|Title||Gut microbiota and feather pecking : Integrating microbiota, behaviour, stress, serotonin and the immune system|
|Author(s)||Eijk, Jerine Alexandra Johanna van der|
|Source||Wageningen University. Promotor(en): M. Naguib; B. Kemp, co-promotor(en): T.B. Rodenburg; A. Lammers. - Wageningen : Wageningen University - ISBN 9789463951562 - 242|
|Publication type||Dissertation, internally prepared|
Early-life factors can have a profound impact on an animal’s behavioural development. An important moment early in life is the rapid microbial colonization of the gut, leading to the establishment of the gut microbiota. From rodent studies it is clear that the gut microbiota influences host behaviour and physiology, such as anxiety, stress, and the serotonergic and immune systems. First indications show that microbiota affects similar behavioural and physiological characteristics in poultry. Through these effects microbiota could alter an animal’s ability to cope with environmental and social challenges, such as those encountered in animal production systems, and could thereby affect the development of damaging behaviours in production animals.
Fearfulness, stress, and the serotonergic and immune systems have been related to severe feather pecking (FP), a damaging behaviour in chickens which involves pecking and pulling out feathers of conspecifics, negatively affecting animal welfare and productivity. Furthermore, high FP (HFP) and low FP (LFP) selection lines were shown to differ in gut microbial metabolites and microbiota composition determined from caecal droppings. These findings suggest a link between the gut microbiota and FP. Yet, it is unknown whether gut microbiota influences the development of FP. Therefore, the aim of this thesis was to identify effects of gut microbiota on the development of FP. First, I identified behavioural and physiological characteristics in FP genotypes (i.e. HFP and LFP lines) and FP phenotypes (i.e. feather pecker, victim, feather pecker-victim and neutral) that were related to FP and shown to be influenced by microbiota. Second, I identified whether microbiota influences FP, and behavioural and physiological characteristics related to FP.
Feather pecking genotypes
FP genotypes differed in behavioural responses, where HFP birds had more active behavioural responses compared to LFP birds (chapter 2, 3 and 6), especially at young age. The active behavioural responses suggest lower fearfulness, higher social and exploration motivation, or higher activity in HFP birds compared to LFP birds. For the stress response, HFP birds struggled later and less, but vocalized sooner and more compared to LFP birds during restraint (chapter 3 and 6). However, FP genotypes did not differ in corticosterone (CORT, the major stress hormone) level after restraint (chapter 3 and 6), suggesting differences in behavioural findings might not be related to stress. With regard to the serotonergic system, whole blood serotonin (5-Hydroxytryptamine or 5-HT) level was measured as indicator for central 5-HT and HFP birds had lower whole blood 5-HT levels compared to LFP birds (chapter 3 and 6). For the immune system, nitric oxide production by monocytes was measured as indicator for the innate immune system, specific antibody level was measured as part of the adaptive immune system, and natural (auto)antibody level was measured, as natural antibodies play an essential role in both innate and adaptive immunity. HFP birds had lower IgM and higher IgG natural (auto)antibody levels, higher nitric oxide production by monocytes, and a tendency for higher IgM and IgG specific antibody levels compared to LFP birds, but did not differ in relative abundances of immune cell subsets (chapter 3, 4 and 6). Moreover, FP genotypes had distinct luminal microbiota composition, where HFP birds had a higher relative abundance of genera of the order Clostridiales, but lower relative abundance of Lactobacillus compared to LFP birds (chapter 5). Yet, FP genotypes did not differ in mucosa-associated microbiota composition. In summary, these findings indicate that divergent selection on FP not only affects FP but also (in)directly affects behavioural responses, peripheral 5-HT level, different arms of the immune system and microbiota composition, but did not affect CORT level.
Feather pecking phenotypes
FP phenotypes differed in behavioural responses, where feather peckers tended to have more active behavioural responses compared to victims and neutrals at young age (chapter 2), which suggests lower fearfulness, higher exploration motivation or activity in feather peckers. Furthermore, victims had more active responses compared to neutrals at young age (chapter 2), which suggests lower fearfulness or higher activity in victims. For the stress response, feather peckers tended to have less active behavioural responses compared to neutrals, while victims had more active behavioural responses compared to other phenotypes during restraint (chapter 3). However, FP phenotypes did not differ in CORT level after restraint (chapter 3), suggesting differences in behavioural findings might not be related to stress. With regard to the serotonergic system, feather peckers had higher whole blood 5-HT levels compared to neutrals at adult age (chapter 3). However, FP phenotypes did not differ in natural antibody level (chapter 3) or gut microbiota composition (chapter 5). In summary, these findings indicate that performing and receiving FP is related to more active behavioural responses and that performing FP is further related to high peripheral 5-HT level.
Feather pecking genotypes vs. phenotypes
When comparing findings from FP genotypes to those from FP phenotypes, there is a similar relation between high FP and behavioural responses. HFP birds had more active responses compared to LFP birds (chapter 2, 3 and 6) and similarly feather peckers tended to have more active responses compared to victims and neutrals (chapter 2), especially at young age. Furthermore, victims had more active responses compared to other phenotypes at adult age (chapter 3). Thus, activity level might be used as potential indicator for FP at group level or even as indicator for individuals that perform or receive FP. Since feather peckers seem to have more active responses at young age (chapter 2), it would be interesting to identify whether activity level at young age could be used to predict which individuals will become feather peckers.
There is an opposite relation between high FP and whole blood 5-HT level, where HFP birds had lower whole blood 5-HT levels compared to LFP birds (chapter 3 and 6), while feather peckers had higher whole blood 5-HT levels compared to neutrals (chapter 3). The actual performance of FP might increase peripheral 5-HT level in feather peckers, possibly due to feather eating. Feather peckers often ingest feathers, which may increase peripheral 5-HT level by providing structural components, as the gut releases 5-HT in reaction to these structural components. However, this relation between feather eating and increased peripheral 5-HT level needs further investigation.
Similar to findings for FP genotypes, FP phenotypes did not differ in CORT level after restraint, indicating that the stress response might not be related to FP in FP genotypes and phenotypes. Furthermore, although differences between FP genotypes were found for the immune system and gut microbiota composition, no such differences were identified for FP phenotypes. This might indicate that differences in immune characteristics and gut microbiota composition are more related to genotype than to actual FP behaviour. Yet, cause and consequence cannot be disentangled from each other based on these findings. Therefore, microbiota transplantation was used to identify gut microbiota effects on the development of FP.
Microbiota and the development of feather pecking
Since FP genotypes differed in gut microbiota composition, but FP phenotypes did not, I focussed on FP genotypes for the second objective. The difference in microbiota composition was used to create a HFP and LFP microbiota pool. I identified effects of early-life microbiota transplantation on FP and on the same behavioural and physiological characteristics that were identified in chapter 2, 3 and 5. Newly hatched HFP and LFP chicks received a control treatment, HFP or LFP microbiota daily during the first two weeks post hatch.
Although limited effects of early-life microbiota transplantation on microbiota composition were found, microbiota transplantation did affect behavioural responses, natural antibody level and whole blood 5-HT level. Thus, microbiota transplantation may have influenced brain, immune and serotonergic system functioning, which (in)directly resulted in differences in behavioural responses, natural antibody level and whole blood 5-HT level.
With regard to behavioural responses, birds receiving microbiota from their own line (i.e. homologous transplantation) had more active behavioural responses compared to birds receiving microbiota from the other line or control treatment. These active behavioural responses suggest low fearfulness, high exploration and social motivation or activity in birds receiving homologous transplantation. For the stress response, LFP birds receiving homologous transplantation had more active stress responses compared to LFP birds that received HFP microbiota or control treatment. However, microbiota transplantation did not influence CORT level after restraint, suggesting these behavioural findings might not be related to stress.
With regard to the serotonergic system, LFP birds receiving HFP microbiota tended to have lower whole blood 5-HT level compared to LFP birds receiving control treatment. Yet, microbiota transplantation effects on whole blood 5-HT level do not seem to be explained by the HFP pools’ microbiota composition. Especially since microbiota composition did not differ between treatments within the LFP line.
For the immune system, birds receiving LFP microbiota had higher IgM natural antibody level compared to birds receiving control treatment, but microbiota transplantation did not affect IgG natural antibody level. Thus, being exposed to an adult microbiota composition might be sufficient to increase IgM natural antibody level. Further research is needed to identify whether microbiota transplantation could influence other immune characteristics in poultry, such as innate and adaptive immune characteristics.
For the first time, effects of early-life microbiota transplantation on FP were investigated. However, early-life microbiota transplantation had limited effects on FP at young age (till 15 weeks of age) (chapter 6), which might be explained by FP usually increasing from the egg laying period onwards (around 20 weeks of age). Thus, further research is needed to identify effects of microbiota transplantation on FP at adult age.
Effects of microbiota transplantation depend on genotype
During treatment, microbiota transplantation influenced behavioural responses in the HFP line, while after treatment it influenced behavioural responses in the LFP line. A potential explanation for this could be that the HFP line has a more responsive immune system (chapter 3, 4 and 6), which responds more strongly to the environment or in this case to microbiota transplantation, with the synthesis and release of pro-inflammatory cytokines. These cytokines in turn act on the brain and alter neurotransmission, thereby potentially influencing behavioural responses. After treatment, microbiota transplantation influenced behavioural responses in the LFP line. These effects do not seem to be explained by the difference in whole blood 5-HT level. Still, it is interesting that LFP birds receiving HFP microbiota had lower whole blood 5-HT levels, as HFP birds had lower whole blood 5-HT levels compared to LFP birds (chapter 3 and 6). This might increase the risk for developing FP in LFP birds receiving HFP microbiota, as high FP is usually related to low whole blood 5-HT level. However, it remains unknown through which pathway microbiota transplantation influences behavioural responses in the LFP line.
It is interesting to note that homologous transplantation resulted in birds having more active responses, suggesting reduced fearfulness. Therefore, homologous transplantation could be a potential approach to reduce fearfulness in chickens. High FP is usually related to high fearfulness, indicating that receiving homologous transplantation might reduce FP. Homologous transplantation might result in reduced fearfulness because of a match between transplanted microbiota composition and host genotype as opposed to a mismatch or control treatment. Homologous transplantation could be seen as a type of vertical transmission, where microbiota is transferred from mother hens to chicks. Vertical transmission might play an important role in initiating a host-specific gut microbiota, which might improve host immune system and brain development. Thus, homologous transplantation might have improved immune system and brain development, thereby altering behavioural responses. It would be interesting to identify whether homologous transplantation can be used to reduce fearfulness in poultry and FP in laying hens.
Role for the immune system in feather pecking?
There is increasing evidence for a role of the immune system in FP. For FP genotypes, HFP birds had higher specific antibody levels, IgG natural (auto)antibody levels and nitric oxide production by monocytes (chapter 3, 4 and 6), suggesting that high FP is related to a more responsive innate and adaptive immune system. Although it should be noted that FP phenotypes did not differ in natural antibody level (chapter 3). The immune system could be a potential route through which microbiota transplantation affects behavioural responses, especially in HFP birds as they seem to have a more responsive immune system (chapter 3, 4 and 6) and microbiota transplantation had immediate but no long-term effects on behavioural responses in HFP birds (chapter 6). Yet, the exact mechanisms through which the immune system affects the development of FP remain unknown, providing an interesting avenue for further research.
Feather pecking selection lines as model system
The HFP and LFP lines were used throughout this thesis as a model system to identify effects of gut microbiota on FP. As these lines are specifically selected on high and low FP, findings with regard to FP should be interpreted with caution when transferring them to other experimental or commercial lines. Overall, high FP was related to low fearfulness, low whole blood 5-HT level and a more responsive immune system in the FP selection lines (chapter 2-6). Previous studies in other experimental and commercial lines show that high FP is related to high fearfulness, low whole blood 5-HT level and a more responsive immune system. Findings with regard to CORT level after restraint are less consistent, with high FP being related to low CORT level after restraint or not. Thus, the FP selection lines seem to show similar relations between high FP, whole blood 5-HT level and immune responsiveness as commercial lines, but an opposite relation between high FP and fearfulness as other experimental and commercial lines. Furthermore, there is inconsistency with regard to the relation between high FP and the stress response. Therefore, findings from the FP selection lines should be used with caution when developing control or preventive methods that are to be applied in production systems. Still, this thesis provides new interesting insights into the relation between FP, behavioural and physiological characteristics related to FP and the gut microbiota.
Divergent selection on FP affects fearfulness, activity, peripheral serotonin, immune characteristics and gut microbiota composition, but not the physiological stress response (i.e. corticosterone). FP phenotypes differ in fearfulness, activity and peripheral serotonin, but not in the physiological stress response, immune characteristics or gut microbiota composition. Yet, relations between high FP and behavioural or physiological characteristics are not always similar for FP genotypes and phenotypes, indicating the importance of taking FP genotype and phenotype into account when studying FP.
Gut microbiota could influence the development of FP, as early-life microbiota transplantation affects fearfulness, activity, peripheral serotonin and immune characteristics, with effects being either immediate or long-term. However, effects depend on age, donor’s and recipient’s genotype, indicating the importance of taking donor’s and recipient’s genotype into account when studying microbiota transplantation effects on behaviour. Overall, this thesis provides new interesting insights into the relationship between gut microbiota, host behaviour and physiology in poultry, which could further be of interest for other species.