THE EFFECT OF ACTIVE IMMUNIZATION AGAINST GHRELIN IN MALE BROILER BREEDERS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 0216208
• Project Start Date: Oct 1, 2008
• Project End Date: Oct 1, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762

Performing Department
FOOD SCIENCE & ANIMAL INDUSTRY

Non Technical Summary
Optimizing feed intake, metabolism and reproduction in farm animals has always been a high priority goal, but with the currently increasing price of animal feed, this goal has become even more important. Therefore, the objective of this proposal is to develop tools to study and manipulate the normal control mechanisms of energy balance and reproduction in commercial poultry. The development of new tools to manipulate feeding could improve production and reduce feeding costs in two different commercial branches of the poultry industry. Enhancing appetite and feed intake, thereby reducing the growing period and saving on feeding and maintenance costs, is desirable within the turkey industry. On the other hand enhancing satiety, and thereby improving production and reducing feeding expenses, is desirable within broiler breeder companies. Our approach is to use the affordable and simple technique of active immunization to attenuate the activity of endogenous serum factors. The approach presented here will enable the in-vivo study of long-term loss-of-function effects. In fact, active immunization treatments can be used for long periods of time if booster immunizations are given periodically according to recorded antibody titers. This approach provides a potent research tool that can also be developed for use in commercial husbandries. Successful results, showing a clear benefit of ghrelin active immunization on commercially important traits, will provide the necessary platform and validation for the development of cost-effective compounds either for active immunization of commercial birds or by using cost-effective synthetic agonists and antagonists such as those currently developed for human therapy. Therefore, understanding the normal control of energy balance and creating tools for its manipulation, could fulfill a number of specific needs in the poultry industry.

Animal Health Component

30%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3220	1020	40%
305	3220	1090	30%
302	3220	1010	30%

Knowledge Area
302 - Nutrient Utilization in Animals; 305 - Animal Physiological Processes;

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1090 - Immunology; 1010 - Nutrition and metabolism; 1020 - Physiology;

Keywords

ghrelin

active immunization

feed intake

appetite

obesity

poultry

broiler breeders

Goals / Objectives
The Poultry Industry is organized in a hierarchy that depends on high reproductive performance of chickens to supply large number of progeny to the market. Intense selection for growth rate has decreased the time required to produce a broiler chicken from approximately 12 to 6 weeks since 1955. This dramatic reduction of the interval from hatching to slaughter has been coupled with a rigorous growth restriction program for the development of parent stock. Food restriction of broiler breeders is practiced commercially to prevent obesity, which results in improved fertility. However, a life-time food restriction is perceived by some as contrary to present attitudes toward animal welfare. We have developed a protocol to immunize animals against ghrelin that results in a significant decrease in voluntary feed intake. If this protocol can be tailored for chickens, obesity may be prevented in broiler breeders, and the public perception of the poultry industry will be enhanced. Additionally (and equally important), chickens are monogastric (similar to humans) and this animal may provide a valid model to better understand human obesity. Ghrelin, a peripheral orexigenic signal, is predominantly produced in the stomach and induces increased feed intake in all species studied to date. We hypothesize that active immunization against ghrelin will impair the ability of this peptide to signal the orexigenic pathways in the hypothalamus. We rationalize that immunized chickens will decrease voluntary feed consumption. Therefore, the objective of this proposal is to evaluate the ability of active immunization against ghrelin to decrease voluntary feed intake in broiler breeders.

Project Methods
One-day-old broiler breeder male chicks (n = 24) will be reared as a single group using the feeding and lighting management recommended by the industry. Briefly, chicks will be placed on a 23 h light (L):1 h dark (D) photoperiod and ad libitum food intake. At 3 weeks of age (WOA), males will be placed on a restricted diet to maintain a weight gain of approximately 75% of their initial body weight per week (standard used in the industry). At the same time, photoperiod will be reduced to 8L:16D. At 8 WOA, 18 birds that are within the target weight will be selected and individually caged. At 10 WOA, animals will be randomly assigned to three treatment groups (n = 6/treatment). In treatment group 1, cockerels will be immunized against ghrelin (see below), while males in treatment groups 2 (negative control) and 3 (positive control) will serve as control and will be immunized against the carrier protein. Booster immunizations will be given at 13 and 16 WOA. Starting at 16 WOA, all chickens will be provided with a photoperiod of 16L:8D. Birds in treatment group 1 and 2 will be placed in an unrestricted diet while negative control birds (treatment 2) will remain in on the original restricted diet. At 21 WOA the experiment will be terminated. Starting at 9 WOA, and until the end of the experiment plasma samples will be obtained every week via a brachial wing vein. Samples will be placed on ice and centrifuged (1800 x g for 15 min) within 4 h, and plasma will be stored at -20 C until concentrations of various hormones are determined. Body weight will be recorded every week starting at 3 WOA. Starting at 16 WOA, daily feed consumption will be measured for five consecutive weeks. On week 21, animals will be euthanized and testis will be removed and weighted. The left testis will be placed in Bouin's solution for future histological analysis. The right testis will be placed immediately on dry ice for later analysis of daily sperm production. Birds on treatment group 1 will be actively immunized against ghrelin conjugated to bovine serum albumin (BSA; 50 μg of conjugated peptide). Control animals will be actively immunized against the carrier protein (BSA). Synthetic ghrelin will be conjugated to BSA by the carbodiimide reaction. The conjugate will be emulsified in Freunds Incomplete Adjuvant (FIA) and Diethylaminoethyl-dextran (DEAE; Sigma Chemical Co., St. Louis Mo). Approximately 4 mL of the emulsion will be given i.m. in four locations of breast muscle. The proposed doses for primary and booster immunizations were chosen from doses used previously by the PI. Expected results and potential problems: We anticipate that the results from the proposed experiment will confirm our hypothesis. Immunized animals will develop antibodies specific for ghrelin, will decrease voluntary feed intake and will attained a reduced body weight gain. No problems are expected while conducting the actual experiment since the PI is familiar with all the techniques proposed in this study including immunization, chicken management, and hormone assays.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Five-week-old turkeys were used to evaluate the effect of passive immunization against ghrelin on feed and water intake and animal behavior. In Experiment 1, females were reared using normal feeding and lighting management recommended by the industry. At 5 wk of age (day 0 of experiment 1), birds (n = 40) were individually caged (0.65 x 0.4 x 0.4 m) with free access to feed and water. Feed and water intake was measured 3 times a day (0800; 1200 and 1700 h) by recording the weight of feed or water offered minus any unconsumed feed or water remaining. After 3 days of adaptation to the cages (day 3), animals were stratified by body weight and feed consumption and randomly assigned to a 2 x 5 factorial arrangement of treatment. Starting on day 3, turkeys were given intravenous (iv) injections (0.5, 1.0, 2.0 4.0 or 8.0 mL) of pooled undiluted plasma obtained from pigs that were previously actively immunized against ghrelin (IMM), or iv injections (0.5, 1.0, 2.0 4.0 or 8.0 mL) of pooled undiluted plasma, obtained from non-immunized pigs (CTRL). In experiment 2, the two highest doses (i.e. 4.0 and 8.0 mL; n=4/treatment) were repeated in a 2 x 2 factorial arrangement as described in experiment 1. A laptop computer with a built-in color camera and appropriate software was used to record birds for 9 consecutive hours, starting 4 h before treatments were applied. Video clips were saved and a human observer watched and annotated animal behavior associated with feeding, drinking, and standing. Passively immunized birds increased feed consumption (P = 0.04) compared with control animals. Water intake was not affected by treatments. There was a tendency for immunized birds to increase the number of pecks per hour and the amount of time devoted for feeding. Our data indicates that the effect of ghrelin in turkeys is similar to that reported in chickens, and opposite of that observed in mammalian species. PARTICIPANTS: D. Washington (undergraduate student) H. Wright (undergraduate student) A. Vizcarra (undergraduate student) K. Bellingheri (undergraduate student) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The animal phase of the project outlined above was completed. However, all samples and antibodies associated with this project were lost after the tornadoes of April 27, 2011. Reagents and blood samples kept at -20 C were lost due to the power outage in the laboratory. A related research project entitled "Preprandial concentrations of ghrelin in turkeys" is being conducted to complement the limited data previously obtained. Rationale: We previously reported that pre-prandial concentrations of ghrelin are increased in pigs. However, our preliminary data (using passive immunization against ghrelin), suggest that the effect of ghrelin on appetite in turkeys is the opposite of that observed in mammalian species. Therefore, we propose to evaluate the orexigenic pathways associated with feed restriction in poultry species. Brief description of project plan: Turkeys will be reared as recommended by the industry. Starting at 5 weeks of age, animals will be weighed and placed in individual cages with free access to feed and water. After 4 days of adaptation to the cages, feed will be removed and animals will be fasted for 24 h. After fasting, a blood sample will be obtained from each animal. Subsequently all animals will be fed and a second blood sample will be obtained 1.0 h after feeding. Blood samples will be collected via venipuncture in 5-mL evacuated tubes containing EDTA (Fisher Scientific, Pittsburgh, PA). Immediately after blood collection, aprotinin (500 KIU/mL of blood) will be added to the collection tube to inhibit the activity of proteases. Tubes will be gently rocked several times, placed on ice, and centrifuged (1,800 x g for 15 min) within 30 min. Plasma samples will be maintained at -20 C until concentration of glucose, insulin, free fatty acids, ghrelin, ACTH and corticosterone are evaluated.

Impacts
Water intake was not affected by treatment; however, feed intake was significantly increased in birds that were passively immunized against ghrelin. Additionally, the number of pecks and the time devoted to feeding was significantly increased in treated birds. Our data suggests that in turkeys, the response to ghrelin is the opposite of that described in mammalian species. In the US, the turkey market is in its growing phase. In fact, per capita turkey consumption has increased 44% during the last 20 years. Therefore, farmers are seeking ways to enhance feed efficiency, which is one of the potential benefits of the ghrelin immunization protocol described in this project. An additional impact of this research is the involvement of undergraduate students. Several undergraduate students have been involved in this research and published results (see below).

Publications

• Vizcarra, J., H. Wright, and A. Vizcarra. 2012. The effect of passive immunization against ghrelin on feed and water intake in turkeys. Poult. Sci. 91 (9) 2305-2309.
• Vizcarra, J.A. 2012. Differential feed intake in mammalian and avian species: Immunization against ghrelin. Arch.Lat.Prod. Anim. 20(3-4):113-117.
• Vizcarra, A., H. Wright, L. Shackelford, G. Abdelrahim, D. Washington, and J.A. Vizcarra. 2011. Orexigenic Effects of Anti-Ghrelin Antibodies in Broad-Breasted White Turkeys. Association of Research Directors. Atlanta, Georgia. (Abs P-SP-U-30 p 173).
• Wright, H., A. Vizcarra, L. Shackelford, G. Abdelrahim, and J. Vizcarra. 2011. The Effect of Passive Immunization Against Ghrelin on Animal Behavior. Association of Research Directors. Atlanta, Georgia. (Abs P-SP-U-34 p 174).
• Vizcarra, A., H. Wright, L. Shackelford, G. Abdelrahim, and J. Vizcarra (advisor). 2011. The effect of passive immunization against ghrelin on feed intake in turkeys. STEM Day 2011. Alabama A&M University. Abstract #58.
• Wright, H., A. Vizcarra, L, Shackelford, G. Abdelrahim, and J. Vizcarra (advisor). 2011. The effect of anti-ghrelin antibodies on turkey behavior. STEM Day 2011. Alabama A&M University. Abstract #59.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: One-day-old female Broad-Breasted-White turkeys were obtained from Eagles's Nest Poultry (Oceola, Ohio) and were reared using the normal feeding and lighting management recommended by the industry. Starting at 5 WOA, birds were randomly assigned to 2 treatment groups. In treatment group 1, turkeys were given intravenous (iv) injections (0.5, 1.0, 2.0 4.0 or 8.0 mL) of pooled undiluted plasma, obtained from pigs actively immunized against ghrelin (passive immunization). In treatment group 2 birds were given iv injections (0.5, 1.0, 2.0 4.0 or 8.0 mL) of undiluted normal pig plasma. Four days before treatments were applied animals were weighed and placed in individual cages with free access to feed and water. Feed and water consumption was recorded at 6 to 8 h intervals. On day 0, immunizations were given. Feed and water intake was determined by recording the weight of feed or water offered minus any unconsumed feed or water remaining. Blood samples were obtained before and 2 h after treatments were applied. In addition, on day 0, animal behavior was evaluated by videotaping animals. The amount of time eating, drinking, lying or standing will be evaluated. The animal phase of this project will continue during the fall semester and laboratory analysis will follow. PARTICIPANTS: D. Washington (undergraduate student) H. Wright (undergraduate student) A. Vizcarra (undergraduate student) L. Shackelford (PhD candidate) J. Khatiwada (Post-Doc fellow) G. Abdelrahim (Associate Professor) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Water intake was not affected by treatment; however, feed intake was significantly increased in birds that were passively immunized against ghrelin. Additionally, the number of pecks and the time devoted to feeding was significantly increased in treated birds. Our data suggests that in turkeys, the response to ghrelin is the opposite of that described in mammalian species. In the US, the turkey market is in its growing phase. In fact, per capita turkey consumption has increased 44% during the last 20 years. Therefore, farmers are seeking ways to enhance feed efficiency, which is one of the potential benefits of the ghrelin immunization protocol described in this project. An additional impact of this research is the involvement of undergraduate students. Up to now three undergraduate students (2 from the Animal Sciences Department and 1 from the Biology Department) have been involved in this research and published results (see below).

Publications

• Orexigenic Effects of Anti-Ghrelin Antibodies in Broad-Breasted White Turkeys (Meleagris gallopavo). A. Vizcarra*, H. Wright, L. Shackelford, G. Abdelrahim, D. Washington, and J. Vizcarra. 2011. Department of Food and Animal Sciences, Department of Biology; Alabama A&M University, Normal Al 35762.
• The Effect of Passive Immunization Against Ghrelin on Feed Intake in Turkeys. 2010. D. Washington, G. Abdelrahim, J. Khatiwada, L. Shackelford and J. Vizcarra
• The Effect of Passive Immunization Against Ghrelin on Animal Behavior. H. Wright*, A. Vizcarra, L. Shackelford, G. Abdelrahim, and J. Vizcarra. 2011. Department of Food and Animal Sciences, Department of Biology; Alabama A&M University, Normal Al 35762.