Productive performance, thermal and blood parameters of Japanese laying quails at different cage stocking densities

This study aimed to evaluate the effects of different stocking densities on the performance, eggshell quality, surface body temperature and hematological parameters of Japanese laying quails based on physiological indicators of animal welfare. Two hundred and thirty seven-week-old Japanese quails were used in this experiment. The quails were completely randomized to four stocking densities: 112.2 (T1), 102 (T2), 93.5 (T3), and 86.31 (T4) cm2/quail and five replicates each. Hematological parameters were analyzed as a 4x4 factorial design (stocking density X time) over four periods of blood sampling (25, 50, 75, and 100 days). To obtain the body surface temperature (Ts, °C) three thermograms (head, core and shin) were captured from each repetition per plot (2 repetitions per experimental plot) every 25 days (25, 50, 75, and 100 days). Feed intake, feed conversion per egg mass, feed conversion per dozen eggs, egg mass, egg production rate, and eggshell quality-related variables were not affected by treatments. However, egg weight (p = 0.023) and core temperature (p = 0.003) were influenced by different cage stocking densities. The heterophil/lymphocyte ratio increased (p = 0.01) with increasing time and stocking density. The mean corpuscular volume (p = 0.0001) as well as the total leukocyte count (p = 0.001) increased until the third blood sampling period (75 days) and then decreased in the last period. Different stocking densities do not interfere with the performance and eggshell quality of Japanese quails. However, the hematological parameters and head temperature are affected by different cage stocking densities and time.


Introduction
The short reproductive cycle of Japanese quails is one reason for the growing domestic production. According to Albino and Barreto (2003), quails start laying eggs around six weeks of age and continue laying until approximately 60 weeks of age.
Farmers resort to increasing stocking density in order to intensify production and profitability per area and reduce costs with equipment and cages. This common practice can lead to reduced quail performance caused by competition for space and food (Leandro et al., 2005).
Stocking density is crucial to successful quail production, i.e., it will allow determining the animal yield per area. In general, high-density stocking associated with climatic challenges, such as high temperatures, reduces livestock performance (Guimarães et al., 2014). Some authors with Albino et al. (2014) state that birds' ability to direct energy towards maintenance, weight gain, and egg production is directly associated with the environmental conditions to which they are subjected. Therefore, stressful situations, whether environmental, pathogenic, or spatial, may affect this ability.
Under heat stress condition, neuroendocrine changes occur in an attempt to resume homeothermia. The hypothalamus-pituitary-adrenal axis initiates the response through the corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and glucocorticoid hormones. CRH is generated in the hypothalamus and depicts the mandatory stimulus to sensitize the pituitary gland to secrete ACTH, which in turn stimulates the adrenal gland to release glucocorticoids (Dhabhar, 2009). The activation of the hypothalamus-pituitary-adrenal axis under conditions of continuous stress, therefore, increases in circulating levels of glucocorticoids, resulting in greater protein catabolism, hyperglycemia, depression, immunosuppression and increased susceptibility to infections (Matteri et al., 2000;Dhabhar, 2009;Rauw, 2012).
However, the neuroendocrine changes that occur in birds under heat stress, provide changes in the immune system that are not yet well understood, because in some situations instead of immunosuppression, immunostimulation can happen (Silva et al., 2009).
Physiological changes inversely associated with well-being and increased levels of corticosterone, blood glucose, and heterophil/lymphocyte ratio (Kodaira et al., 2015) may alter feed intake, egg production rate (Vercese et al., 2012), egg weight (Pereira et al., 2008) and weaken the immune system (On Aşila & Aksoy, 2005). On the other hand, other authors have demonstrated that stocking density does not affect the performance of Japanese quails (Lopes et al., 2006).
Physiological and behavioral indicators of stress are useful as they can be quantified and broadly fit with preconceived views of well-being (Scanes, 2016). Castilho et al. (2015) analyzed physiological and productive parameters (egg production) of laying hens under different stocking densities and reported no effect of stocking density on the studied parameters because birds have adapted themselves to that condition.
Hematology allows monitoring of general health status in livestock and evaluating the animal's ability to transport oxygen and defend against infectious agents under stress (Voigt, 2003;Schmidt et al., 2007). Blood contains specific defense cells, antibodies, and other immune system components that act as the body's defense mechanism against infections (Junqueira & Carneiro, 2004).
It is well known that stress evokes significant changes in the heterophil/lymphocyte ratio (H/L) in poultry (Davis et al., 2008). On the one hand, birds housed with reduced space show increases in circulating hemoglobin concentration (Bedánová et al., 2007). On the other hand, Fahey and Cheng (2008) reported no changes in hematological parameters and subpopulations of T lymphocytes in layers submitted to simultaneous changes in group size and cage stocking density.
This study aimed to evaluate the effects of different stocking densities on the performance, eggshell quality, surface temperature of the quail regions (head, core and shin) and hematological parameters of Japanese laying quails based on physiological indicators of animal welfare.

Methodology
This study was assessed and approved by the Ethics Committee on Animal Experimentation of the Federal University

Animals and management
Two hundred and thirty seven-week-old Japanese quails with an initial mean body weight of 166 ± 8.3 g were randomly divided into four groups with 10, 11, 12, and 13 birds per experimental unit. Each group was subdivided into five replicates and submitted to the following stocking densities: 112.2 (T1), 102 (T2), 93.5 (T3), and 86.31 (T4) cm²/quail. The quails were housed in cages with dimensions of 100 cm x 34 cm x 16 cm divided into three compartments of 33 x 34 cm with an area per compartment of 1.122 cm². Each cage was equipped with a feeder and trough-type drinker located at the cage's back and front, respectively.
During the experimental period (March/2019 to August/2019), the quails were exposed to ambient temperature conditions (subtropical) of Northern Tocantins (Table 1). Quails were exposed to seventeen hours of light per day. Supplemental lighting was used as needed, after nightfall 60W incandescent lamps were used, for maximum egg production. the lights in the shed were on until 10 pm (posture light program). Locally purchased diet for Japanese laying quails was provided (

Variables studied
Eggs of each cage were counted and collected daily in the morning to estimate the egg production rate. Feed intake, egg production rate, egg weight, egg mass, and feed conversion per egg mass and dozen eggs were calculated at the end of every 25 experimental days. The eggs were individually weighed using a digital scale with 0.001 g accuracy, and then the average egg weight was calculated. Five eggs per experimental unit within the average weight range were selected for analysis of the eggshell quality (eggshell weight and thickness, and specific gravity).
Specific gravity was determined according to the methodology of Araújo and Albino (2011). The eggshells were washed under running water, oven-dried at 105ºC for 24 hours, and weighed on a digital scale with 0.001 g accuracy to obtain the average eggshell weight. Eggshell thickness was determined according to Nordstrom and Ousterhout (1982).
To obtain the surface temperature of the quail regions (head, core and shin) surface temperature (Ts, °C) of quails, an infrared thermographic camera (Flir Systems, model Flir E60, resolution 320x240 (76.800 pixels), was used, accuracy +/-2% or 2 °C). Three thermograms (head, core and shin) were captured from each repetition per plot (2 repetitions per experimental plot) every 25 days (25, 50, 75, and 100 days), 0.4m away from the animals, as recommended by the manufacturer. The generated thermographic images had a resolution of 320x240 pixels, where each pixel represents a temperature point. The camera has an internal automatic temperature calibrator. An emissivity of 0.98 was considered, which is indicated by the manufacturer for biological tissues. Subsequently, these thermographic images were analyzed by the software Flir Tools © 1.2, to obtain the average surface temperature of each distinct body region: head, core and shin (figure 1).
Blood samples were taken as a function of time. Every 25 days (25, 50, 75, and 100 days), two birds from each replicate were randomly selected, totaling 40 birds, and blood samples (1-1.5 mL) were drawn using a needle and syringe by ulnar venipuncture. Blood samples were taken within 2 minutes after the initial disturbance (removal from the cage), collected into heparin-coated tubes, and stored in an icebox. Then, the samples were transferred to the laboratory for determination of heterophil/lymphocyte ratio, erythrocyte count (RBC), total leukocyte count (TLC) (Figure 2), hemoglobin (HEM), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Hematocrit level was obtained using microcapillary tubes filled with blood samples, which were sealed and centrifuged at 12,000 rpm for 5 minutes. Microhematocrit concentrations were expressed as a percentage, obtained by specific reading for the hematocrit values.
For estimation of the total leukocyte count ( Figure 2), a drop of blood was used to prepare blood smears. After drying, blood smears were fixed in methanol for 10 minutes and stained with fast panoptic stain. The total number of heterophils was divided by the total number of lymphocytes to estimate the heterophil/lymphocyte ratio (Campbell, 2004;Thrall et al., 2006).
Slides were read by a single person, and subsequently, the images were captured.
Source: Own research.

Statistical analysis
Errors were analyzed by the Kolmogorov-Smirnov's normality test (α=0.05). The homogeneity of variances was evaluated by Levene's test (α=0.05), and all variables showed a normal distribution of errors and homoscedasticity (SAS, 9.0, proc GLM). Then, an analysis of variance (SAS 9.0 Proc GLM; α = 0.05) was performed (performance, external egg quality and thermographic variables; α=0.05). Hematological-related and surface temperature of the quail regions (head, core and shin) variables were analyzed as a 4x4 factorial design with four stocking densities and four blood sampling periods. The comparison of means between treatments and treatments vs. blood sampling periods was performed using Tukey's test (α=0.05).

Quail performance
There was no significant effect of stocking density ( During the whole experimental period (Table 1), the quails were exposed to temperature conditions outside the thermal comfort zone (22 -24ºC), which was determined by Castro et al. (2017) in a study with Japanese laying quails.
In the present study, eggs of quails housed at 86.31 cm²/animal were 310 mg lighter than eggs of quails housed at 93.5 cm²/animal.

Temperatures of the head, body core, and shank
There was a significant interaction between time and stocking density on head temperature (p=0.02). However, there was no interaction between time and stocking density on the core (p=0.198) and shank temperatures (p=0.658), although time densities alone have significantly affected core (p=0.0001) and shank temperatures (p=0.001) ( Table 4).
However, there was no interaction between time and stocking density on heterophil/lymphocyte ratio (p=0.361), total leukocyte count (p=0.516) and mean corpuscular volume (p=0.184), although time significantly affected these variables (Table   5).
In the last sampling period (100 days of the experiment), the results of hematocrit concentration for quails housed at 112.2 and 102 cm²/quail were within the reference range, which corresponds to 35 to 55% of the red cell mass. It may be related to animals' adaptation to cage size and the greater temperature range recorded in the fourth experimental period (Table   1). Hematocrit concentration increased sharply at 75 days of the experiment (96.90%), although it did not differ between treatments. However, there was a significant difference in hematocrit levels between sampling times.
Regardless of treatment, the mean hemoglobin concentration of quails at 25 days of sampling reached 19.82 g/dL, then decreased at the second period (11.57 g/dL), increased at 75 days of the experiment (28.90 g/dL), and decreased again at 100 days (11.65 g/dL). Mean hemoglobin concentrations, regardless of the sampling period, were 17.45, 18.09, 16.9, and 19.5 g/dL for quails housed at stocking densities of 112.2, 102, 93.5, and 86.31 cm²/quail, respectively. The effect of sampling time on hemoglobin concentrations for each experimental treatment (112.2, 102, 93.5, and 86.31 cm²/quail) was similar to that of hematocrit concentration (Table 6).
There was a significant interaction between sampling time and stocking density on mean corpuscular hemoglobin (p=0.002).
There was a significant interaction between sampling time and stocking density on mean corpuscular hemoglobin concentration (p=0.02).
A significant difference was observed (p=0.001) for heterophil/lymphocyte ratio (H/L), as shown in Table 5. Dantzer and Kelly (1989) suggested that stress suppresses gonadotropin-releasing hormone pulse generator activity.

Discussion
In turn, it compromises the reproductive functions of the axis due to impaired secretion of follicle-stimulating and luteinizing hormones in laying birds, which may compromise egg size.
In the present study, the quails were able to maintain physiological homeostasis to the point that the stress caused by the reduced space due to high density (86.31 cm²/quail) was not enough to elicit competition between birds, which could somehow affect the evaluated parameters. Although a significant difference in egg weight was observed, this difference between the heaviest and lightest egg weight was only 0.3 g on average.
In animals, corticosterone levels are influenced by stress (Quinteiro Filho et al., 2010;Shini et al., 2010;Calefi et al., 2014), particularly by temperature (Soares et al., 2019). However, the short evaluation period (100 experimental days), adaptation to adaptation to housing density and ad libitum feed offer may not have been sufficient to alter corticosterone concentration because quails housed at the highest (86.31 cm²/quail) and lowest stocking densities (112.2 cm²/quail) showed similar feed intake, and subsequently, egg production rate, feed conversion per mass and per dozen eggs.
These findings differ from those of Lima et al. (2012), who tested different stocking densities (121.4, 106.2, 94.4, and 85 cm²/quail) for Japanese laying quails. The authors reported a significant effect of stocking density on feed intake, egg weight, feed conversion by egg mass, and feed conversion by dozen eggs. Moreover, Lima et al. (2012) observed that quails housed at 85 cm²/quail had lower feed intake and egg weight.
It is known that egg-laying poultry exposed to increasing stocking density may experience thermal stress and reduced capacity to dissipate heat. Therefore, birds tend to distance themselves from each other to maximize sensible heat loss. Birds use mechanisms of physiological regulation such as increased respiratory rate during eggshell formation in the uterus, with a consequent increase in CO2 loss and then reduced availability of carbonate ions (HCO3). Therefore, although calcium is available, eggshell quality decreases. However, quails housed at high stocking densities (86.31 cm²/quail) had similar results for eggshell thickness, eggshell weight, and specific gravity compared with quails housed at lower stocking densities.
On the one hand, Soares et al. (2018) reported no significant differences for eggshell thickness, specific gravity, and eggshell weight in Japanese quails housed at different stocking densities (121.43, 106.25, 94.44, and 85.00 cm²/quail). On the other hand, Vercese et al. (2012) reported increased respiratory rate and decreased eggshell strength in Japanese quails exposed to temperatures above 21°C. Moreover, the eggshell thickness was reduced when birds were exposed to temperatures above 27°C. This response was not observed in this study, although birds were exposed to conditions of moderate stress (Soares et al., 2019) with temperatures reaching 27ºC and maximum amplitude of 14.78 ºC.
Given that the mean minimum temperatures were around 19.5 ºC and the light was turned off at 10 p.m. (lighting program), the quails may have changed the feed intake pattern (Pereira et al., 2013;Guimarães et al., 2014), with more feeding activities during the evening. It may explain the lack of difference in animal performance and eggshell quality. Owing to nighttime food compensation The means for head temperature as a function of time decreased at 100 days of the experiment (35.21, 35.30, 34.78 and 36.36 ºC). It indicates adaptation to temperature conditions in the last period, which averaged 26.7 ºC (Table 1). When quails are not under heat stress, this area remains in thermal homeostasis. In a temperature mapping study using infrared thermography in Japanese quails , Souza Júnior et al. (2013) concluded that the region of the head had higher surface temperature than the other regions analyzed.
Quails housed at the highest stocking density (86.31 cm²/quail) had the highest head temperature (36.36ºC) at the end of the experimental period compared with quails housed at 112.20 cm²/quail (35.21ºC), 102 cm²/quail (35.30ºC), and 93.5 cm²/quail (34.78 º C). High-density stocked quails released more heat than quails with smaller cage space due to heat stress.
During periods of heat stress, featherless areas such as comb, wattles, and shins are typically vasodilated (Richards, 1971;Hillman et al., 1982). According to Dahlke et al. (2005), the comb, wattle, and shinks' total surface area correspond to 16% of the total body surface, which confirms the importance of these body regions to heat dissipation. This result corroborates the findings for mean core (33.68ºC) and shin temperatures ( The imposed stocking conditions was not the only factor influencing quail hematology (Table 1). We observed that the increase in ambient temperature up to 33.7ºC at 75 days of sampling, with minimums of 18.9ºC, coincides with the period in which the highest hematocrit concentration was reported (Table 6). Although the maximum temperature reached 34.9ºC at 100 days, the amplitude in this period (16.9ºC) was the largest (Table 1). Therefore, it indicates that the minimum temperatures may have some compensating effect, promoting adaptation of quails to the imposed conditions.
In situations of acute environmental and/or thermal stress, physiological changes such as decreased blood viscosity, increased hematocrit and hematimetric indices, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration are typical in birds (Laganá et al., 2005;Yahav et al., 2007). The decreased viscosity can alter blood flow in vessels and reduce the distribution of blood to body tissues (Borges, 2001), which compromises quail performance. Wein et al. (2017) evaluated the effects of cage size and different stocking densities in hens and observed increased hematocrit levels in high-density stocked animals as a response to stress. According to Bounous and Stedman (2000), the mean hemoglobin concentration in poultry (broilers and laying hens) is 9 g/dL, ranging from 7.0 to 13.0 g/dL. El-Kholy et al. (2017)  reaction may be present in poultry exposed to stress. This biphasic leucocytic response to stress may be unique to poultry (Campbell, 2004). Therefore, this reduction in heterophil counts may be related to the increase in lymphocyte count (lymphocytosis), which is associated with immune stimulation. Moreover, it is also associated with some types of heterophils under conditions of stress and disease, such as toxic heterophils, as seen in Figure 3. There is a relationship between stressful conditions and increased lymphocytic cells in quails. Some theories suggest that the increase in circulating lymphocytes depends on the demand of the tissues for lymphocytes. In response to circulating glucocorticoids, circulating lymphocytes bind to endothelial cells that line the walls of blood vessels, and migrate from the blood circulation to other tissues such as the spleen, lymph nodes, bone marrow, and skin, where they are rescued (Dhabhar, 2002). Scanes and Christensen (2014) observed no significant difference in differential leukocyte count and heterophil/lymphocyte ratio under conditions of moderate stress. This result differs from that found in the present study. On the other hand, other authors such as Rosa et al. (2011), Prieto andCampo (2010), and Soleimani et al. (2011) reported increased heterophil/lymphocyte ratio in birds under stress conditions. The H/L ratio is a less variable stress indicator than individual cells, and it is more reliable than plasma corticosteroid levels. However, this rule applies only under mild and moderate stress (Gross and Siegel, 1983). The results showed that the heterophil/lymphocyte ratio increased over time.
The CTL was lowest (14245 µL) during the last sampling period (100 days). With the experience of stress, leukocyte reserves tend to decline, and this effect has a profound influence on the number of leukocytes entering the blood circulation.
According to Jain (1993), the reference values of leukocytes in poultry ranges from 12000 to 30000 leukocytes/mm 3 .
The mean corpuscular volume or mean globular volume was significantly affected (p = 0.0001) by time. The MCV was significantly higher in the last sampling periods (75 and 100 days; 3,101 and 2,411 µm 3 , respectively) compared to 25 and 50 days, which were statistically similar among each other. The present study does not agree with Porto et al. (2015), who observed that chronic heat stress does not interfere with the production of serum anti-Newcastle antibody titers in pre-starter and starter chicks. But corroborates with the findings of Souza et al. (2010) in a study with stress-induced laying hens. The MCV is used as an indicator of anemia in animals.

Conclusion
Different stocking densities do not interfere with the performance and eggshell quality but affect the core and shin temperatures of Japanese quails. Hematological parameters and head temperature are affected by stocking density and time.
High-density stocked Japanese quails exposed to prolonged stress can become chronically stressed. However much the quail tries to adapt to the adverse condition, the organism reaches exhaustion, which leads to severe consequences on physiological and behavioral parameters. The stocking density of 102 cm²/quail is the most recommended because it does not differ from the control treatment.