Acute stressors experienced by layer breeders do not affect measures of stress and fear in their offspring

Stressors experienced by layer breeders during egg production can lead to changes in the egg hormone content, potentially impacting their offspring, the commercial layers. Genetic differences might also affect the offspring’s susceptibility to maternal experiences. In this study, we tested if maternal stress affects measures of stress and fear in five strains of layer breeders: commercial brown 1 & 2, commercial white 1 & 2 and a pure line White Leghorn. Each strain was equally separated into two groups: “Maternal Stress” (MS), where hens were subjected to a series of 8 consecutive days of acute psychological stressors, and “Control,” which received routine husbandry. Additional eggs from Control were injected either with corticosterone diluted in a vehicle solution (“CORT”) or just “Vehicle.” Stress- and fear-responses of the offspring were measured in a plasma corticosterone test and a combined human approach and novel object test. Both MS and CORT treatments failed to affect the measured endpoints in the offspring, but significant strain differences were found. The offspring of the white strains showed a higher physiological response compared to brown strains, but the White 2 offspring was consistently the least fearful strain in the behaviour tests. Our study found that the acute psychological stressors experienced by layer breeders did not affect the parameters tested in their offspring and that corticosterone does not seem to be the primary mediator of maternal stress in laying hens. This is highly important, as in poultry production, layer breeders are often subjected to short-term stressors. In addition, we successfully dissociated the physiological and behavioural parameters of stress response in laying hens, showing that increased concentrations of plasma corticosterone in response to stress is not directly associated with high levels of fear.


Introduction 39
The organizational structure of commercial layer breeding companies follows a pyramidal 40 model; at the top of the pyramid is a relatively small population of pure line elite stock, followed 41 by the grandparent stock, the parent stock, and the commercial layers at the base (Fig 1). In their 42 lifetimes, layer breeders from the parent stock can produce approximately 115 commercial laying 43 hens (1,2); therefore, a comparatively small number of breeders are needed to provide the total 44 number of commercial layers in the egg system. Parent stocks are typically raised in mixed-sex 45 groups of approximately 100 females per 8 males, either in floor systems housing thousands of 46 birds or in smaller groups in colony breeder cages (2). Similar to the commercial layers, the parent 47 stock may be exposed to stressful procedures and events, such as beak trimming (regulations vary 48 across regions), human handling and social conflicts with other birds.

54
Once believed that only genetic selection could shape the phenotype of the progeny, it is 55 now known that the over-activation of the hypothalamic-pituitary-adrenal (HPA) axis (i.e., stress 56 response) experienced by the female during egg production can have long-lasting effects in the 57 offspring (3). It has been suggested that these "maternal effects" may shape the phenotype of 58 offspring, which could be adaptive if mother and offspring share similar environments; or 59 detrimental if their environments differ ("environmental matching hypothesis" (3)). Likewise, 60 given that the global population of commercial layers is 7.6 billion birds (4), stressors experienced 7 127 Parent stock: Experimental design 128 Each strain of the parent stock had two pens assigned to the control treatment and two pens 129 assigned to the MS treatment. The control groups were strictly submitted to regular husbandry, 130 while MS hens were subjected to 8 consecutive daily sessions of acute psychological procedures 131 at 3 different ages: 32, 52 and 72 weeks. At the end of the 8 th day of stressors, fertile eggs from 132 both treatments were collected, and additional eggs from the control treatment were either injected 133 with corticosterone diluted in vehicle (10ng/mL egg content) ("CORT") of just vehicle 134 ("Vehicle"). All eggs were incubated, and the offspring flocks from each maternal age were treated 135 as replicates over time (Fig 2). This experimental design allowed us to work with a larger sample 232 test, we measured the duration of time that the birds spent in zones located a distance away from 233 either a person and a novel object (umbrella) (47). The longer the duration of time spent away 234 from the stressor, the more fearful the bird was considered to be.

235
Birds at 16 weeks of age (N = 160; Table 1) were tested in same-sex pairs. Testing was

253
The birds were marked with livestock paint two days before testing to allow for individual 254 identification on video analyses. On the test day, each pair of animals was quietly removed from 255 their home-pen and moved into the start box attached to the arena. After 30 seconds, a pull-up door 256 was opened, giving the birds access to the arena. At this moment, the "observer" (a person who 257 had not been seen by the birds on that day), was sitting on the chair, facing the arena, but avoiding     The baseline corticosterone concentration was similar for all strains; however, the White    355 during the novel object test; however, strain differences were found (Table 5)

373
Descriptive statistics of the birds' location at the moment when the umbrella was opened are 374 presented in Table 6, with 61.9% in Zone 1. Behaviour response to sudden movement (umbrella 375 opening) varied among strains; commercial-and pure line-white strains flew away from the 376 umbrella more frequently than both brown strains (Fig 9).
377 Table 6. Location of the birds at the moment when the umbrella was opened.

390
Our results show that the acute stressors experienced by layer breeders during egg 391 production did not shape the behaviour or the stress response of commercial layers. Previous work 392 in poultry that subjected females to long-term, chronic stressors, report carry-over effects on 393 behaviour and physiology of offspring (13,31,49), however ours is the first test of acute stress.
394 Moreover, the mothers of the birds tested in the previous studies were housed in cages, a 395 contributing factor to the occurrence of maternal effects (50). Our work is highly important, as in 396 poultry production, breeders are often subjected to acute stressors (such as handling, vaccination 397 and loud noises) and these data suggest that these experiences do not seem to affect the offspring.

398
The lack of results in the CORT treatment suggests a weak or inexistent biological link 399 between maternal corticosterone and the offspring's phenotype, as previously reported in a 400 passerine species (18). It is possible that the corticosterone concentration used in this study was 401 not sufficient to shape the phenotype of the offspring. However, it is most likely that puncturing 402 and injecting the fertile egg moments prior to incubation, as well as applying a silicon sealant onto 403 the egg surface, are highly invasive and unnatural procedures, which might have exposed the 404 progeny to an additional stressor and unintentionally selected a subset of birds that were more 405 resilient to the adverse effects of the injection; thus, limiting the generalization of these results.

406
Although no treatment effects were observed, our study revealed significant differences in 407 both behavioural and physiological measures among strains. Results show that the white strains 408 produced more corticosterone in response to physical restraint, approached the person and the 409 novel object more frequently and showed greater flight response (avoidance behaviour) when 410 startled by the umbrella than the brown strains.

411
Behavioural and physiological responses to stress are commonly interpreted along a 412 proactive-reactive continuum (51). Proactive animals tend to produce lower physiological 413 response to stress (e.g., corticosterone) and have a fast response to a novel stimulus (e.g. fast 414 approach, more aggressive); whereas reactive animals produce higher physiological response to a 415 stressor and display a slow and shy behaviour response (e.g., slower approach, more passive 416 towards a stimulus) (52,53). Our study showed that all white strains produced more corticosterone 417 in response to physical restraint, suggesting a reactive physiological profile. However, the White 418 2 strain approached both novel stimuli more rapidly than the other strains and displayed a faster 419 avoidance response to the startling stimulus, thus characterizing a proactive behavioural profile.

420
Mammals and passerines typically display the same profile for both behavioural and 421 physiological responses to stress, thus characterizing the concept of "coping style" (51). In 422 agreement with previous studies (36,53-56), our results show that this concept does not apply to 423 laying hens. Although the majority of birds from the White 2 strain flew away from the umbrella 424 and showed a high concentration of corticosterone in response to physical restraint, they 425 consistently spent more time close the human and the novel object, standing out as the least fearful 426 strain in the behaviour tests. In a previous publication, we also reported that these same white 427 strains produced fewer distress calls in response to social isolation and spent less time in tonic 428 immobility, a measure of fearfulness in chickens, compared to the brown strains (11). Our results, 429 thus, suggest a dissociation between stress reactivity and fearfulness in laying hens.

430
In many species, physiological traits are associated with pigmentation (57,58) and melanism 431 has been shown to signal the ability to cope with elevated stress hormones in barn owls (59).
432 Effects on aggressive behaviour have also been found (60) This research successfully shows that acute stressors such as brief transportation, physical 487 restraint or loud auditory noise experienced by layer breeders during egg production, do not shape 488 the behaviour or the stress response of their offspring, the commercial layers. This finding has 489 significant importance in the agricultural field, as layers breeders are constantly subjected to 490 different types of acute stressors in their lifetimes.

491
Our results also suggest the disconnection between stress reactivity and fearfulness in 497 when determining the overall adaptability of a strain to a specific housing system; therefore, having 498 significant welfare and economic impacts in the egg production system.