4. Discussion
In the present study, brown-shell eggs were used, which differs from other studies found in the literature, where white-shell eggs are mostly used. The differences found may be related to the light absorption capacity of different eggshells, since eggshells are a critical factor that influences the incubation performance of eggs [
15,
23]. As in the present study, the color of the eggshell was brown, so the filtration capacity of the lights may have been different.
The light-absorbing capacity of the eggshell is related to the shell’s color, strength and thickness. Generally, brown eggs have larger shell thickness, on average 0.37 mm [
24], while the light-pink shells have an average thickness of 0.32 mm in the intermediate period of egg laying, which affects the light absorption capacity [
25].
In broilers, Archer [
26] suggested that white light from LEDs is filtered so that the wavelengths of the light are red-shifted, while red and green lights are not filtered or shifted that way. The different transparency of the eggshell for different wavelengths of light can be environmentally important, since different colors of the shell can selectively filter the light to which the embryos are exposed. Eggshell pigments admit penetration or block different wavelengths of the light spectrum [
27]. However, considering the particularities of each species of birds, the effects of light during the incubation period may be different [
28]. Thus, more studies are needed to understand the transmission of light waves in different colors and species of birds.
Incubation temperature is the most important factor during embryo development [
4]. Thus, the eggshell temperature has been used as an indicator of embryonic temperature. According to Meijerhof and Van Beek [
29] and French [
30], the eggshell temperature deviates around 0.1 to 0.2 °C from the embryo temperature.
Different eggshell temperatures were compared by Lourens et al. [
31], in which embryos exposed to a temperature of 37.8 °C throughout incubation showed better hatchability rates (88.1%) and embryonic development when compared to embryos submitted to a high temperature (38.9 °C), which obtained 86.2% hatchability.
Willemsen et al. [
32] observed reduced hatchability (74.2%) in treatment with high shell temperature (40.6 °C) when compared to a temperature of 37.8 °C (93.1%), possibly caused by increased final embryonic mortality (13.8% vs. 2.5%, respectively). Van der Pol et al. [
33] also found lower hatching values (78.4%) and organ development in embryos exposed to high temperatures (38.6 °C) when compared to the control group (95%).
High or low incubation temperatures can influence embryogenesis, affect blood biochemical levels and chick quality and, consequently, productive performance during breeding [
34]. The three eggshell temperatures were 34.6 °C (low), 37.6 °C (control), or 40.6 °C (high) during incubation days [
32].
According to data from our study, the minimum temperature of the eggs is low (34.1 °C treatment without lighting; 35.1 °C treatment with white lighting and 34.3 °C treatment with red lighting) in all treatments with and without lighting, so the low temperature provided thermal stress in the embryonic development of the chicks. The temperature data of the eggs were determined using an infrared thermographic camera, while in the studies mentioned above it was determined by a thermohygrometer coupled to the eggshell, so values may be slightly different because of the technology employed.
Regarding YFBM (yolk free body mass), this is a quality parameter that represents the body weight of the chick without the weight of the residual yolk sac. Lower yolk sac weight and higher YFBM are desirable, since they indicate ideal environmental conditions in incubators and hatcheries, in addition to the embryo’s ability to use yolk components for structural formation [
9]. Joseph et al. [
35], while studying the effect of high incubation temperatures (39.5 °C) on chick weight, observed a reduction in body weight (39 g) and lower YFBM (34.7 g) compared to a temperature of 37.8 °C, where the results were 40 g of body weight and 35.9 g of YFBM. However, in the present study, no differences were observed for chick weight, yolk sac weight, chick weight without yolk sac and chick relative body weight with the use of monochrome lights in the artificial incubation process.
Relative humidity should also be controlled within the recommended limit, as its deviations affect the quality of the newborn chick [
36]. When the relative humidity (RH) is above ideal during incubation, the chick’s weight increases, as excess water is incorporated into the embryonic tissues, impairing its initial performance [
37]. Thus, the relative humidity found in the present study is considered high (maximum RH was 72% and minimum RH was 68%), but no significant difference was found for egg weight and weight loss in this study.
The loss of egg mass is directly linked to water loss, and, under normal conditions, the egg loses 13 to 15% of its mass between the day of laying and the day of internal pecking [
30]. The egg starts to lose water after oviposition. For optimal hatchability, egg weight loss is expected to be close to 6.5–12.0% [
38,
39]. Water loss is also an indicator of the embryo’s metabolic rate and a high metabolic rate accelerates water loss [
40]. In the present experiment, weight loss is within the normal range, with an overall average of 10.33%, and not impairing the embryonic development of the eggs.
When eggs are exposed to periodic photo lighting, melatonin secretion is stimulated and begins to establish a circadian rhythm in the pineal gland from the sixteenth day of embryonic development [
41]. The provision of photoperiod during incubation can stimulate pineal melatonin secretion and regulate growth hormone synthesis. However, the increase in overall embryo weight as well as muscle weight was also found when there was continuous light exposure in chicken embryos [
42,
43]. Such results indicated that muscle growth may depend on light exposure, but it is not associated only with circadian rhythms commanded by the photoperiod. The wavelength and intensity of light can influence the amount of light that can pass through the eggshell and reach the embryo. Light intensity may be one of the main factors affecting mitosis in the mesoderm of the neural crest during the early stage of embryonic development [
44]. However, in the present study, no difference was found for increased embryo weight.
Increase in hatching may be attributed to positive changes in hormones related to hatching, especially thyroid and corticosterone, in response to lighting treatments. Such improvement may also be due to the positive impact on the rate of embryo development, and the study by Ghatpande et al. [
45] suggested that light during incubation increases the rate of embryonic development. Furthermore, it may also be due to the change in the rhythm of melatonin in the embryos provided by light during incubation [
46]. No improvement in hatching was observed in the current study.
Geng et al. [
47] found that birds which did not receive light stimulus during the 21 days of incubation showed the highest percentage of hatching, demonstrating that instead of promoting benefits, the exposure of embryos to green light resulted in hatchability impairment. However, in the present study, the lights used were white and red, with no effect on hatching between treatments.
Serum biochemical indices may partially reflect the metabolism and health status of the organism, especially serum immunological and antioxidant indices, which indirectly reflect the animal’s health status [
47]. Responses to stressors are evidenced first at the biochemical level, followed by physiological responses and, finally, manifested at morphological level. Thus, changes in all these levels are indicative of stress and, therefore, parameters such as plasma glucose are important physiological indicators of stress levels [
48]. In the present study, differences were observed in biochemical parameters (Calcium, Phosphorus, Cholesterol, Amylase, Glucose, Urea and TGP), which may be explained by low temperature stress in conjunction with the use of monochromatic lights in eggs.
Heat stress causes multiple changes in the neuroendocrine physiology of birds. Several studies report continuous activation of the hypothalamic–pituitary–adrenal axis, which promotes increased circulating levels of corticosterone, resulting in greater protein catabolism, hyperglycemia, and immunosuppression and increased susceptibility to infections [
49,
50].
Studies reveal that thermal manipulation during incubation, in the development periods of the hypothalamic–pituitary–thyroid and hypothalamic–pituitary–adrenal axes of the embryo, promotes epigenetic and metabolic changes, which allow birds to adapt to high and low environmental temperatures [
51,
52]. Such changes are in the stress regulatory pathways, decreasing metabolic rate and increasing the expression of pro-angiogenic genes in muscle. This process enhances the vasomotor response and, consequently, heat loss as well, as it increases the expression of anti-apoptotic genes, preserving the integrity of cells during thermal challenge after hatching [
53].
Guest et al. [
54] show increases and decreases in several hormones and compounds whose metabolisms are directly impaired in each phase of stress. In the alert phase, there is an increase in noradrenaline and adrenaline and in glucose, and therefore, in the individual’s heartbeat, in addition to vasoconstriction and mydriasis in individuals. In the exhaustion phase, an increase was observed for levels of fatty acids, such as triglycerides and cholesterol, for glycerol and leukocytes. Such an increase can directly affect the physiology, causing increased heart rate, vasoconstriction, inhibition of insulin, and increase in blood glucose, cholesterol and triglycerides.
Photostimulation with green monochromatic light during the late phase of incubation (between ED18 and ED20), which is considered a critical phase in embryonic development, increases the activity of the somatotropic axis at the level of the positive control; thus, a critical period for photostimulation of broiler embryos can be considered. The critical period is defined by several physiological mechanisms, including the transition to pulmonary respiration and increased hepatic gluconeogenesis to increase glucose levels and provide sufficient energy for the embryo to hatch [
55], with this pattern presented in the white and red light used in the present study.
According to Artacho et al. [
56], the physiological state can be reflected according to plasma metabolites, which are intermediate products of metabolism. All physiological and metabolic mechanisms carried out during embryogenesis, hatching and post-hatching can be directly affected by temperature at incubation. Studies show that situations of high and low temperatures and/or low oxygen availability can interfere with the physiology and metabolism of the chicken embryo and, consequently, promote harmful effects on embryo development and survival [
31,
57,
58].
At the beginning of incubation, the oxygen support of the embryo is extremely limited due to the immature state of the vascular system. Thus, anaerobic glycolysis becomes active, but in a restricted way, as glucose levels are minimal at this stage [
59]. In the final third of incubation, the embryo also uses alternative routes, such as gluconeogenesis for glucose production, which will be stored as liver and muscle glycogen for use in the final moments of incubation [
60].
The hatching process requires high energy demand and the fatty acids can no longer efficiently supply all the necessary fuel. Glucose is released from glycogen and the embryo performs anaerobic glycolysis until external pecking, increasing the circulating lactate [
61]. Hoiby et al. [
62] stated that after internal pecking, the embryo comes into contact with the tube, and the supply of oxygen for the metabolism is resumed. The amount of lactate decreases and the catabolism of fatty acids starts the energy supply again. In the period shortly after the hatching of chicks, the synthesis of glucose from the oxidation of fatty acids is intensified [
63].
Regarding the storage of glucose in the animal organism, this occurs through the glycogenic route with the formation of glycogen, which is stored primarily in the liver, in addition to the membrane of the yolk sac, breast muscle and intestines [
64,
65].
Glycogen is the energy reserve polysaccharide of animal organisms and it stores glucose in a readily mobilizable form. Therefore, in the absence of plasma glucose, hepatic and renal glycogen is mobilized for glucose release by glycogenolysis. In the case of muscle glycogen, degradation occurs in response to tissue energy expenditure. When there are limiting levels of glycogen, some tissues produce glucose from amino acids of the residual proteins. The main storages of this compound are found in the liver and skeletal muscle. In the liver, glycogen plays a role in maintaining the concentration of glucose in the blood, especially in the early stages of fasting, while muscle glycogen acts as a fuel reserve to synthesize ATP molecules [
66].
Glycogen reserves in the final incubation period are abundantly mobilized, indicating the essentiality of glucose in the artificial incubation of chicken eggs [
67]. According to Moran Jr [
59], the storage of hepatic glycogen reserves is of paramount importance, as it is a preparation for the hatching process. One mechanism to conserve glycogen is the gluconeogenesis process by the liver from non-glycosidic compounds such as glycerol and amino acids [
68].
During the first days of incubation, the embryo has low oxygen supply due to the immaturity of blood cells and the vascular chorionic system [
69]. According to Moran Jr [
59], the embryonic compensatory mechanism in those situations is to perform anaerobic glycolysis, and therefore, the lactate increase is pronounced and considered normal in those stages. In addition, glycogen is degraded to release glucose molecules that will be oxidized to pyruvate. Pyruvate is transformed into lactate in anaerobiosis [
32,
70].
Harms and Harms [
71] related high plasma lactate concentrations in response to stressful stimuli. Referring to the hatching process, a large amount of energy is required for the rotational movements of the body and breaking of the shell. Glycogenic stores are depressed and a low glucose concentration forces the embryo to mobilize body protein. At the end of incubation, the metabolism is increased to obtain glucose and, due to the lack of oxygen in those last stages, the embryo cannot produce energy from lipid metabolism, becoming dependent on the gluconeogenic pathway [
59].
Then, the anaerobic production of lactate is intensified, and this is converted to glucose. Accordingly, there is a progression of lactate levels at high and low incubation temperatures as muscle activity is high and oxygen availability is low during the hatching process. The pyruvate formed during anaerobic glycolysis for lactate formation increases the blood hydrogen proton and is buffered by bicarbonate (HCO
3), avoiding a reduction in pH [
55].
Although amylase is not directly related to the gluconeogenic pathway, it is noteworthy that glucose is one of the products of starch digestion by amylase. The resulting glucose can be used by the body’s cells as an energy source, and the excess is stored as glycogen in the liver and muscles, to be used later. In situations of low glucose availability, such as prolonged fasts, gluconeogenesis is activated to maintain blood glucose levels. Amylase is an enzyme present in the body whose main function is to break down starch into smaller sugar molecules, such as glucose [
68]. However, no direct relationship between amylase and the gluconeogenic pathway could be detected in this study.
The serum decrease in urea is not indicative of kidney injury, because it originates from the hepatic metabolism of nitrogenous compounds. It can also be an indicator of the use of gluconeogenesis as an alternative route for energy production [
69]. In this study, the increased use of gluconeogenesis was due to increased glucose and decreased urea for lighting treatments.
During the transition from the pre-eclosion phase to the post-eclosion phase, metabolic heat is rapidly produced by the rapid rate of fatty acid oxidation [
59]. Yalcin et al. [
70] reported changes in fatty acid composition and concentrations in high-yield broilers when eggs were incubated at high and low temperatures. This probably occurs as a cell protection mechanism, since many of these lipids are involved in the permeability and structure of developing cells.
Connor et al. [
71] found that 98% of the chicks’ cholesterol at hatching comes from the yolk. Thus, the increase in the cholesterol rate promoted by the low incubation temperature must have resulted from greater use of the yolk sac recorded for those chicks. The results corroborate those found by Willemsen et al. [
32], who also recorded higher cholesterol values for egg chicks incubated at a lower-than-usual temperature. The white and red lights followed this profile for cholesterol.
Calcium is the mineral with the highest concentration in the bird’s organism, being a relevant part in the formation of eggs and eggshells, in addition to participating in many important biochemical reactions [
72]. Phosphorus, like calcium, is an important part of bone formation and participates in the regulation of acid–base metabolism and energy production [
73]. This last function, due to the presence of large amounts of phosphate compounds in red blood cells, gives birds a reduction in oxygen affinity, offering an advantage for the regulation of oxygen transport [
74]. There is interdependence between calcium and phosphorus values, so that the deficiency or excess of one of them can impair the absorption or use of the other [
75].
Glutamate pyruvate transaminase is an enzyme present in high concentration in the kidney, heart, skeletal muscles, liver and lung, and therefore the interpretation of its value in poultry liver diseases is controversial [
76,
77]. Glutamate oxaloacetate transaminase is widely distributed in poultry and is present in high concentration in various organs and tissues, mainly heart, liver, skeletal muscles, kidney and brain. However, the enzyme’s distribution differs among avian species. Thus, it cannot be considered as a hepato-specific enzyme, as it also indicates muscular sensibility [
78].
The metabolites of AST and ALT provide information about the functional capacity of the liver that is involved in a given metabolic pathway. AST is the most sensitive indicator of liver disease in poultry [
79]. Liver damage causes the release of AST or ALT enzymes into the bloodstream. Hassan et al. [
80] mentioned that monochromatic lights in broiler chicks did not alter liver enzymes (AST and ALT) in a healthy batch. Olanrewaju et al. [
81] reported that the use of two-color temperatures of LED lamps did not influence the plasma concentrations of AST and ALT in broilers. The result differs from the present study in terms of ALT in the blood of chicks in the first hours of birth submitted to monochromatic white and red lighting and to no light, in which ALT was not detected in the interaction.