pigs did not affect the interval between SS
events (P>0.10). Age of the pigs affected the outcome of the nursing events. The odds of

a SS were lower (P<0.05) on day 3 (3.2), day 6 (3.2), day 12 (6.2) and day 15 (4.0) than

119

day 21 (23.0), for sows housed in the TN environment. There were no differences in the
number of nursing attempts from sows housed in the TN environment (P>0. 10).

In the HT environment, the interval between SS events was longer (P<0.01) on
day 3 (82.7 min) than any other day (ranged from 54.0 on day 12 to 56.2 on day 15). The
odds of a SS were lower (P<0.05) on day 3 (1.8), than day 6 (7.2), day 12 (7.9), and day
21 (12.8), but similar (P>0.10) to day 15 (3.2), while the odds of a SS were similar for
day 6, 12, and 21 of age (P>0.10). The number of nursing attempts of sows housed in the
HT environment was higher (P<0.05) on day 6 (11.3) than on day 21 (10.2).

Interestingly, the odds of a SS was lower (P<0.05) for 6 day old pigs from TN
sows (3.2) than 6 day old pigs from HT sows (7.2), with an odds ratio of 0.45 for TN
sows versus HT sows.

In both environments, piglets slept more oﬁen before a nursing event than
engaging in any other activity (P<0.01; Fig. 5.2). In the TN environment, 347 (77.3 %)
out of 449 nursing events, pigs were sleeping before nursing, while in 102 (22.7 %) out
of 449 nursing events, the piglets were active (P<0.001). In the HT environment, 468
(81.8 %) out of 572 nursing events, pigs were sleeping before nursing, while in 104 (18.2
%) out of 572 nursing events, the piglets were active (P<0.001). Level of activity was
not different between environments, 102 (49.5 %) of 206 in the TN, and 104 (50.5 %) of
206 in the HT environment (P>0.10). However, the frequency of being asleep before a
nursing event was lower in the TN environment, 347 (42.6 %) of 815 in the TN and 468
(57.4 %) of 815 in the HT environment (P<0.001). As lactation progresses, piglets spent

less time sleeping and more time active before a nursing event (P<0.001).

120

In the TN and HT rooms, a SS was associated with the pigs being asleep at the
udder before a nursing event (P<0.01); 332 of 416 (79.8 %) in TN and 386 of 465 (83 %)
in HT sows.

The termination of a nursing event was different between environments (Fig. 5.3
and 5.4). The number of nursings terminated by the sow (TN) sitting up were 217 (43.9
%), standing up were 53 (10.7 %), and moving to restrict the piglets access to the
mammary gland were 8 (1.6 %). When TN sows did not terminate a nursing by postural
adjustments, 207 (41.9 %) of the nursings the piglets fell asleep at the sow’s udder, while
9 (1.8 %) of the nursings the pigs continue massaging and suckling the sow’s udder. The
terminations of 569 nursing events were recorded in the HT environment. The numbers
of nursings terminated by the sow (HT) sitting up were 161 (28.3 %), standing up were
53 (9.3 %), and moving to restrict the piglets access to the mammary gland were 10 (1.8
%). As lactation progressed, the sow terminates a nursing event by postural changes
more often in both environments (P<0.001).

Environmental temperature had no effect on the frequency of massaging the udder
after a suckling (P>0.10); 9 of 19 (47.4 %) in the TN and 10 of 19 (52.6 %) in the HT.
The piglets in the HT environment slept more often at the udder than TN pigs (P<0.001);
207 of 542 (38.2 %) in the TN and 335 of 542 (61.8 %) in the HT environment.
Environmental temperature did not affect postural changes such as moving to restrict the
piglets’ access to the udder (P>0. 10), or the times the sow stood up (P>0.10). However,
sows in the TN environment more often sat down to terminate a suckling event than sows
in the HT environment (P<0.01); 217 of 378 (57.4 %) in the TN and 161 of 378 (43.6 %)

in the HT.

121

The suckling success was not associated with the piglets’ behavior alter a
suckling event in any environment (P>0.10). However, a SS event in the TN
environment was associated with the sow terminating the event by sitting up (P<0.001).
Discussion

To our knowledge, this is the ﬁrst investigation on the nursing behavior of sows
exposed to heat stress and their piglets. Success of milk ejection during lactation is an
alternative indicator of maternal investment and lactation performance (Cameron, 1998).
Lactation is a highly energy demanding process (Spaaij et al., 1994). Curtis (1987)
applied Maslow’s “hierarchy of needs” to farm animals and concluded that the animal
tend to fulﬁll their needs in order of priorities. Physiological needs being the most
important followed by safety needs, and at last, behavioral needs. According to this
model a sow exposed to heat stress will prioritize her own physiological and health needs
before lactation. Therefore, in order to diminish heat production under heat stress, it
seems reasonable to speculate that the sow will decrease milk production or transfer if it
is detrimental to her health.

We hypothesized that sows under heat stress may decrease milk production by
altering nursing behavior. We predicted that the odds of a SS would be lower in HT
sows. We found no difference in the odds of a successful suckling by environmental
treatment. The number of attempts to nurse was also not different between
environmental temperatures. The interval (60 min) between successﬁil sucklings was not
altered by environmental temperature. However, the interval between SS in this study is
longer that those reported in previous studies; 53.2 min (Fraser, 1977), 50 min (Castrén et

al., 1989), 46.4 min (Whatson and Bertram, 1980), 45.6 min (Farmer and Robert, 2000),

122

and 44.3 min (Ellendorff et al., 1982). From these results, we conclude contrary to our
hypothesis, that high ambient temperatures does not alter in any way the maternal
investment of the sow in her litter. However, we observed a decrease in litter weight gain
from pigs suckling sows exposed to heat stress (211.7 g/d for TN compared to 196.8 g/d
' for HT; P<0.05, Chapter 1). Interestingly, 3 day old pigs in the HT environment had
longer intervals between SS and a lower chance of receiving a SS. We cannot discard the
possibility that a decrease in milk intake during the ﬁrst days of life could potentially
affect later growth. In addition, we did not measure milk intake directly, therefore, we
cannot be certain that milk intake was similar in all pigs. Furthermore, similar milk
ingestion does not translate in similar grth rates, since other factors, such as
differences in milk composition (Old ham and Fliggers, 1989), metabolic rates of
offspring (Kretzmann et al., 1993) and efﬁciency of nutrient utilization (Verme, 1989),
can alter growth rates. The ﬁrst week of lactation is crucial to piglet survival. The
piglets’ ability to suckle is one of the main factors determining their survival (English and
Smiths, 1975). Starvation is the most prevalent cause of pre-weaning death (English and
Smith, 1975). Piglet mortality is higher within the ﬁrst 3 days of lactation (Bille et al.,
1974; English and Smith, 1975). The higher number of nursing attempts on day 6 in the
HT environment, in addition to higher odds of a SS compared to day 6 in the TN
environment, could suggest that HT piglets are trying to compensate for the higher US on
day 3 of lactation.

Furthermore, we found a higher probability of receiving a SS on day 21 of
lactation versus any other day for sows in the TN environment. Our results are in

agreement with Whatson and Bertram (1980) who reported lower frequency of US by

123

day 20 of lactation compared to day 6 to 12. During the ﬁrst day of lactation, the
transition from copious nursing to cyclical nursing is achieved by reinforcement learning
of the piglet by the sow. Milk, the reward, is only provided for the right stimuli at the
appropriate time (Lewis and Hurnik, 1985). Our results and the results of Whatson and
Bertram (1980) suggest that 20-21 day piglets are more efﬁcient in giving the appropriate
stimuli for milk ejection. Perhaps the piglets have become more successful in
recognizing the stimuli that will gain them the reward.

Several behavior studies have shown that the sow may restrict the pigs’ access to
the udder when disturbed (Whatson and Bertram, 1980; dePasillé and Robert, 1989;
Gotz, 1991; Hartmann et al., 1997). Under heat stress, a possible conﬂict of motivations
may arise. First, as thermoregulation is sub-optimal, the sow may be less active to
prevent heat production by reducing physical activity. Second, the sows needs to
decrease the access to the udder as piglets are more active. We hypothesized that sows
under heat stress could be more disturbed by their piglets than TN sows. In this study,
the piglets’ behavior before a nursing was affected by high ambient temperature. Piglets
in the HT environment were more often asleep before a suckling than piglets in the TN
environment. Le Dividich and Noblet (1981) reported less activity in nursing pigs kept at
18-20° C compared to piglets kept at 30-32° C, as a mean to conserve heat. However, in
our study the frequency of activity before a suckling was not different between
environments. This is in agreement with the similarity of SS frequency in both
environments, since the activity of the pigs increases during the time previous to an US

(Fraser, 1977; Ellendorff et al., 1982; Castrén et al., 1989). This also supports our

124

ﬁnding that in the TN environment, sleeping before a suckling was associated to higher
SS frequency.

The behaviors recorded for the nursing pig after a suckling event were aimed to
identify the level of hunger. Massaging the udder after the termination of a suckling
event was identiﬁed as the piglets being hungry. An increased time spent massaging the
udder has been observed in piglets nursing sows treated with bromocriptine, a prolactin
inhibitor (Farmer et al., 1998). On the contrary, sleeping at the udder after a suckling
event was identiﬁed as satiety. Therefore, the lack of difference in the frequency of
massaging the udder after a nursing event corresponds with no difference in frequency of
SS. This suggests that piglets in both environments were receiving enough milk to not
alter this behavior.

The difference in frequency of falling asleep at the udder between environments
(lower in TN) corresponds to the sow behavior after a nursing event in the TN
environment. Sows terminate a nursing event by sitting down more often in the TN
environment. These two events are mutually exclusive, as if one occurs (sitting by the
sow) the other (pigs falling asleep at udder) cannot occur. Although we reported no
differences in the frequency of the sow standing up aﬁer a nursing event between
environments, a SS was associated with sitting up after the nursing event in the TN
environment.

Our results suggest that the sows in the TN environment depend more on
behaviors to actively control the nursing events. On the contrary, lactating sows in the
HT environment may be reducing the energy costing behaviors while her physiological

needs are increased under heat stress. Furthermore, sows did not seem to be more

125

disturbed by the piglets in the HT environment, as they did not increase the restriction to
the udder. However, this behavior could be justiﬁed if it has an advantage towards heat
dissipation. Lateral recumbency may be a better position to dissipate heat in sows.

The decreased milk availability during the ﬁrst days of life could decrease the
growth potential of nursing pigs of sows exposed to heat stress. Sows under heat stress
seem to take less advantage of behavior to terminate nursings. However, the nursing
behavior of lactating sows under heat stress is not greatly altered compared to
thermoneutral sows in a way that could explain the decrease in litter weight gain
observed in this environment.

Implications

The decrease litter weight gain observed in pigs from sows exposed to heat stress

is not the result of increased frequency of unsuccessful nursing events. However, other

alterations in sow behavior may inﬂuence the transfer of milk to the nursing pigs.

126

Fig. 5.1 Overall frequency (%) of SS and US is sows exposed to 20° C (TN) or 29.4° C
(HT). Total number of occurrences observed was 1021. Statistical signiﬁcance of
P<0.05 is indicated by an “*”.

127

 

 

 

 

 

 

 

 

 

 

 

100 -

0
60

0
20‘

0050030000 .«0 away 55:00::

128

Fig. 5.2 Overall frequency (%) of behavior of litter (80 % of pigs) before a nursing event
by sows exposed to 20° C (TN) or 29.4° C (HT). Total number of occurrences observed
was 1021. Statistical signiﬁcance of P<0.05 compared to TN is indicated by an “”*.

129

60

n
0
5

 

0
4 3 2

05.503030 do Aged 5:00.000”—

Sleeping

130

Fig. 5.3 Overall frequency (%) of behavior of litter (80 % of pigs) after a nursing event
by sows exposed to 20° C (TN) or 29.4° C (HT). Massaging udder (MU) and sleeping at
udder (SU). Total number of occurrences observed was 1021. Statistical signiﬁcance of
P<0.05 compared to TN is indicated by an “*”.

131

 

 

 

 

 

 

 

 

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0
7

w

0 0 0 0
5 4 3 2

30.5anon .«o 3% xocozcocm

 

132

Fig. 5.4 Overall frequency (%) of behavior of sows exposed to 20° C (TN) or 29.4° C
(HT) after a nursing event. Sow sits (ST), sow stands (SST) or sow restricts access to
udder (SR). Total number of occurrences observed was 1021. Statistical signiﬁcance of
P<0.05 compared to TN is indicated by an “*”.

133

 

 

 

 

 

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SST

ST

134

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Farmer, C. and Robert, S. 2000. Multiple crossfostering: effects on prolactin, grth
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137

Summary and Conclusions

Exposure to high ambient temperatures depresses lactation performance of sows,
i.e., decrease feed intake, increase weight loss, and decrease litter growth rate. Although
several management practices have been used to alleviate heat stress, they contribute
little to improving litter growth rate. Few nutritional approaches to improve performance
in sows exposed to high temperatures have been used. In this thesis, we investigated
whether dietary L-arginine supplementation improve lactation performance of sows
under heat stress. Arginine requirement for lactation has not been established, and the
current published requirement may be lower than the amount required by lactating sows
under heat stress. Arginine is the precursor of nitric oxide, which is responsible for
increasing vasodilation and blood ﬂow. In this experiment, sows were exposed to 29.4°
C in order to achieve a state of heat stress.

Heat exposure depressed sow voluntary feed intake, increased body weight loss,
and depressed litter growth rate. Supplementing L-arginine to heat stressed sows
decreased sow weight loss and decreased voluntary feed intake. However, arginine
supplementation did not improve litter growth rate.

Of the hormones and metabolites measured, heat exposure increased plasma
glucose concentration with no changes in plasma insulin or salivary cortisol
concentrations. Plasma growth hormone concentration was increased, while plasma
prolactin concentration was not affected. Supplementing L-arginine to heat stressed sows
increased plasma insulin concentration with no changes in plasma glucose or salivary
cortisol concentrations. Plasma growth hormone concentration was decreased, while

plasma prolactin concentration was not affected by L-arginine supplementation. The

138

results suggest that L-arginine supplementation may decrease sow body weight loss via
an increase in insulin concentration.

Heat stress decreased plasma and milk concentration of arginine as well as other
amino acids. L-arginine supplementation increased arginine concentration in plasma and
proline concentration in milk. Plasma nitric oxide concentration was not increased by
heat exposure or L-arginine supplementation.

High environmental temperatures did not decrease number of successful suckling
events. Overall, interval between successful sucklings or number of nursing attempts was
not decreased in heat stressed sows. The interval between successful sucklings was
longer and the odds of a successful suckling lower on day 3 of lactation in heat stressed
sows. These changes in nursing behavior under heat stress during the ﬁrst days of life
may play a role in the decreased litter growth rate, as food deprivation is the ﬁrst cause of
mortality during this stage.

In conclusion, the depressed lactation performance observed in sows under heat
stress may occur via a depressed insulin utilization and (or) glucose utilization.

Depressed litter growth rate may be the result of decreased milk amino acid concentration
and not the result of alterations in nursing behavior. Dietary L-arginine supplementation
may maintain sow body condition via an increase in insulin concentration, which
promotes anabolism. The increase in plasma arginine concentration and proline milk
concentration by dietary L-arginine supplementation does not improve litter growth rate.
Arginine requirement for lactating sows under heat stress may be higher to maintain sow

body condition.

139

   
  
      

   

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