Progress 10/01/01 to 09/30/04
Outputs
We reported that pigs weaned between 10-12 days of age (early-weaned: EW) were more aggressive and showed deficits in their social behavior when compared to pigs weaned at 21 days of age (CW) (Yuan et al., 2004). We proposed that excessive aggression, which persisted for up to 9 weeks of age in EW pigs, was caused by memory deficits. We adapted the Morris-Water Maze to assess spatial memory (Laughlin, Wilson and Zanella, in review, AABS) and developed a novel test to assess social recognition (Souza et al., in review, AABS). We reported that EW pigs showed deficits in spatial memory when socially isolated (Laughlin and Zanella, in review, Physiol. and Behav.) and had their ability to recognize familiar pigs compromised (Souza et al., in review, AABS). We developed the pig brain cDNA library (Nobis et al., 2003) and used this unique resource to investigate the association between aggression and cognitive deficits with gene expression profile in the frontal cortex (FC)
(Poletto et al., in review, Behavioral Neuroscience). EW pigs and non-weaned pigs (NW) were either socially isolated (SI) for 15 minutes, two days post-weaning, immediately prior to euthanasia, or were not isolated (C). Microarray hybridization and Q-RT-PCR examined the effects of EW and social isolation on gene expression in the FC. Social isolation of NW and EW piglets decreased biosynthesis of neurotransmitters and neuropeptides, affected neurosteroidogenesis, and caused deficits in neuronal development and protection in the FC of pigs. Q-RT-PCR was used to further investigate 6 brain-related genes from the microarray results. A significant suppression of carboxypeptidase E, tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein mRNA, and phosphoprotein enriched in astrocytes 15kD (PEA-15) mRNA as an effect of SI in NW isolated compared to NW controls. Early-weaning in addition to SI significantly suppressed diazepam-binding inhibitor (DBI) and actin related
protein 2/3 complex (Apr2/3) mRNA in EWI compared to EWC animals. We investigated the potential neuroprotective role of RU486, a glucocorticoid antagonist, in EW and CW pigs. The oral administration of the compound was the most successful route for maintaining consistent levels of the compound in the central nervous system,with minimal disturbance. We administered 1, 5 and 10 mg/kg BW of RU486 every 8 hours, during 48 hours post-weaning. RU486 did not prevent the spatial memory deficits experienced by early-weaned pigs. Q-RT-PCR was used to examine the expression of the stress related genes 11 beta-hydroxysteroid dehydrogenases 1 and 2 (11 beta-HSD1 and 11 beta-HSD2), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) in the hippocampus and FC (Poletto et al.,in preparation). Social isolation decreased the expression of all tested genes (p < 0.05) in the FC of both 12 and 23 days old pigs. When compared with non-weaned at age 12, hippocampus of early-weaned piglets
showed a significant decrease in expression of the four genes (p = 0.004). Immunohistochemistry studies is currently being performed on a subset of hippocampal slides using antibodies against GR, MR, 11 beta-HSD1 and 11 beta-HSD2.
Impacts
Stress induced disorganization of glucocorticoid sensitive brain pathways is likely to be the source of neuropathology in animals, including humans. We demonstrated that stress associated with depriving pigs of maternal nourishment and care, at an early age, affects the brain and behavioral development. Aggression in pigs, is a very important animal welfare issue. We demonstrated that aggression can be decreased by not weaning animals before three weeks of age. We presented this information at national and international meetings and supervised one MS and one Ph.D. graduate student in this project. Our studies will enhance our ability to design production systems and treatments that will reduce stresses and resulting aberrant and potentially damaging behaviors and excessive aggression. These changes will result in improved gains in production and increased profitability. The understanding of the changes in the brain associated with the onset of behavioral problems and
consequent disorganization of neural pathways will provide scientific information regarding the validity of such measures as welfare indicators. Our goal is to develop intervention strategies to allow the reversal of stress induced pathological processes and to diminish the consequent developmental problems reported in several animal species.
Publications
- Laughlin K, Wilson E, & Zanella AJ. (2004 - submission) In review. A. Modification of the water maze procedure to examine cognitive processes in nursing and newly weaned pigs. Appl Anim Behav Sci.
- Laughlin K & Zanella AJ. (2004 - submission) In review. B. Stress-induced deficits in pig spatial learning: influence of weaning age. Physiol Behav.
- Poletto R, Siegford J, Nobis W, Zanella AJ. 2003. Differential expression of genes in the hippocampus of early-weaned piglets when examined using a cDNA microarray. International Symposium for Animal Functional Genomics, Michigan State University.
- Poletto R, Siegford JM, Stiebel JP, & Zanella AJ. 2004. Differential expression of genes in frontal cortex of pigs following weaning and social isolation: implications for neural underpinnings of welfare. Proceedings of the 38th International Congress of the ISAE. 38:114.
- Poletto R, Siegford JM, Steibel JP, Coussens PM, Zanella AJ. (2005 -submission)In review. Effects of early weaning and social isolation on gene expression in the frontal cortex of piglets. Behav Neurosci
- Siegford JM, Souza AS, Jansen J, Zanella AJ. 2004. Correlating spatial learning, social recognition, and aggression in young pigs. J Anim Sci 82(Supplement 1):276.
- Souza AS, Laughlin K, Siegford JM, Zanella AJ. 2004. Acute stress impairs spatial learning and social recognition in early-weaned pigs. J Anim Sci 82(Supplement 1):276.
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Progress 01/01/03 to 12/31/03
Outputs
Our overall goals are to understand and alleviate health and well-being problems associated with weaning stress in piglets. In situ hybridization work on GR and MR has been performed using a radioactive protocol in hippocampi of 12 and 23 day old piglets subjected to combinations of early weaning (EW), conventional weaning (CW), and social isolation (SI). A pig brain cDNA library (PBL) has been developed with collaborators (Nobis et al., 2003) and used to create a cDNA microarray to simultaneously show EW and SI influence expression in 866 genes in areas involved in emotional regulation (i.e., frontal cortex) and learning and memory (i.e., hippocampus: Siegford et al., 2003). Frontal cortex of 12 day old animals experiencing combinations of EW and SI shows changes in expression of proteins involved in structure, protection, and translation. Preliminary microarray work in hippocampal tissue also suggests EW and SI piglets show decreased expression in the hippocampal
genes for protein synthesis and cell function. Our second aim was to monitor the consequences of age and glucocorticoid antagonist and agonist injection on A) behavioral development, B) spatial memory, and C) social behavior of young pigs. A) Piglets experiencing EW had higher basal cortisol levels prior to mixing at weaning with piglets from other litters than CW piglets, however only CW animals showed increases in cortisol levels after mixing. CW piglets fought more than EW piglets on morning of the day of mixing, but groups were not different at subsequent times, while average duration of fights was longer for EW than CW piglets at mixing (Leite et al. in prep). B) The Morris water maze (MWM) was adapted to measure spatial learning and memory in young pigs. Unweaned (UW), EW, and CW pigs showed similar learning curves in the MWM (Laughlin et al., in press). Further trials examined whether SI immediately prior to testing affected the performance of weaned pigs in the MWM compared to
unstressed controls. Both CW SI and control pigs showed significant and comparable rates of learning while EW SI pigs showed no evidence of learning across exposures (Laughlin & Zanella, in press). The effects of RU486 administration near weaning on the performance in MWM at 14 and 23 days of age and SI-induced deficits in spatial learning of EW pigs are being examined. Long-term effects of weaning age and RU486 on cognitive functioning are being investigated by testing EW and CW pigs at 100-130 days of age in the holeboard, a land-based spatial learning paradigm. C) We successfully adapted a social recognition (SR) test for use in young pigs (Souza et al., in prep a), however excessive handling and fighting during the familiarization period led us to develop a novel protocol to familiarize piglets to minimize these potential confounds (Souza et al., in prep b). Using the novel SR protocol, we then showed that EW (11 days of age) plus SI prior to testing disrupted social recognition
of familiar piglets (Souza & Zanella, in prep).
Impacts
Stress induced disorganization of glucocorticoid sensitive brain pathways is likely to be the source of neuropathology in animals, including humans. Pathological conditions may be expressed by compromised emotional regulation, behavioral abnormalities, excessive aggression and memory impairment. There is evidence that the stress associated with depriving pigs of maternal nourishment and care at an early age affects physiology and behavior and can be a major challenge to piglet health and well-being. Our studies can enhance our ability to design production systems and treatments that will reduce stresses and resulting aberrant and potentially damaging behaviors. These changes will result in improved gains in production and increased profitability. The occurrence of behavioral abnormalities, particularly in animals kept in suboptimal conditions, has been used as an indicator of poor welfare. The understanding of the changes in the brain associated with the onset of
behavioral problems and consequent disorganization of neural pathways will provide scientific information regarding the validity of such measures as welfare indicators. MSU pig brain cDNA library has already proven to be a unique resource to study gene expression in animals, including humans, subjected to stress (Poletto et al., 2003 and Siegford et al., 2003). Our goal is to develop interventions strategies to allow the reversal of stress induced pathological processes and to diminish the consequent developmental problems reported in several animal species.
Publications
- Laughlin, K. and Zanella, A.J. 2004. Stress-induced deficits in pig spatial learning: influence of weaning age. Physiol. Behav.
- Nobis, W., Ren, X., Suchyta, S.P., Suchyta, T.R., Zanella, A.J. and Coussens, P.M. 2003. Development of a porcine brain cDNA library, EST database and microarray resource. Physiol. Genomics 16:153-159.
- Poletto, R., Siegford, J., Nobis, W. and Zanella, A. 2003. Differential expression of genes in the hippocampus of early-weaned pigs when examined using a cDNA microarray. Int. Symp. Anim. Func. Genom. online:http://www.isafg.msu.edu/posters.html.
- Siegford, J., Nobis, W., Poletto, R., Ren, X., Coussens, P. and Zanella, A.J. 2003. Development and validation of a porcine brain cDNA library and microarray resource using the hippocampus of early-weaned pigs. Soc. Neurosci. Abstr. 758.19.
- Silveira de Souza, A. and Zanella, A.J. 2003. A novel approach to testing social recognition in pigs and the modulating effects of relocation. Proc. 37th Int. Cong. ISAE 54:162.
- Jansen, J., Yuan, Y. and Zanella, A.J. 2003. The influence of weaning age on post-mixing agonistic interactions in growing pigs. Proc. 37th Int. Cong. ISAE 54:133.
- Zanella, A.J., Hodges, H. and Palme, R. 2003. Glucocorticoids and their metabolites in the hippocampus of weaned and unweaned piglets. Proc. 37th. Int. Cong. ISAE 54:116.
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Progress 01/01/02 to 12/31/02
Outputs
Using a modification of the water maze paradigm, and observation of social behavior post-mixing we assessed the spatial learning abilities (Laughlin & Zanella, 2002) and social skills of nursing and newly weaned pigs, and examined how these were affected by stress and weaning age. The water maze is an escape task, in which pigs are placed in a large pool of opaque water and required to locate a submerged platform. The latency of pigs to reach the platform from a pre-determined release point is recorded over 7 exposures to the maze, separated by 10-min intervals. We examined whether the presentation of a stressor (15-min social isolation), immediately prior to testing, affects the performance of 14- and 23-day old pigs in the maze. Forty-eight female pigs were weaned at either d12 (early-weaning, EW, n=24) or d21 (conventional-weaning, CW, n=24). Trial analysis showed a highly significant correlation between length of the swimming path and latency to find the platform
(Pearson's product moment correlation; n=382, r = 0.929, p<0.0001), indicating that a decrease in latency to locate the platform across a trial is a reliable measure of spatial learning. Isolating the pigs prior to testing caused a significant impairment in the performance of EW pigs compared to non-isolated control animals, when tested at d14 (GLM; F1, 70= 26.03, p<0.0001) and d23 (GLM; F1, 70= 26.7, p<0.0001). However, isolation treatment resulted in only a slight (non-significant) impairment in performance of CW pigs, tested two days after weaning (GLM; d14, F1, 70= 0.01, NS; d23, F1, 70= 3.31, p=0.073) compared to control pigs, indicating that the stress of the weaning process itself does not account for the level of performance impairment observed in the earlier-weaned pigs. Our results suggest that the deficits in spatial learning, indicative of some cognitive disruption, may be affected by age at which the developing brain is exposed to increases in stress hormones. We also
investigated the effects of weaning age on agonistic interactions after mixing. 47 piglets from 6 litters were subjected to either an early weaning treatment (weaning at 10 days of age, n=24, EW) or a conventional weaning treatment (weaning at 21 days of age, n=23, CW). At 9 weeks of age, animals, early- and conventional weaned were regrouped based on their weight and all agonistic interactions and subsequent outcomes were monitored for 3 days. Results show no significant effect of weaning age on the number of agonistic interactions after mixing (GLM, F1, 45=1.63; p=0.21). EW pigs were shown to initiate more fights than CW animals (GLM, F1, 45=4.20; p<0.05). Spearman correlations show that, on the first day after mixing, initiating and winning a fight were correlated for CW pigs (r=0.57, p<0.01) while this correlation was not found for EW animals (r=0.27, p=0.20), suggesting that the increase in initiated fights may be caused by an impairment of social skills or a deficit in the
memory or recognition of dominant animals. Oral administration of RU486, a glucocorticoid antagonist, will be used to investigate the role of glucocorticoid hormones on the cognitive deficits experienced by early-weaned pigs.
Impacts
The techniques that we developed to assess some aspects of cognitive abilities, in early and conventionally weaned pigs, have practical applications for the pig industry. Husbandry procedures can now be tested using an index of cognitive impact. The objectivity of scientific welfare assessment exercises, using the index of cognitive impact, will be greatly enhanced. We predict that the North-American Pig Industry will re-evaluate the acceptability of weaning practices in pigs.
Publications
- K. Laughlin, E. Wilson & A.J. Zanella (2003). Modification of the water-maze procedure to examine cognitive processes in nursing and newly weaned pigs. Applied Animal Behaviour Science (in press)
- K. Laughlin & A.J. Zanella (2002) Weaning age impairs spatial memory of pigs at increased but not basal levels of stress. Proceedings of the 2002 Annual Meeting of the American Society for Animal Science, Quebec City, Canada.
- C.C. Coe, R. Dantzer, R, P. Jensen, S.L. Lightman, R.J. Nelson, S, Porges, J.M.H.M Reul, J. Rushen, V. Stefanski & A.J. Zanella (2002) Key elements of coping. In Broom, D.M (editor) Dahlem Workshop on Coping with challenge: welfare in animals including humans.
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Progress 01/01/01 to 12/31/01
Outputs
Two unique protocols were developed at Michigan State University to assess some aspects of cognitive function in very young pigs. Sress hormones may alter brain organization, diminishing the ability of animals to develop optimal coping strategies to respond to their social and physical environment. Dysfunctional neural-pathways, altered by early exposure to stress hormones, can be associated with excessive levels of aggression and behavioral abnormalities. The welfare and productivity of pigs that show behavior abnormalities and aggressive behavior can be severely compromised. We validated novel protocols to assess spatial memory, using the water-maze, and social recognition, monitoring pig behavior following exposures to familiar and unfamiliar animals. Both tests can be performed with minimal training (1 to 2 hours) and yield consistent results which are sensitive to exposure to stressful situations.
Impacts
(N/A)
Publications
- Souza, A.S. & Zanella, A.J. (2001) Investigating indicators of pig social recognition and how these are affected by cortisol and age. In Garner, J.; Mench, J. and Heekin, S. (Editors). Proceedings of the 35th International Congress of the International Society for Applied Ethology. Unversity of California, Davis, August 4-9, 2001
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