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DATE DUE DATE DUE DATE DUE 1/” Wu EFFECT OF IMMUNIZATION AGAINST CHOLECYSTOKININ (CCK) ON PERFORMANCE OF PRIMIPAROUS SOWS AND NURSERY PIGS By Josep Garcia-Sirera A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1 999 ABSTRACT EFFECTS OF IMMUNIZATION AGAINST CHOLECYSTOKININ (CCK) ON PERFORMANCE OF PRIMAPAROUS SOWS AND NURSERY PIGS By Josep Garcia-Sirera Three experiments were designed to test the effect of immunization against CCK on parity-one sow productivity, plasma CCK concentrations during lactation and growth performance of nursery pigs. in Experiment 1, seventy-two gilts were either vaccinated against CCK or with a placebo during gestation. Sow performance was monitored for the parity-one lactation period (26 :l: 1 d). Analysis by RIA showed a large range of anti-CCK titer for sows in the CCK group (log serum titer values ranged between 0 and 4.15). There were no differences (P>.05) between CCK and control sows, respectively, in ADFI (kg) in wk 1 (4.26 vs 4.25) wk 2 (5.00 vs 4.94), wk 3 (5.70 vs 5.46), and for the total duration of the experiment (4.99 vs 4.88). Likewise, there were no differences (P>.05) in piglet ADG (CCK vs Control, respectively) in wk 1 (.17 vs. .17), wk 2 (.23 vs .24), wk 3 (.21 vs .21) and for the duration of the experiment (.21 vs .21 ). Weaning-to—estrus interval (days) was similar for sows in CCK and control groups (4.84 vs 5.20, respectively). Active immunization against CCK did not improve the sow’s lactation and reproductive performance in parity one. In Experiment 2 fourteen gilts were used. A similar protocol as in Experiment 1 was followed. In addition, gilts were fitted with an ear vein catheter and blood samples obtained at 15 minutes interval during a six-hour collection period, to determine free and total CCK concentration in plasma. Feed intake pattern was also visually recorded during the six-hour period. Anti-CCK antibody titer, total and free CCK concentration were analyzed by RIA. Analysis of plasma free-CCK showed sows immunized against CCK had similar (P>.05) peak values as control sows (12 i 3 pmollL CCK vs 19 pmol/L : 5 Control ). Feed intake pattern was not altered by CCK vaccination. in Experiment 3, forty-eight, early-weaned crossbred pigs (10 to 12 d of age) from sows vaccinated against CCK or with a placebo (Control), were used to test the benefits of passive immunization on performance of nursery pigs. Pigs were housed in pens of four. Individual pig weights were recorded on d 0, 7, 14, 21, 28, and 35. Feed disappearance was monitored daily. Blood samples for serum were collected from each pig on days of age 14, and 21 and anti-CCK titer was determined by RIA. There was a difference in ADG (P<.01) between immunized (CCK) and control pigs during week 1 (.23 kg vs .20 kg,respectiveiy). Values for ADG for individual wk 2, 3, 4 and 5 were not different between treatments. Values for ADFI among treatments differed during wk 2 (.42 kg; vs .35 kg; P< .05). Values for ADFI for individual wk 1,3,4 and 5 were not different between treatments. For the entire experiment, ADFI (.44 vs .41) was greater (P<.05) and ADG (.67 vs .63) tended to be greater (P<.1) for pigs in the CCK treatment. There were no differences in Feed/Gain ratio (FIG) for any week or entire experiment. Passive immunization against CCK improved growth performance of early-weaned nursery pigs. Als meus amics, tinc la sort de tenir els millors. To my friends, I'm very lucky to have the best. ACKNOWLEDGMENTS This work would not have been possible without the involvement of many people. I’d like to thank my advisor, Dale Rozeboom for his guidance and support. Thanks also to my committee members, Dr Barb Straw, Dr Gretchen Hill, Dr Nathalie Trottier, and Dr Adroaldo Zanella for their criticism of this work. Thanks to Dr Jerome Pekas for his help and guidance with the titer analysis, and Sue Wise for her lab help at MARC. Thanks to all the people who help numerous ' times when I had to vaccinate and collect samples. Thanks Marcia Carlson, not only for all your help with this project, but for allways being a great friend. Thank you Pasha Lyvers for putting aside everything else to spend endless hours helping me collect samples, and doing it always with a smile. Thank you Daniel “Sows are Silly” Nelson, Ross Santell, Felix Nuflez and Leonor Martin, Luis and Carolina Rodriguez, Abner Rodriguez, Mike Schlegel, Michelle Mater, Savi and Dilum Dunuwila for your help and for your friendship. Thanks Jane Link for your help, for teaching me how “not to mess up everything” in the lab and your friendship. Thanks Pao Ku, Larry Chapin for your help with the CCK analysis. Thanks Barb Sweeney for all your help with every “last minute order” and your patience with all my requests. Thanks to my family, in particular my parents for their love and support and my sister Carmen, for allways being available when I needed someone to talk about “everything but work” and “en catala”. Thanks to my friends from Catalonia for providing those important vacation times to relax. Most of all, thank you Monu, for your help, support, friendship, and your love. I would not have done it without you. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION CHAPTER 1 REVIEW OF LITERATURE lntroduction.. Cholecystokinin- introduction Anatomical Distribution of CCK. .. CCK Receptors: types and anatomical dIstrIbutIon CCK as a satiety factor” ... Peripheral CCK and Food Intake. CCK action in the CNS... CCK and other hormones and neuropeptIdes Role of CCK In lactation... .. Immunization against endogenous CCK Summary. Model ... ... ... ... Hypothesis CHAPTER 2 ACTIVE IMMUNIZATION OF GILTS AGAINST CHOLECYSTOKININ AND ITS EFFECT ON PARITY-ONE LACTATION PERFORMANCE, POSTWEANING RETURN-TO-ESTRUS AND PLASMA FREE-CCK CONCENTRATION Abstract” Introduction Materials and Methods . General Procedures for Pigs Vaccine Preparation and LaboratoryAnalySIs Statistical Analysis” Results” Discussion ImplIcatIons CHAPTER 3 PASSIVE IMMUNIZATION AGAINST CHOLECYSTOKININ (CCK) AND ITS EFFECT ON GROWTH PERFORMANCE OF NURSERY PIGS Abstract .. vi viii LONG-50000 11 13 14 15 18 19 20 22 22 23 24 25 27 29 33 4O 41 43 43 introduction Materialsand Methods GeneralProceduresfor Pigs StatisticalAnalysis Results Discussion implications CHAPTER 4 CONCLUSIONS Future Research APPENDIX A Determination of Antisera Titer Against Cholecystokinin ...... APPENDIX 8 Ear Vein Catheterization Procedure... APPENDIXC Free and Total CCKAnalysis APPENDIXD Experiment 1 Group Data BIBLIOGRAPHY vii Sifififi 49 50 51 58 59 63 66 68 7O LIST OF TABLES Table 1. Diet Composition Table 2. Effect of immunization against CCK on sow's lactation performance Table 3. Effect of immunization against CCK 0 on weaning-to-estrus interval Table 4. Effect of immunization against CCK on eating behaVIor Table 5. Composition of diets fed in different phases of nursery ...... Table 6. Effect of passive immunization against CCK of nursery pigs on growth performance Table 7. Exp1: Effect of replicate (group) on sow ADFI and pigletADG Table 8. Exp1: Effect of replicate (group) on weaning-to-estrus interval viii 32 35 36 39 48 53 68 69 LIST OF FIGURES Figure 1. Suggested mechanism of immunization against CCK and its effect on feed intake Figure 2. Exp 1 time sequence of vaccination and sample collection Figure 3. Effect of immunization against CCK on weaning-to-estrusinterval Figure 4. Effect of immunization against CCK on free and total CCK concentration in plasma Figure 5. Time sequence of nursery trial showing time sow vaccinations and sample collection .. 21 31 37 38 47 INTRODUCTION Modern swine production requires that sows wean large, heavy litters, and have short weaning-to-estrus intervals. Averages of 2.5 Iitters/sow/year, 25 pigs weaned/sow/year and litter weights of 65 to 75 kg are attainable. Maximum lactation feed intake is essential to achieve this level of production. Concurrent with an increase in sow productivity has been an increase in consumer demand for lean pork, which has forced the swine industry to select for animals with reduced fat reserves. This selection has coincidentally led to a reduction in voluntary feed intake of sows (Riley, 1989). Most lactating sows do not consume enough feed to maintain body weight during a 23-week lactation period. Sows typically lose more weight during the last stages of lactation, when milk output is maximized. A problem associated with excessive weight loss and body condition is delayed return to estrus (>10 days). Extended weaning-to- estrus intervals results in fewer litters per year. This problem is especially present in the primiparous sow. The limitation that voluntary feed intake puts on maximizing productivity has been studied with the lactating sow and with growing pigs (Pekas, 1985;Matzat, 1990). Lactating sows, which were superalimented via a gastric fistula and provided with 20% more feed than those sows allowed ad libitum access to feed, were able to preserve a greater amount of body mass and were also able to synthesize greater quantities of milk (Matzat, 1990). This study proved that appetite is a limiting factor in increasing sow productivity, and that increasing feed intake would result in better performance of sows. One approach to increase sow feed intake has involved immunoneutralization of systemic Cholecystokinin (CCK), a hormone involved in regulation of appetite and feed intake. in a series of studies (Pekas and Trout, 1990; Pekas, 1993; Pekas, 1996) with growing pigs, active immunization against CCK stimulated feed intake and enhanced growth, in proportion to the anti-CCK antibody (CCK-Ab) titer elicited in the animal. The same technique was used to increase the feed intake of lactating sows (Nelson, 1996). The studies with CCK immunization show promising results but leave their approach to increasing feed intake in sows in need of future elucidation. The positive correlation between feed intake and anti-CCK titer in serum of immunized sows shown by Nelson(1996), indicates a potential benefit of immunizing sows against the hormone, but a true comparison of feed intake between immunized and control animals needs to be examined. Furthermore, the potential effects of CCK immunization on subsequent reproduction performance of the sows needs to be addressed. Lastly, a possible improvement in growth performance of the sow’s offspring via passive immunization has been postulated (Nelson,1996) but not investigated. Chapter 1 REVIEW OF LITERATURE Introduction Eating behavior can be influenced by several factors such as environmental conditions, sensory cues (sight, smell, taste), nutrients in the diet, gastrointestinal factors, hormones, neurotransmitters and metabolites. Pen density, feeder design, water intake, alternative feedstuffs (fiber, fat), genotype, photoperiod and temperature are all examples of environmental factors which can influence feed intake. Alternatively, distention of the stomach due to feeding, activates pressure sensitive receptors that signal the brain to induce satiety. Metabolites, hormones and neurotransmitters are released when food enters the gastrointestinal tract (GI), and also influence appetite. The primary site responsible for the integrated control of feed intake and energy balance is the central nervous system (CNS). it is generally accepted that swine eat to meet their energy requirements. This is not followed in a period of great biological demand such as lactation, where the animal does not eat enough to obtain the energy necessary for maintenance and lactation. Also, when fed diets with higher nutrient density, swine tend to eat more total nutrients, indicating that there are other factors that influence appetite. One of the hormones involved in control of feed intake is CCK. A review on CCK structure, mechanism(s) and site(s) of action and possible ways to increase feed intake by means of altering CCK action follows. Cholecystokinin — Introduction The first studies involving what later would be known as Cholecystokinin were undertaken by ivy and Oldberg (1927,1928). The authors reported that infusion of lipid into the duodenum of experimental animals stimulated the release of a chemical substance that caused contractions of the gall bladder. They proposed the substance be termed Cholecystokinin as “it excites or moves the gall bladder”. in 1943, Harper and Raper isolated a substance from extracts of the mucosa of the upper intestine which, upon intravenous injection, stimulated the release of pancreatic hormones. They called it pancreazymin. Over two decades later, researchers (Jorpes and Mutt, 1966; Mutt and Jorpes, 1971) purified Cholecystokinin extracts from the mucosal cells of the porcine small intestine and characterized it as a 33 amino acid peptide. They demonstrated that, in addition to producing contraction of the gallbladder, Cholecystokinin also caused secretion of pancreatic hormones (Mutt and Jorpes, 1968). Thus, it became apparent that Cholecystokinin and pancreazymin were one and the same substance. For many years thereafter, the gut peptide was referred to as Cholecystokinin-pancreozymin, but more recently it has been referred to as Cholecystokinin (CCK). Subsequent work has revealed the existence of several other molecular forms of CCK besides CCK-33. Larger molecular forms of the peptide with 39 and 58 amino acids, and smaller forms with 4, 5 and 8 amino acids have been found in the periphery and brain of several species, including rat, pig, dog and man (Calam et al., 1982; Walsh et al.,1982; Eberlien et al., 1988). The peripheral biological activity of CCK appears to be contained within the C-terminal octapeptide with an O-sulphated tyrosine residue (CCK-88) as reported by Ondetti and coworkers (1970), and is well conserved across mammalian species. More recently, a cDNA fragment encoding a 115 amino acid precursor to CCK has been reported (Deschenes et al., 1984). Various molecular forms of CCK are obtained from this precursor by post-translational processing (Gubler et al., 1984). With regard to degradation, a number of peptidases have been shown to degrade the various forms of CCK into inactive fragments by cleavage, including membrane-bound aminopeptidases (Deschodt- Lanckman et al., 1981) and an enkephalinase enzyme (Zuzel et al., 1985). Anatomical Distribution of CCK Cholecystokinin is widely produced and distributed, both in the periphery and in the central nervous system. Sjolund and co-workers (1983) revealed the presence of CCK-like immunoreactivity (CCK-Ll) in high concentrations in the upper small intestine. The main site for CCK-Ll localization is within mucosal l- ceils of the duodenum and jejunum. Depending on the species, the forms of CCK present in the small intestine can vary. Other forms of CCK such as CCK- 58, CCK-39, CCK-33 and CCK-8 are abundant in dogs, cats, and humans, while in rats and pigs, smaller forms such as CCK-33, CCK-22, CCK12 and CCKB predominate (Eberlien et al., 1988). The different forms of CCK can also be found in enteric nerves, mucosa and smooth muscle of the lower gastrointestinal tract (Larsson and Rehfeld, 1979), in ascending afferent fibers of the vagus nerve (Dockray et al., 1981 ), and in the testis (Persson et al., 1988; Pelto-Huikko et al., 1989). in addition, CCK-Ll is also found in blood plasma, and it is generally believed that CCK-88 is the most abundant circulating form, although other forms, such as CCK-33, CCK-39 and CCK-58 are also present (Calam et al., 1982; Walsh et al., 1982). Vanderhaeghen and co-workers (1975) first demonstrated the possible existence of CCK-Li in the CNS, using antibodies against gastrin. A year later Dockray (1976) showed that most of this immunoreactivity corresponded to the carboxyl terminal octapeptide of CCK. Subsequent work revealed that CCK—88 is the main molecular form of the peptide in the CNS (Docray, 1978; Robberecht et al., 1978). Other forms of CCK also present in significant amounts in the CNS include CCK-8 unsulfated (US), CCK-5 and CCK-4 (Rehfeld, 1978, 1986; Shively et al., 1987). Larger forms of CCK are almost absent in the CNS except CCK-58 which has been isolated both from dog and pig brains (Eysselein et al., 1984; Tatemoto et al., 1984). Techniques such as immunohistochemistry, radioimmunoassay and in situ hybridization have helped to provide strong evidence that CCK-83 is released from nerve terminals and acts as a neurotransmitter within the CNS (Schick et al., 1994). There is also evidence that CCK may co-exist with other neurotransmitters within the CNS, such as dopamine (Hokfelt et al., 1980) and substance P (Skirboll et al., 1982). Cholecystokinin is widely distributed within the CNS and studies in rats have demonstrated the existence of the peptide in neurons of cerebral cortex, hippocampus, septum, amygdala, olfactory bulb, hypothalamus, thalamus, parbrachiai nucleus, raphe nucleus, substancia nigra, ventral mesencephalon, nucleus tractus solatarious, ventral medulla, and spinal cord (Vanderhaeghen et al., 1975; Hokfelt et al., 1988; Vanderhaeghen and Schiffman, 1992). CCK Receptors: types and anatomical distribution Different studies have been performed to determine the location and structure of CCK receptors. Innis and Snyder (1980) carried out radioligand binding studies with different fragments of CCK in tissue homogenates from brain and pancreas. They found that the unsulphated form of CCK-8 (CCK-BUS), pentagastrin (CCK-5), and CCK-4 inhibited the binding of [mi] Bolton-Hunter CCK-33 to brain homogenates with much greater efficiency than to pancreatic homogenates. These results suggested the possibility of two classes of CCK receptors and led to the use of the terms “peripheral type” receptors and “ brain type” receptors. Subsequent studies by Moran and co-workers (1986) provided evidence for the presence of both subtypes of receptors in the brain. They reclassified the two subtypes of CCK receptors as CCKA and CCKa, respectively. Type A receptors are found mainly in the periphery, but also in some areas of the CNS. They have a high affinity for CCK-88. Type B receptors are found mainly in the CNS and have a high affinity for CCK-88, CCK-BUS, CCK-5 and CCK-4. Wank and co-workers (1992a,b) have sequenced and cloned the genes of the two subtypes of CCK receptors from rat pancreas and rat brain. Similarly, the genes for the two CCK receptors have also been cloned from canine and human stomach and human brain (Pisegna et al., 1992). Sequence analysis of rat pancreatic and rat brain CCK receptor genes has revealed a 48% homology between the two receptors. The cloning of the receptors has also confirmed that both peripheral and CNS CCKA receptors are identical and that the CCKa receptor has high homology to gastrin receptors (Wank et al., 1994). Autoradiography binding studies have been used to determine the distribution of CCKA and CCKa receptors in peripheral tissue and in the CNS. As expected, studies in rats have indicated high populations of CCKA receptors in peripheral tissue, and high populations of CCK; receptors in the CNS. Type A receptors have been found in the pancreatic acinar cells, gall bladder, smooth muscle of the pylorus and vagal afferents. Local populations of CCKA receptors have been found in the CNS in the area postrema (AP), the dorsal medial hypothalamus, the interpenduncular nucleus and the havenulum (Moran et al., 1986; Hill et al., 1987, 1990, 1992; Mercer and Lawrence, 1992). Type B receptors are found on vagal afferents and in the CNS regions, such as the cerebral cortex, olfactory bulb, nucleus accumbens, caudate nucleus, hippocampus, hypothalamus, amygdala, substantia nigra, dorsal raphe nucleus and the dorsal horn of the spinal cord (Gaudreau et al., 1983; Hill et al., 1992; Corp et al.,1993). There are also some species differences in the distribution of CCK receptors subtypes. For example, CCKA receptors are the predominant subtype in rat pancreatic acinar cells, CCKB receptors predominate in the pig pancreatic acinar cells (Morisset et al., 1996). CCK as a satiety factor The role of Cholecystokinin as a satiety factor has been studied extensively. CCK participates in rapid, preabsorptive satiety in pigs (Anika et al., 1981). lntraperitoneal injections of partially-purified CCK produces a dose related suppression of feed intake (Gibbs et al., 1973; Stallone et al., 1989). Lateral cerebral ventricle injections of CCK also depresses feed intake in rats (Tsai et al., 1984). Systemic infusions of CCK-8 reduce meal size in pigs (Houpt, 1983) Evidence from Kow and Pfaff (1986) suggest that the role of CCK-8 in satiety induction is two fold: CCK-8 serves as a satiety agent in the periphery (non CNS), mediated through vagal afferent nerves to the brain, and as a neurotransmitter in the brain to convey the information originating in the periphery. The results of Linden (1989) concur with these findings, indicating that peripheral CCK receptor mechanisms induce a release of CCK in the brain. In the brain and in the periphery CCK may serve as a neurotransmitter (Kow and Pfaff, 1986; Linden, 1989). Ultimately, the satiety center of the hypothalamus is stimulated by these endogenous compounds, and the animal terminates the meal. Artificial synthesis of the specific CCKA receptor antagonist devazepide, has made possible the testing of the CCK’s role in control of satiety. Studies with different species indicate that the inhibitory effects of exogenous peripheral CCK on food intake could be completely abolished by intraperitoneal pretreatment with devazepide (Hewson et al., 1988; Ebenezer et al., 1990a; Ebenezer et al., 1993). These results suggest that a peripheral CCKA receptor mechanism is involved in the suppression of feeding produced by the peptide. Furthermore, it was found that devazepide, on its own, increased the size of a test meal when administered systemically to several species, including the rat, pig, mouse, monkey, dog, cat and chicken. These tests have been conducted under a number of different feeding schedules and dietary conditions (Hewson et al., 1988; Silver et al., 1989; Ebenezer et al., 1990a; Bado et al. 1991; Cheng et al., 1993; Moran et al., 1993; Weatherford et al., 1993; Covasa and Forbes, 1994; Ebenezer and Baldwin, 1995). These experiments have provided another clear indication that endogenous CCK, acting via CCKA receptors, plays an important role in the control of food intake. Some researchers question whether CCK “satiety‘ action is due to creating discomfort to the animal such as vomiting and nausea, and not directly by controlling food intake. Activation of both, CCKA and CCKa, receptors have been described as being responsible for interruption of food intake. In addition, activation of CCKA receptors seems to be responsible for release of vasopresin 1O shortly after activation, and release of cortisol after a longer period of time after receptor activation (Parrot et al., 1991 ). Recent research questions the involvement of CCKA in cortisol release (Parrot and Forsling, 1992). The authors used a CCKA receptor antagonist to block the receptor action, and still observed release of cortisol after infusion of CCK in the circulation. CCKe receptors have been associated with stress-related disorders. in general, these studies promote the idea that the animal stops eating due to the discomfort produced by CCK action. The first indications in favor of CCK as a true satiation agent are that CCK decreases food intake but not water intake, suggesting that the decrease in intake is not secondary to general malaise (Gibbs et al., 1973). Other research also shows that CCK’s action on satiety are more focused on fat intake than other ingredients which would rule out the “discomfort” theory, since discomfort would generate a rejection of any food. Peripheral CCK and Food Intake Cholecystokinin cannot penetrate the blood brain barrier (Passaro et al., 1982). Thus it is likely that systemic CCK acts at a peripheral site to inhibit feeding (Weller et al., 1990). The target site for the hypophagic actions of the peptide is not clear. Recent studies have suggested that peripheral exogenous CCK-8S may activate vagal afferent fibers that relay the signal to brain areas concerned with feeding where it is translated into behavior consistent with satiety 11 (Smith et al., 1981, 1985; Crawley et al., 1984; Crawley and Kiss, 1985; Smith and Gibbs, 1992). It has also been proposed that peripheral CCK may act at certain brain sites where the blood brain barrier is interrupted, for example AP and NST. Indeed peripheral CCK-A receptors have been identified in AP as mentioned before in this review. Van der Kooy (1984) showed that lesions of the AP specifically blocked the satiety effect induced by CCK. CCK action in the CNS Cholecystokinin has been shown to act in the CNS affecting behaviour of pigs trained to make operant responses for food and water (Parrot and Baldwing, 1981). injections of bolus doses of CCK-88 into the lateral cerebral ventricles of pigs decreased operant responding for food in a dose-related manner. The study was done with several doses of CCK-88, and none of them inhibited water intake in thirsty animal indicating the specificity of the peptide for food. Later studies using specific CCK receptor agonist has shown that the hypophagic effect of CCK in pigs in the CNS is mediated by CCKA receptors (Parrot, 1994; Ebenezer et al., 1996). 12 CCK and other hormones and neuropeptides in addition to CCK direct action on feed intake, there are different hormones and peptides related to feed intake control that appear to be related to CCK. Bombensin, a hormone that has been isolated in the GI tract, induces a satiety effect in the pig. Injection of bombensin both, peripherally and directly in the brain has been shown to induce satiety. Bombensin also induces a release of peripheral CCK, raising the question whether bombensin action is mediated by CCK. The use of antagonists for CCK receptors has shown that bombensin has a satiety effect by itself, independent of its action through CCK (Parrot and Baldwin, 1982). Neuropeptide Y (NPY) has been isolated in the pig’s brain (Tatemoto et al.,1982). in the brain, CCK induces a reduction of NPY levels, suggesting that NPY could be in the sequential chain of events brought on by CCK (Gourch et al., 1990). Because the majority of studies on feeding behavior have been conducted in satiated rats, the question emerged whether NPY actually stimulates feeding behavior or, rather, attenuates satiety signals. Schick and coworkers (1991) showed that intracerebrai NPY did not augment food intake initially, but rather delayed the reduction in feeding that occurs normally with satiation, suggesting that NPY does not act to stimulate feeding behavior per se, but rather to suppress satiety signals. 13 Role of CCK in lactation In lactating rats and sows, daily ad libitum feed intake generally increases as lactation progresses. it has been suggested that the increase in energy spent associated with increasing amounts of milk synthesis is responsible for a large proportion of hyperphagia observed in rats and sows (Flemming, 1976). However, there could be other mechanisms associated with this hyperphagia. Daily feed intake is the sum of the number of meals eaten within a day and the size of each meal. Meal size is the combination of the duration of time spent eating, and the rate of ingestion. Cholecystokinin mediates its effects on feed intake by reducing meal size (Leibowitz, 1986). In lactating rats, the gradual increase in feed intake occurs primarely because of an increase in meal size (Strubbe and Gorissen, 1980; McLaughlin et al., 1983), rather that an increase in meal frequency. Meal size becomes larger in lactating rats in spite of the fact that plasma CCK concentrations are elevated (McLaughlin et al., 1983; Linden, 1989). A possible explanations for the elevated CCK concentrations in lactating rats could be that CCK is released in lactating rats (and dogs) in response to suckling stimulus (Linden et al., 1990). This release of CCK is immediate and short lasting, and in rats, of smaller magnitude that the release produced after feeding (Linden et al., 1990). Even with elevated CCK concentrations as lactation progresses, feed intake gradually increases also during lactation. For this reason, several studies (McLaughlin et al., 1983; Wager-Srdar et al., 1986, Linden, 1989) speculate with the idea that marked hyperphagia of lactating rats reflects an insensitivity of the animals to the inhibitory effects of CCK on food 14 intake as lactation length progresses. However, pancreatic hypertrophy, a known bilogical response to elevated CCK concentrations still occurs in lactating rats (McLaughlin et al., 1983). This seems to indicate that CCK-insensitivity during lactation is restricted to satiety. In should be noted that some research did not observe a decrease in sensitivity to the anorexigenic effects of CCK as lactation progressed in rats (Helmereich et al., 1991). Immunization against endogenous CCK Several studies involving CCK antibodies (CCK-Ab) have been conducted in order to prove involvement of endogenous CCK in feeding behavior. Circulating CCK-Ab do not cross the blood-brain barrier, but are believed to sequester the peripheral, free circulating endogenous CCK, making it unavailable to the CCK receptor. This phenomenon is known as immunoneutralization. Exogenous CCK-Ab, administered via continuous lateral cerebral ventricular injection, increases feed intake in whethers (Della-Fara et al., 1981). Feed intake of Zucker rats has also been increased by exogenous CCK-Ab administration, and also by endogenous CCK-Ab following active immunization against CCK (McLaughlin et al., 1985). Similarly, the feed intake and growth rate of growing pigs has been elevated (8.2 and 10.6% respectively) following immunization against desulfated CCK-9 conjugated to human serum globulin (Pekas and Trout, 1990). in another experiment, Pekas (1993) reported elevated feed intakes of growing pigs following immunization against desulfated CCK-8 15 conjugated to one of four different haptens; bovine serum albumin (BSA), human serum globulin (HSG), Keyhole limpet hemocyanin (KLH), or purified protein derivative (PPD). Nelson (1996) reported a correlation between serum titer and titer of colostrum of lactating sows and their average feed intake (ADF i) in week three of lactation (R2=.43 to .67, P<.01 in all cases). Two other studies with sheep have reported no differences between immunized animals and controls (Trout et al., 1989; Spencer, 1992). In both studies feed intake was slightly depressed on the days following booster vaccinations. Pekas and Trout (1990) reported a similar observation in pigs. Spencer, when explaining the results of the study, hypothesized that potential reasons for immunized sheep failing to show a response were: (1) Insufficient amount of CCK-Ab raised to effectively neutralize the endogenously produced CCK, (2) CCK-Ab raised may have had too low of an affinity to effectively prevent the hormone from binding to the receptor, (3) There is an elevated production and secretion rate of endogenous CCK due to decreased negative feedback as CCK-Ab sequestered CCK. (4) CCK action is primarily autocrine or paracrine, and less affected by circulating CCK, (5) The CCK-Ab may act like a plasma binding protein and protect the hormone from degradation thereby extending it’s biological half-life, 16 (6) The Ab-bound hormone may be presented to the receptor in such a way as to enhance it’s orientation at the receptor binding sites, (7) The Ab-bound hormone may extend the hormone’s transmembrane effectiveness by inhibiting internalization and clearance of the honnone. The only report to date on immunized sows is that of Nelson (1996). His results appear to indicate an advantage of immunizing against CCK to obtain a better ADFI during lactation. The study though raises several questions. The statistical approach was that of a regression model. The author did not truly compare control against immunized animals. The regression (r) coefficients between values for ADFI and mean log titer of colostrum are low (ranging between .057 and .123) when comparing ADFI in weeks 1 and 2 of lactation with log titer of serum at d 7 and. d 14. Values for r are higher when comparing ADFI in week 3 with log titer of serum in d 21 (.705). The highest r value reported was obtained by comparing ADFI on week 3 with a combination of serum log titer of samples taken during late gestation, d 7, 14, 21 of lactation and colostrum. While an r value of .705 appears to indicate a good correlation between immunization against CCK and feed intake, one could question the physiological significance of correlating feed intake during the third week of lactation with serum values obtained as far as 36 days earlier (day 92 of gestation, booster 2). 17 Summary Cholecystokinin is involved in the control of feed intake. It’s mechanism of action appears to involve both the periphery and the CNS. Active immunization against CCK has shown different results depending on the species and the phase of production, growing or lactating. The report on active immunization against CCK on lactating sows (Nelson, 1996) showed promising results but did not truly compare a control set of sows against CCK immunized sows. Furthermore, it did not addressed potential implications on post weaning reproductive performance. Other aspects of the problem that need to be addressed are the possibility that vaccinated animals could compensate the sequestering of free-CCK by CCK-Ab by increasing production or decreasing clearance. As a result, the free- CCK concentration on a given time could remain the same between immunized and non immunized animals, and the anorexic effect would remain unchanged. This hypothesis could explain why in some studies immunization against CCK failed to achieve greater feed intake than control animals (Trout et al., 1989; Spencer, 1992). Effects of passive immunity against CCK on nursery pigs by transfer of immunity from sows to piglets through colostrum is another aspect of the problem in need of study. Weaning piglets at less than three weeks of age (Early Weaning) has become a standard practice in swine production because of health considerations and improved growth performance. Early-weaning main advantage is to stop bacterial and viral spread from the sow to the piglets. Piglets are. passively immunized against disease. Sows immunized against CCK transfer 18 their immunity to CCK to their piglets as well (Nelson, 1996), and thus, could produce an improvement in piglets feed intake by stopping CCK action on satiety. Model The model proposed in this study for the mechanism of action of CCK is shown in Figure 1. The model shows CCK action in the periphery. This study will focus in the peripheral action of CCK. The stop symbol .“ shows the sections of the model which could be affected by CCK immunization. When food enters the small intestine, it triggers a response on CCK- producing cells, which increase production and release it into circulation. Systemic CCK reaches receptors that trigger a signal, which in turn, is relayed to the brain trough the peripheral nervous system. The signal reaches the area of the brain in charge of control of feed intake and the animal stops eating. immunization against CCK could produce antibodies against the hormone. The antibodies could sequester free peripheral CCK and stop it from binding to the CCK receptors. As a consequence, the signal triggered by free-CCK would be less strong in immunized animals and they would continue eating. 19 Hypothesis The hypothesis proposed for each experiment are as follows. Experiment 1. Sows actively immunized against CCK will increase feed intake during lactation when compared to control sows vaccinated with a placebo. The immunized sows produce anti-CCK antibodies which sequester the hormone and prevent it from binding to the receptor and exert its action on feed intake. Experiment 2. Sows actively immunized against CCK have the same eating pattern (number of meals, duration of meals, and amount of feed eaten) than sows vaccinated with a placebo. Free CCK concentration remains the same for sows immunized against CCK or vaccinated with a placebo. The CCK-immunized sows compensate sequestering of free-CCK by anti-CCK antibodies by increasing production. Experiment 3. Passively immunized nursery pigs increase feed intake and growth performance when compared to pigs from non-immunized sows. Antibodies against CCK present in immunized piglets sequester free-CCK in circulation and stop it from exerting its effect on feed intake. 20 Peripheral Nervous System r s i (If) 69) Blood Ve::(l @O a”; $3.1m» ........... _o 0 QOR Q d d 0 5} O CCK-producing V " cells with food receptors D D D D Food particles DQ’U D 9 Small intestine Figure 1. Suggested mechanism of immunization against CCK and its effect on feed intake. 21 Chapter 2 ACTIVE IMMUNIZATION OF GILTS AGAINST CHOLECYSTOKININ (CCK) AND ITS EFFECT ON PARITY-ONE LACTATION PERFORMANCE, POSTWEANING RETURN-TO-ESTRUS AND PLASMA CCK CONCENTRATION Abstract Two experiments were designed to test the effect of active immunization against Cholecystokinin (CCK) on parity-one sow productivity (Exp 1) and plasma CCK concentrations during lactation (Exp 2). in Exp 1, seventy-two gestating, Yorkshire x Landrace, gilts were vaccinated against CCK or with a placebo (Control). Sow performance was monitored the parity-one lactation (26 w: 1 d). A similar protocol was employed in Exp 2 with fourteen gilts, however, in addition to performance measurements, feed intake pattern was studied and females were catheterized to analyze for free-CCK and total-CCK concentration in plasma. Anti-CCK antibody titer, free and total CCK concentration in plasma were analyzed by RIA. in both experiments, RIA analysis showed a great range of anti-CCK titer for sows in the CCK group (log serum titer values ranged between 0 and 4.15). There were no differences (P>.05) between CCK and Control sows, respectively, in ADFI (kg) in week 1 (4.26 i .149 vs 4.25 i .169) week 2 (5.00 i .150 vs 4.93 i .170), week 3 (5.70 :t .179 vs 5.46 i .204), and for the total duration of the experiment (4.99 i: .134 vs 4.88 i- .152). Likewise, there were no differences 22 (P>.05) in piglet ADG (CCK vs. Control, respectively) in week 1 (.17 i .009 vs .17 :r .008), week 2 (.23 j: .006 vs .24 i .007), week 3 (.22 i .007 vs .22 i .008) and for the duration of the experiment (.21 ._+. .005 vs .21 i .006). Weaning-to-estrus interval (days) was similar for sows in CCK and Control groups (4.84 i .21 vs 5.20 i .25, respectively). In Experiment 2, RIA analysis of plasma free-CCK showed sows immunized against CCK had similar (P>.05) peak values as control sows (12 i 3 pmollL CCK vs 19 pM/L i 5 Control ). Feed intake pattern was not altered by CCK vaccination. Active immunization against CCK did not improve the sow’s lactation and reproductive performance in parity one. Sows may compensate for the sequestering of endogenous CCK by increasing synthesis of free-CCK. Key words: CCK, Active Immunization, Sows, Lactation. introduction Cholecystokinin (CCK) is a hormone involved in regulation of appetite and feed intake. In a series of studies with growing pigs (Pekas and Trout, 1990; Pekas, 1993; Pekas, 1996), active immunization against CCK stimulated feed intake and growth in proportion to the anti-CCK antibody (CCK-Ab) titer elicited by the animal. Because of the sows general inability to consume enough feed in lactation to maintain body condition, Nelson (1996) actively immunized parity one sows against CCK to try to improve sow feed intake. He observed a correlation between feed intake and CCK-Ab titer. 23 While the report on active immunization against CCK in parity one lactating sows (Nelson, 1996) showed promising results, the study utilized a regression model approach and a relatively small number of animals. A control set of sows were not included and immunization resulted in widely-variable titer responses. Lastly, the study did not address potential implications on post- weaning reproductive performance. Because immunization against CCK has produced inconsistent titers in sows and has failed to elicit greater feed intake in sheep (Trout et al., 1989; Spencer, 1992), effects of this technique need further study. In particular overproduction of CCK as a feed back mechanism could explain results of studies in which immunization against CCK failed to achieve greater feed intake. The objectives of this study were, in Exp 1, to determine the effect of active immunization against endogenous CCK on the voluntary feed intake, lactation productivity, and weaning-to-estrus interval of primiparous sows; in Exp 2, to evaluate systemic concentrations of free-CCK to test for possible overproduction of free-CCK by sows immunized against CCK. Materials and Methods All procedures for this study were approved by the Michigan State University Committee for Animal Use and Care. 24 General Procedures for Pigs Experiment 1. Seventy-two gilts, divided in nine different groups, were used from March 1996 to June 1998. Gilts were observed for signs of estrus twice daily and artificially inseminated 12 hrs after being observed in standing heat, and at 12 hours interval thereafter for a maximum of three inseminations. Gilts were housed in individual gestation crates over partially slotted floors throughout gestation, and were offered 2.5 kg per head daily of a standard com-soybean meal diet (table 1). Room temperature ranged between 20°C and 25°C, heat was provided by gas heaters and ventilation by manually-controlled wall fans. Artificial light was provided between 8 am and 7 pm each day. On about d 64 of gestation (as an average of breeding dates for the entire group) gilts were randomly divided into two treatments: 3) control or vaccinated with placebo and b) CCK or vaccinated with Cholecystokinin, and administered the primary vaccination (see vaccine preparation below). The primary dose contained KLH- KLH conjugate for control sows and CCK-8-KLH conjugate for CCK sows. Both conjugates were emulsified in a solution of 50% Freund's complete adjuvant and 50% phosphate buffer (Pekas and Trout, 1990). Each 1 mL dose contained 1mg of antigen. The dose was administered subcutaneous on one side of the neck. On d 78, 92 and 106 of gestation gilts were administered booster vaccines (Figure 1) containing the same amount of either conjugate as the primary vaccine, except administered in Freund’s incomplete adjuvant. Booster doses were alternatively administered on the opposite side of the neck. Gilts were also vaccinated prebreeding for parvovirus, leptospirosis and erysipelas; prefarrowing 25 for bordetella, E. coli, pasteurella, Transmisible Gastroenteritis, erysipelas and clostridium. Prior to each CCK vaccination, and also on d 7, 14, and 21 of lactation, blood samples were taken from sows'via external jugular vein puncture. Serum was harvested and frozen for future anti-CCK titer analysis. One week before scheduled farrowing time, gilts were moved to partially slotted farrowing crates. Room temperature ranged between 20°C and 25°C, heat was provided by gas heaters and ventilation by manually-controlled wall fans. Artificial light was provided between 8 am and 7 pm each day. During lactation (26 i 1 d), sows were provided ad libitum access to a standard lactation corn-soybean meal diet (Table 1) and water. Feed disappearance during lactation was recorded daily. Sows were weighed at about d 110 i 2, 12 h post-farrow and on d 7, 14 and 21 of lactation. The return-to- estrus interval was recorded following weaning. Sows were observed for estrus twice daily (morning and evening). Because suckling stimulus is known to drive feed intake and may stimulate CCK release (Linden et al., 1990), litter size was standardized at 11 pigs per litter by d 2. Non-experimental sows which farrowed at the same time as the test gilts provided additional piglets as needed. Creep feed was not offered to piglets during lactation. Stillbom and live piglets were individually weighed within 12 h of birth and the number of mummified fetuses was recorded. Piglets were weighed on d 7, 14 and 21 of lactation. Litter gains were used to assess sow milking ability. 26 Experiment 2. Fourteen gilts (n=7 for each treatment), were used. In addition to the procedures mentioned above, blood samples were taken on d 18 of lactation for total (bound to CCK-Ab + free) and free-CCK determination. On the evening of d 17 of lactation, gilts were catheterized through the ear vein using a 0.1 mm diameter catheter (Tygon® lD 0.1mm Fisher Cat # 14170-15D). Gilts were fasted until the next morning. On d 18 of lactation (sampling day), a pre- prandial blood sample was taken, and immediately afterwards, gilts were fed 6.8 kg of their lactation diet. Gilts were monitored for number of meals taken, duration of each meal, amount of feed eaten in first meal, and total amount of feed eaten during a 6 h period. In addition, blood samples were obtained at 15 min intervals during the 6h period. Blood samples were collected (5 mL) into a ice-chilled syringe and transferred into a polypropylene tube containing EDTA (1 mg/mL of blood) at 0°C. Blood was centrifuged at 1600xg for 15 min at 4°C and plasma was collected and stored at -70°C until laboratory analysis. Vaccine Preparation and Laboratory Analysis The CCK immunogen was purchased from Cambridge Research Biochemicals (Cambridge, UK). it was produced by conjugating the desulfated C-terminal octapeptide of Cholecystokinin (CCK-8) to keyhole limpet hemocyanin (KLH) via glutaraldehyde condensation. Although the sulfated form of CCK-8 is needed for biological activity, antibodies have been raised to the unsulfated form (Pekas and Trout, 1990) at much lower expense. For this reason the desulfated form was used. The placebo immunogen consisted of only KLH-KLH conjugate. 27 On the same day of vaccination, the antigen was dissolved in phosphate buffer and emulsified with Freund’s adjuvant using a tissue homogenizer (Ultra Turrax T25, Janke and Kunkel®, Frankfurt, Germany). Determination of serum, anti-CCK titers was performed using the radioimmunoassay method described by Pekas and Trout (1990) as revised by Pekas (1996). Bolton-Hunter 125l-labeled CCKBS (sulfated; 2200 Ci/mmol; Dupont, Boston, MA) was the radiolabeled antigen. Each assay tube contained 4,885 dpm or 1 fmol of Bolton-Hunter 125l-labeled CCKBS. Antiserum titers were computed from the specific binding of Bolton-Hunter 12‘r’l-labeled CCK8S at four dilutions (1 :50, 1:325, 122113, and 1:13731) in phosphate buffered saline (.01 M, pH 7.5) containing 05% gelatin (Sigma cat # G-2500). The antiserum titer is defined as that serum dilution that gives 50% specific binding of Bolton-Hunter ”SI-labeled CCKBS. Determination of free-CCK in Experiment 2 was performed by filtering 1.5 mL of plasma (Centriplus 100 filter, cat It 4414 Amicon lnc., MA) using a centrifuge with a 34-degree fixed-angle rotor at a speed of 3000 xg for 2 h at 4°C. Alliquots of 0.7 mL of filtered plasma were extracted with 1.4 mL of 100% ethanol. Samples were stored at room temperature for 30 min after vigorous vortexing for 10 s. The mixture was centrifuged for 30 min (1500 x g) at 4°C. The supernatant was decanted into clean 12 x 75 mm polypropilene tubes and then evaporated in 3 Speed Vac Concentrator (Heto VR-1, ATR, Laurel, MA) at room temperature. The dried extracts were reconstituted to original volume with assay 28 buffer and analyzed for CCK (RlK-7181, Peninsula Laboratories, Belmont, CA) following manufacturer's instructions. Determination of total CCK was performed by extracting 0.4 mL of plasma with 0.8 mL of 100 % ethanol and following the same procedure as described for free-CCK determination with the exception of the filtering step. Statistical Analysis Experiment 1. Data was analyzed as a randomized block design using the GLM procedures of SAS (1996). Sows were blocked by time (group). Analysis of Variance was used to test immunization treatment (CCK and Control) on the independent performance variables (sow ADFI and piglet ADG during wk 1,2,3, and for the total 3 wk experimental period, weaning-to-estrus interval, number of piglets born alive, litter weight at birth, litter size at d 21 of lactation, and litter weight at d 21 of lactation). The mean differences between treatments were detected by comparison of least square means. Percent of sows expressing estrus by d 7 postweaning was also analyzed using Chi-square. Regression analysis was used to correlate ADFI and log mean dilution titer during wk 1, 2, 3 and for the total 3 wk experimental period. Difference between means was considered significant at P<.05, and P<.1 was considered a trend. Experiment 2. Statistical analysis was performed using the GLM procedure of SAS (1996). Analysis of Variance was used to test immunization treatment (CCK and Control) effect on peak free-CCK concentration, number of meals, average duration of meal, duration of first meal, amount of feed eaten in first meal, and 29 amount of feed eaten for the duration of the experimental period (6h). The mean differences between immunization treatment (CCK and Control) were detected by comparison of least squares means. Difference between means was considered significant at P<.05, and P<.1 was considered a trend. 30 Blood collection for titer analysis .33:- + ”aim Gestation d64 d78 d92 d106 d14 Farrow d 7 £ £ £ Ii Lactation Estrus Farrow Figure 2. Exp 1 time sequence of vaccination and sample collection. 31 Table 1. Diet Composition (as fed) Diets Gestation Lactation ingredients % in diet Corn dent yellow 68.32 63.92 Soybean meal 44% 14.59 28.97 Wheat Bran 10.00 —- Choice White Grease 2.00 2.00 Ca Phos Monocal 21% 1.88 2.05 Vitamin Premixa 0.60 0.60 Salt. 0.50 0.50 Limestone 1 .30 1 .15 Trace Min premixb 0.50 0.50 Sow Pack° 0.30 0.30 Calculated Analysis Lysine % 0.65 1.00 Ca % 0.90 0.90 Total P % 0.80 0.80 a Supplied per kilogram of premix: vitamin A, 918583 lU; vitamin 03, 91858 IU; vitamin E, 11023 lU; vitamin K (as menadione sodium bisuifite complex) 735 mg; riboflavin, 735 mg; pantothenic acid 2939 mg; niacin, 4409 mg; vitamin B12, 5512 mcg; thiamin, 184 mg; pyridoxine, 165 mg; Ethoxyquin, 0 .15%; Mineral Oil, 2%. ° Supplied per kilogram of premix: Zn, 2000 mg; Cu, 2000 mg; Fe, 2000 mg; Mn, 2000 mg; I, 30 mg; Se, 60 mg, mineral oil 2%. ° Supplied per kilogram of premix: Vit A, 918583 lU; Biotin, 73487 mcg; Choline, 128602 mg; Folic Acid, 551 mg; Ethoxyquin, 0.15%; Mineral Oil, 2%. 32 Results Experiment 1. Three sows did not finish the experiment because of health considerations (two of them, one per treatment, did not farrow, and the other, Control, failed to milk). Thus, data was obtained from sixty-nine sows. Serum titer analysis by RIA yielded a great range of titer on farrow (d0), d7, d14, and d21 of lactation. Log dilution values ranged between 0 and 4.15. Variation was similar on any sampling day. Coefficient of Variation for each sampling day were .247, .250, .244, .258 for samples at farrow, d7, d14, and d21 of lactation, respectively. Samples from eleven CCK sows did not reach 50% of specific binding activity at the lower dilution (1:50, definition of titer). Analysis of lactation and reproduction data showed no differences between treatments in sow ADFI in wk 1, 2, 3 of lactation, and for the total duration of the experiment (Table 2). There were no differences between treatments on ADG of piglets for wk 1, 2, 3 and for the total duration of the experiment (Table 3). Number of piglets born alive, litter birth weight, litter size at d 21 of lactation and litter weight at d 21 of lactation were similar for both treatments (Table 3). Differences among sow groups (replicates) were identified for ADFI in wk 1, 2, 3 and for the total duration of the experiment (Table 7, Appendix D). There were also differences in ADG of piglets among replicates in wk 2, 3, and for the total duration of the experiment (Table 7, Appendix D). Analysis of weaning-to-estrus interval data showed no differences between treatments (Figure 3), but some differences (P<.05) among replicates (Table 8, 33 Appendix D). Percentage of sows retuming-to-estrus by d 7 postweaning were similar between treatments (Figure 3). Data was also analyzed without including values belonging to sows that did not develop a titer against CCK. This was done to rule out the potential confounding effect of animals that, belonging initially to the immunized group (CCK treatment), did not raise antibodies against CCK. Again, there were no differences either between treatments in any of the variables analyzed. A regression analysis was also performed, correlating ADFI for each sow, during individual wk 1, 2, 3, and the total 3 wk lactation period, with log titer values of sows for those weeks. There were no correlations (P>. 05) during wk 1, 3 and total 3 wk lactation. There was a significant (P<.05) r2 for ADFI in wk 2 and average log titer for that period, with r2 explaining only 15% of variation. Experiment 2. There were no differences between treatments for free-CCK values (12 i 3 pmol/L CCKivs 19 leL i 5 Control). immunized animals had greater total CCK (CCK-Ab bound and free) than free CCK (70 i- 9 vs. 12 : 3 pmollL, respectively). Animals on the control group had the same values for free and total CCK (Figure 4). There were no differences between treatments in number of meals, total eating time, average meal time, duration of first meal, feed intake of first meal (Table 4). Sows immunized against CCK tended to eat more (P<.1) than controls during the 6 h observation period (3.97 i .41 CCK vs 2.89 i .45 Control). 34 Table 2. Effect of immunization against CCK on sow’s lactation performance“. Treatment Level of Item Control CCK Significance n 34 35 NS° Sow Pre-Farrow Weight (kg) 180.34 15.04 100.34 i4.07 NS Sow Weight Change from Farrow to d 21 (kg) 11.52 :183 11.05 :148 NS 1 Sow ADFI (kg) Wk1 4.25 i .169 4.26 i .149 NS Wk2 4.93 :I: .170 5.00 :I: .150 NS MS 5.46 i .204 5.70 i .179 NS Total3wk 4.88 i .152 4.99 i .134 NS a Least Square Means :1: SE, n=69 ° NS = Non-Significant 35 Table 3. Effect of immunization against CCK on soWs reproductive performance“ Treatment Level of item Control CCK Significance n 34 35 Ns" Piglets Born Alive 8.06 i .421 7.72 i .385 NS Litter Birth Weight (kg) 12.44 :t .63 11.57 1 .58 NS Before Cross-foster Piglet ADG (kg) M1 .176 i .009 .176 :I: .008 NS Wk 2 .236 :I: .007 .233 :t .006 NS Wk 3 .217 i .008 .219 j: .007 NS Total 3 wk .210 :l: .006 .209 :t .005 NS Litter Size atd21 10.58 i .18 10.62 i .16 NS (number of pigs) Litter Weight at d 21(kg) 62.70 11.67 62.00 $1.53 NS ° Least Square Means i SE, n=69 ° NS = Non-Significant 36 I % of sows 0 A. I] days day' 10 9 8 7 6 5 4 3 2 1 0 Control CCK Treatment Figure 3. Effect of immunization against CCK on weaning-to-estrus interval. 37 leL 80 70 60 50 Free ; Total Contrbl CCK Treatment Figure 4. Effect of immunization against CCK on free and total CCK concentration in plasma. 38 Table 4. Effect of immunization against CCK on eating behavior“. Treatment Level of Item Control CCK Significance n 7 7 NS” Number of meals 4.14 t .44 4.48 i .40 NS Total time eating (minutes) 54.48 :5 10.81 61.41 i 9.81 NS Average time per meal 1.82 3: .41 1.87 i .37 NS Duration of 1"t meal (minutes) 30.49 i 5.87 27.72 i 5.32 NS 6 h feed intake (kg) 2.89 i .45 3.97 :t .41 t Feed intake 1"t meal (kg) 1.82 i .41 1.88 :i: .37 NS ° Least Square Means i SE. ° NS=Non-Significant. ' t indicates P<.1 (trend). 39 Discussion The great variation in titer values was expected based on results obtained by Nelson (1996). Results from Exp 1, with no differences across treatments for any of the lactation performance variables, indicate that there’s no advantage of vaccinating sows against CCK. Our results are in agreement with those in sheep (Trout et al., 1989; Spencer, 1992) in which there were no feed intake differences between vaccinated and control animals. The possibility of vaccinated animals not achieving greater performance simply because they did not develop a titer, is ruled out by the analysis of data only from animals with a titer against controls. Again in this case, there were no differences in performance between animals with a titer and controls. Regression analysis results demonstrated also that there’s no advantage in vaccinating against CCK. Our regression analysis included ADFI during wk 1, 2, 3 and the total 3 wk lactation period, with log titer value for the same period, respectively. Nelson (1996) obtained significant r2 values (r2=.7) when correlating ADFI during week 3 with a combination of log titer values including gestation, lactation and colostrum. However, the biological reason for establishing a relationship between ADF I with titer values from periods of sow production as far as 64 days before is weak. Results of Exp 2 help to explain those of Exp 1. Sows immunized against CCK appear to overproduce CCK in response to the sequestering of free-CCK by anti-CCK antibodies. This is shown by the fact that total CCK values were larger than free-CCK values in CCK-immunized animals (Figure 4). Total and 40 free-CCK values for control animals did not differ (Figure 4). A reasonable explanation would be that, in animals vaccinated against CCK, CCK-Ab sequestered almost 2.5 times the amount of free-CCK hormone. The animal then, sensing a decrease of free-CCK, responded with a feed-back mechanism by producing more free-CCK in order to achieve a normal (before vaccination) free-CCK concentration in blood. This was suggested by others (Spencer, 1992) Our values for free-CCK are in agreement with other reports on free-CCK values in pigs (Clutter et al., 1998). Free-CCK values are no different for both treatments. This would explain the lack of response in performance across treatments to CCK immunization in both Exp 1 and Exp 2. In Exp 2, immunized sows tended to eat more during the 6 h period (3.97 i .41 CCK vs 2.89 i .45 Control). This could be explained by the free-CCK values for each treatment. Immunized animals had a lower free-CCK concentration (numerically) than Controls (12 i 3 pmollL vs 19 pM/L i 5, respectively). While these values were not significantly different, the fact that that sows on CCK group had lower free-CCK could result in less anti-eating action of CCK, thus showing a greater feed intake. Implications Vaccinating parity-one sows against CCK did not improve ,lactation performance, nor it affected return-to-estrus interval. Vaccinated animals may compensate for the sequestering of endogenous CCK by overproducing higher 41 amounts of free-CCK, as suggested by Spencer (1992). This suggests that the development of different vaccines which produce similar amounts but more consistent titer responses from sows, would be of little benefit. Further studies testing the overproduction hypothesis could be developed by measuring mRNA, an indicator of peptide production, in CCK-producing cells, such mucosal-l cells of the small intestine. 42 Chapter 3 PASSIVE IMMUNIZATION AGAINST CHOLECYSTOKININ (CCK) AND ITS EFFECT ON GROWTH PERFORMANCE OF NURSERY PIGS ABSTRACT Forty-eight, early-weaned crossbred pigs (10 to 12 d of age) from sows vaccinated against CCK or from control sows, which had been vaccinated with a placebo, were used to test the potential benefits of passive immunization on performance of nursery pigs. Animals were housed in pens containing four animals of the same treatment. Individual pig weights were recorded on d 0, 7, 14, 21, 28, and 35. Feed disappearance was monitored daily for each pen. Serum samples were collected via vena cava puncture from each pig on days of age 14, and 21 and anti-CCK titer on was determined by RIA. There was a difference in ADG (P<.01) between immunized (CCK) and non-immunized (Control) animals during wk 1 (.23 kg vs .20 kg, SE .01 ). Values for ADG for individual wk 2, 3, 4 and 5 were not different between treatments. There was a difference in ADFI (P<.05) between treatments during wk 2 (.42 kg CCK; vs .35 kg Control; SE .02). Values for ADFI for individual wk 1,3,4 and 5 were not different between treatments. Over the 5 wk experimental period, ADF I (.44 vs. .41, SE .01) was greater (P<.05) and ADG (.67 vs .63, SE .01) tended to be greater (P<.1) for the CCK group. Feed/Gain ratio (F/G) did not differ for any individual week or the 5 wk experimental period. Passive immunization against CCK improved growth performance of early-weaned nursery pigs. Keywords: Pigs, CCK, Passive immunization, Nursery, Early-Weaning. 43 Introduction Cholecystokinin (CCK) is a hormone involved in regulation of appetite and feed intake. In a series of studies (Pekas and Trout, 1990; Pekas, 1993; Pekas, 1996) with growing pigs, active immunization against CCK stimulated feed intake and growth in proportion to the anti-CCK antibody (CCK-Ab) titers elicited in the animal. Nelson (1996) demonstrated that piglets from sows vaccinated with CCK were passively immunized, having a titer at d 7 of age equivalent to that of the lactating sow. Weaning piglets at less than three weeks of age (Early Weaning) has become a standard practice in swine production because of health considerations and improved growth performance. Early-weaning main advantage is to stop bacterial and viral spread from the sow to the piglets. Piglets are passively immunized against disease. Sows immunized against CCK transfer their immunity to CCK to their piglets as well, and thus, could produce an improvement in piglets feed intake by stopping CCK action on satiety. This study was designed to determine the effect of passive immunization against CCK on growth performance of early- weaned nursery pigs. Materials and Methods All procedures for this experiment were approved by the Michigan State University Committee for Animal Use and Care. General Procedures for Pigs. A total of 48 early-weaned crossbred pigs Newsham® X (Yorkshire X Landrace)), averaging 11 d i 1 of age were used. Piglets were obtained from sows that had been vaccinated against endogenous CCK on d 64, 78, 92 and 106 of gestation (procedure described by Garcia-Sirera, 1999, previous chapter). Gilts were also vaccinated prebreeding for parvovirus, leptospirosis and erysipelas; prefarrowing for bordetella, E. coli, pasteurella, Transmisible Gastroenteritis, erysipelas and clostridium. Processing pigs on day one after birth included: ear notching, clipping of needle teeth, tail docking, iron shots of 150 mg iron and 0.25 mg ceftiofur hydrochloride. One day prior to weaning, pigs were weighed individually for allotment to treatment. At weaning, 150,000 U benzothine penicillin was given for prevention of Streptococcus suis infection. Pigs were not vaccinated at weaning. At weaning, pigs were allotted to treatments based on litter, sex, and passive immunization treatment (non-immunized vs. immunized). Pigs were housed in the Michigan State University Veterinary Isolation Facility, G-barn. Pigs were housed in three rooms (four pens per room). Pens were 1.22 x 0.9 m, with four pigs per pen. Temperature was controlled at each location so that the ambient room temperature remained between 20° and 28° C. Heat lamps and heat pads (1 in each pen) were used for the first week to provide micro environments. During the first week all pens were fed 50 g per day per pen on 400 cm2 plastic trays, and also had access to fenceline feeders. Pigs had access to one nipple waterer per pen. During wk 1 waterers were set to drip continually to help avoid dehydration and possible navel sucking. Feed intake and growth performance of piglets was monitored for 5 wk. individual weights were recorded 45 on d 0, 7, 14, 21, 28, and 35. Feed disappearance for each pen was monitored daily. Pigs were fed the same diet regardless of treatment (Table 5). The experimental period was divided into four phases. Phase 1 and Phase 2 diets were fed on wk 1 and wk 2, respectively. Phase 3 diet was fed on wk 3 and 4 and Phase 4 was fed on wk 5. Serum samples were collected via vena cava puncture from each pig on days of age 14, and 21 (equivalent to d 2 and d 9 of the feeding trial) . Anti-CCK titer was determined by RIA (Garcia-Sirera, 1999, previous chapter). Statistical Analysis. Data was analyzed as randomized block design using the GLM procedure of SAS (1996). Pigs were blocked by initial weight and equalized for ancestry and sex. Pen was the experimental unit. The mean differences between immunization treatment (CCK and Control) were detected by comparison of least squaremeans. Differences were considered significant at the level of P<.05, P<.1 was considered a trend. Piglets blood collection 66 Days of Age Gestation D 14 D 21 ll DO D7 014 weaning A vaccinations Farrow D 21 D 28 Days on Trial Feed intake Pigs weight 1r Figure 5. Time sequence of nursery trial showing time of sow vaccinations and sample collection. 47 Table 5. Composition of diets fed in different phases of nursery (as fed). Diets Phase 1 Phase 2 Phase 3 Phase 4 ingredients % Corn dent yellow 29.49 40.53 53.77 60.74 Soybean meal 44% 15.30 17.88 27.63 35.07 Plasma AP920 7.50 2.50 --- -- Edible Grade Lactose 7.50 4.00 -- ---- \Mtey, dried 25.00 20.00 10.00 ---- Fish meal 6.62 7.50 5.00 --- Choice White Grease 5.00 4.00 --- -- Mono-Dical Phos 0.62 0.80 1.20 1.45 Vitamin Premixa 0.60 0.60 0.60 0.60 Salt 0.50 0.50 0.50 0.50 Zinc Oxideb 0.38 0.38 -- --- Limestone 0.45 0.35 0.45 0.91 Antibiotic° 0.25 0.25 0.25 0.25 Trace Min premix‘ 0.50 0.50 0.50 0.50 Copper Sulfate“ 0.05 0.05 0.10 0.10 DL-Methionine 0.13 0.06 -- --- L-Lysine HCI 78.8% 0.10 0.09 --- --- Calculated Analysis ME, kcal / kg 3513.29 3457.36 3396.40 3408.00 CP % 23.70 22.11 19.64 19.19 Lysine 1.70 ' 1.45 1.25 1.15 Met + Cys 0.50 0.45 0.36 0.32 Ca % 0.90 0.88 0.90 0.80 Total P % 0.57 0.53 0.54 0.41 a Supplied per kilogram of diet: vitamin A, 5512 lU; vitamin D3, 551 IU; vitamin E, 66 IU; vitamin K (as menadione sodium bisulfite complex) 4.4 mg; riboflavin, 4.4 mg; pantothenic acid 17.6 mg; niacin, 26.4 mg; vitamin B12, 33 mg; thiamin, 1.10 mg; pyridoxine, 1.0 mg. ° Supplied 3000 mg of Zn per kilogram of diet (in addition to that provided by trace mineral premix). ° mecadox-10®. ° Supplied per kilogram of diet: Zn, 10 mg; Cu, 10 mg; Fe, 100 mg; Mn, 10 mg; I, 0.15 mg; Se, 0.3 mg. ° Supplied 125 mg of Cu per kilogram of diet (in addition to that provided by trace mineral premix in Phase 1 and 2). ' Supplied 250 mg of Cu per kilogram of diet fin addition to that provided by trace mineral premix in Phase 3 and 4). 48 Results Analysis by RIA on d 14 of age (d 2 of feeding trial), showed only 12 pigs with a titer against CCK (mean log titer 2.37 i .44). All samples from control animals did not show any specific binding as expected. immunized piglets in the experiment were originally from litters of six sows actively immunized against CCK. Serum from all sibling piglets belonging to one of the six original vaccinated sows, showed very low specific binding (between 15 and 19%) on d 14, and even lower on d 21 (around 10%). At least one piglet in each of the five other litters had serum with specific binding higher than 50% (1 :50 dilution, definition of titer) on d 14. With the exception of piglets from the low-titer litter, all other piglets had serum values showing specific binding values beyond 50% or very close to titer definition (higher than 40% but lower than 50%). Four out of the 12 pigs with a titer on d 14, still had a titer on d 21 (mean log titer 2.54 :t .20). Day 21 of age corresponds to an average of nine days into the feeding experiment. For the remaining 8 pigs with a titer on d 14, the specific binding had dropped beyond titer definition (50%) on d 21. The four pigs with serum samples still showing specific binding higher than 50% on d 21 were from the same original litter. All other samples from immunized animals on d 21 had low specific binding values (around 30%). Table 6 shows ADFI, ADG and FIG. There was a significant difference between immunized (CCK) and non-immunized (Control) animals for ADFI during wk 2 (.42 vs. .35, respectively; P< .05) and the difference tended to be significant for all 5 wk of experiment (.67 vs. .63; P < .1). There was also a significant 49 difference between treatment groups for ADG during wk 1 (.23 CCK vs. .20 Control; P < .01) and for all 5 wk of experiment (.44 CCK vs. .41 Control; P< .05). There were no differences in FIG ratio in any of the 5 weeks individually or when considering the whole experimental period. Discussion Overall ADG during the total duration of the experiment was increased in immunized animals vs. controls (.44 vs. .41, P<.05, respectively), and tended to be increased in ADFI (.67 vs. .63, P<.1, respectively). The results on growth performance can be explained with the results of titer analysis. Analysis of serum titer against CCK showed half the piglets from CCK sows, had a specific binding higher than 50% on d 14 of age, the threshold considered to be called “titer”. All the siblings originally from one of the six vaccinated sows that provided piglets for the experiment did not develop any titer. This was, most likely, a result of the sow not developing good immunity and consequently not passing it down to the piglets. On d 21 of age the percentage of piglets with a titer was even lower (only four pigs). This was expected because of clearance of immunoglobulins. This low percentage of animals with a titer may explain why the ADFI and ADG differences between treatments were not even more obvious. Titer in the piglets is determined by the titer in the colostrum of the sow, which can vary greatly (Nelson, 1996) and clearance of immunoglobulins once they are absorbed by the piglet. 50 it’s generally accepted that passive immunity in piglets reaches its peak after absorption of colostrum, at approximately 2 — 3 d of age and decreases rapidly after that. By week 3 of age immunity from colostrum is very low. For this reason passive immunization against CCK was expected to yield any potential benefits during the firsts weeks of the experiment, when the piglets were still very young. This reasoning was reflected, to a point, by the improved ADFI and ADG of immunized vs. control animals during the first two weeks of the experiment. The fact that ADFI was different between treatments during wk 2 and not wk 1 could be explained by the stress from weaning and adaptation to the experiment diets tended to equalize feed intake across treatments during wk 1. During wk 2, with the animals well adapted, the treatment effect of passive immunization was evident. it is important to notice also that, numerically, CCK-immunized animals outperformed Control animals in each individual week of the experiment for ADFI and ADG (Table 6). Good performance in the nursery is very important for future productivity of pigs, as a rule of thumb, one kilogram of improved weight gain during the nursery phase results in three total days less to achieve market weight. implications Passive immunization against CCK improved growth performance of early-weaned nursery pigs. Development of a sow immunization schedule with emphasis on producing peak titer values at farrowing, in order to increase 51 passive immunity of the piglets, may further improve results seen in this experiment. 52 Table 6. Effect of passive immunization against CCK of nursery pigs on growth performance“. Treatment Level of Item Control” ccr