Maternal behaviour and changes in levels of neuropeptides in the brain of the broody domestic hen

Studies on broodiness and maternal behaviour in birds are directly relevant to welfare. In third world countries where traditional poultry is still prevalent and an important part of the economy, farmers keep predominantly endogenous breeds of chickens. These birds are often naturally more resistant to disease than commercial breeds, more capable to escape predators and may even provide better quality meat and eggs but they have a tendency to stop laying and go broody. While broody hens are used by farmers to hatch and rear chicks, unwanted broodiness still has a detrimental effect on egg production. In addition to that, there is evidence that quail reared by their mothers are more sociable, less fearful and have better growth rates. This confirms the importance of maternal care for the overall welfare of the progeny in birds. Understanding how this behaviour is controlled may help us manipulate it or replicate it for the purpose of improving welfare for birds in the farming industry.
Maternal behaviour in chickens encompasses two separate behaviours - incubating and rearing - each of which is triggered and governed by different hormones and environmental factors. Incubating behaviour is characterised by the cessation of egg-laying, development of the 'brood patch', sitting on the eggs almost continuously for the period necessary for chicks to hatch, diminished movement, food and water intake and social interaction. Rearing behaviour occurs after the eggs hatch and comprises behaviours related to the care of chicks including warming, protecting and interacting with them.
Mesotocin (MT) and vasotocin (VT) are nonapeptides found in birds and other non-mammalian vertebrates. They are very close in structure and localisation to the mammalian neuropeptides oxytocin (OT) and vasopressin (VP), respectively, and perform similar functions. Nonapeptides are expressed in the brain by magnocellular and parvocellular neurones in several brain areas including the paraventricular nucleus of the hypothalamus (PVN), medial preoptic area (POM) and the bed nucleus of the stria terminalis (BnST). The PVN, POM and BnST have all been implicated in a number of social behaviours, as well as the stress response in both mammals and birds. MT and VT, as well as OT and VP, act both centrally within the brain and in other parts of the body. They play an important role in regulating functions such as egg-laying, water balance, learning and memory, the stress response and social behaviours across taxa. Vasopressin and oxytocin, and more recently mesotocin, have been found to regulate maternal behaviour in mammals and birds respectively. MT immunoreactivity increases throughout incubation and is significantly higher in the PVN and POM of rearing compared to laying Thai hens. The expression of c-fos (an early gene routinely used as an indicator of neuronal activity) in several brain areas known to be involved in maternal behaviour including the POM, PVN and medial BnST (BnSTm) is significantly higher in turkey hens rearing chicks than in non-rearing hens and MT antagonists completely abolish rearing behaviour in the turkey.

The possible role of VT in maternal behaviour in birds has not been studied but VT is a known component of the stress axis in the avian brain. Some bird species display an attenuated stress response during certain periods of their reproductive cycle and this lower reactivity to stress is likely a mechanism to ensure that parental birds will prioritise reproduction and care for the offspring over their individual safety.
To determine the roles of MT and VT during incubation and rearing in the chicken, we examined the changes in MT and VT mRNA levels in the brain of the domestic hen throughout the reproductive cycle from laying through to the first day after hatch.
Adult hens from a cross between pure Silkie and White Leghorn lines were divided into four groups (n=8) - Layers (L, laying status confirmed post mortem), Onset of incubation (O, taken as the third day of continuously sitting on eggs), Incubating for two weeks (I) and Rearing chicks for 24 hours (R). Both groups of incubating hens (O and I) and the rearing hens were housed in a quiet experimental room equipped with video cameras in floor pens split in two with a low barrier which the hens could jump over. Each side of the pen was equipped with a nest box filled with ten fertile eggs in order to encourage the birds to incubate. Food and water were provided ad libitum.
On the morning of day 3 of incubation, day 14 of incubation or on the morning after the chicks had hatched, hens were euthanised, brains were collected immediately after death, frozen on dry ice and stored at -70˚ C. Coronal brain sections were cut on a cryostat at 15µm and stored at -70˚C. Radioactive in situ hybridisation was performed with oligonucleotide probes designed for the purpose.
In the PVN of hens, a significant increase of MT mRNA was observed in rearing hens compared to layers. This result is in agreement with the previous data from chickens and turkeys which suggest that the MT system in the PVN plays a role in regulating rearing behaviour in precocial birds.
In the BnSTl MT mRNA levels were the same in laying and rearing birds but decreased significantly in the two incubating groups (onset of incubation and incubating for two weeks). This significant decrease in MT mRNA expression during incubation corresponds to the time hens were spending alone in the nest with limited social interaction. We observed that MT mRNA expression had recovered on the first day after hatch when tactile and auditory social cues would have been provided by the chicks. These results can be explained by the BnSTl regulating social interaction both between adults and between adults and chicks.
There was no difference in VT mRNA levels in the PVN between groups but in the BnSTl there was a significant decrease in both incubating groups and the rearing group compared to layers. The VT system is involved in the stress response in birds and higher c-fos expression has been recorded in the BnSTl of stressed chickens. In addition, in some bird species, the stress response is downregulated when efforts are directed towards preserving the progeny. Therefore we hypothesise that the decrease of VT mRNA during both incubation and rearing compared to egg-laying may be connected to an attenuation of the stress response in favour of focussing efforts towards maternal care.
In conclusion, the results from this study confirm the link between MT and rearing behaviour and demonstrate changes in MT and VT expressing neurones in the BnSTl suggesting a possible role of this nucleus in regulating stress and sociality during incubation.
Future work may include experiments with MT and VT antagonists, central neuropeptide injections, measuring neuronal activation in brain areas of interest during interactions with chicks in the chicken and examining how maternal behaviour in tandem with stress or aggression affects MT and VT in the brain.