Nutrição in ovo usando mel : efeitos sobre eclodibilidade , desempenho e rendimento de carcaça em frangos de corte In ovo nutrition using honey : effects on hatchability , performance and carcass yields in broilers

The objective of this study was to determine whether inoculating honey from Apis mellifera into broiler chick eggs (in ovo nutrition) during incubation would improve hatchability and performance. Initially, four hundred eggs were incubated; at 12 days of incubation, ovoscopy was performed to select the fertile eggs and divide them into three groups: control, saline and honey groups. On the 17th day of incubation we inoculated 0.2 mL of each solution directly into the amniotic cavity. After hatching, the chicks were housed in an experimental house until 28 days of age, when two birds per experimental unit were sacrificed to evaluate carcass yield. The hatchability was not affected by inoculation of saline or honey (p>0.05). However, mortality was higher in the honey and saline groups than in the control group (p 0.05). These results show that the in ovo nutrition with bee honey resulted in lower production costs, that is, the birds consumed less food (feed) and had the same weight gain as other treatments.


Abstract
The objective of this study was to determine whether inoculating honey from Apis mellifera into broiler chick eggs (in ovo nutrition) during incubation would improve hatchability and performance. Initially, four hundred eggs were incubated; at 12 days of incubation, ovoscopy was performed to select the fertile eggs and divide them into three groups: control, saline and honey groups. On the 17 th day of incubation we inoculated 0.2 mL of each solution directly into the amniotic cavity. After hatching, the chicks were housed in an experimental house until 28 days of age, when two birds per experimental unit were sacrificed to evaluate carcass yield. The hatchability was not affected by inoculation of saline or honey (p>0.05). However, mortality was higher in the honey and saline groups than in the control group (p<0.05). The birds that received in ovo nutrition with bee honey had a body weight 11% higher than 28 days compared to the control. The honey group showed better feed conversion and greater heart weight at 28 days (p<0.05). Mortality, carcass yield and bowel variables did not differ between treatments (p>0.05). These results show that the in ovo nutrition with bee honey resulted in lower production costs, that is, the birds consumed less food (feed) and had the same weight gain as other treatments.

Introduction
Because commercial chicken strains are selected for high weight gain and rapid development of the pectoral muscle, there is increased demand for energy and protein.
Consequently, there is an imbalance between demand and reserves in the eggs themselves. In ovo nutrition via amniotic fluid injection can mitigate this deficit and boost chick development (Campos et al., 2010). Bird embryos digest and absorb nutrients before hatching; the development of the gut occurs throughout incubation ; however, functional skills only occur after oral consumption of amniotic fluid between the 17-19-day-old embryo (Campos et al., 2010). The inoculation of an in ovo substance into amniotic fluid causes the embryo to naturally consume supplemental nutrients, orally, before hatching; this supplementation can accelerate enteric development and the ability to digest nutrients (Vieira, 2005;Campos et al., 2010). In so doing, it promotes the development of healthy intestinal flora, an essential condition for a good initial development (Ferket, 2013).
The in ovo nutrition technique aims to provide nutrients (primarily glucose) to the embryo, avoiding gluconeogenesis of endogenous proteins , because most glycogen reserves are used during hatching. Subsequently, the chick must replenish glycogen reserves through gluconeogenesis of body proteins (mostly the pectoral muscle) to support thermoregulation and survival until beginning to consume and use dietary nutrients (Moran, 1985). Because the intestine is the organ that provides nutrients, the sooner it reaches its functional capacity, the faster the bird will be able to use the nutrients in the diet and grow efficiently, expressing its genetic potential and resistance to infectious agents and metabolic diseases .
Carbohydrates are widely used in in ovo nutrition because they increase the level of glucose available to the embryo . It is important to note that the carbohydrate concentration of the egg is only 1% of the total, compared to the other nutrients (Sugino et al., 1997), with blood glucose and glycogen reserves maintained by the gluconeogenesis of amino acids, glycerol and other gluconeogenic components (Sunny and Bequette, 2010), reducing the availability of these nutrients for muscle growth. These explanations are used to justify in ovo nutrition, as seen in injections with glucose, fructose, sucrose, dextrin and maltose (Leitão Research, Society and Development, v. 9, n. 8, e43985178, 2020(CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5178 et al., 2008, carbohydrates (Zhai et al., 2011), amino acids (Al-Murrani, 1982, among others. According to Leitão et al. (2008), glucose-based solutions for in ovo nutrition for the period close to hatching are important energy sources during this phase, reserving proteins and lipids for other specific functions.
Bee honey is a suitable substance for in ovo nutrition because it has considerable amounts of reducing sugars (fructose and glucose) (Gois et al., 2015), with α-and β-glucose and fructose being the most abundant (Campo et al., 2015). Because honey is composed mostly of glucose and fructose, this monosaccharide would not need enzymes to be digested and would be readily available for use. In addition to its nutritional and energetic capacity, honey has antimicrobial and antioxidant effects, and it promotes wound healing (Silva et al., 2006). For these reasons, the objective of this study was to determine whether egg nutrition with injections of honey solution would improve embryonic development, performance and broiler carcass yield.

Materials and Methods
This research had an exploratory and quantitative nature (Pereira et al., 2018). To achieve the objectives of this study, we used a completely randomized design, as detailed below. Four hundred COBB® eggs were incubated in four automatic incubators, model Premium (Ecológica EP160), with automatic turning every 2 hours. Before starting the incubation process, the incubators were washed and disinfected with sodium hypochlorite, and had their temperature and humidity adjusted to 37.5 °C and 60%, respectively. The four hundred eggs were weighed in trays with a capacity of 24 eggs to determine the average egg weight, later disinfected with 70% alcohol and distributed at random in the incubators. The design used was randomized blocks, with four blocks (incubators) and three treatments (types of inoculation or control).
The solutions used to nutrition the eggs were prepared on the same day as the inoculation. Two solutions were used for nutrition, according to each treatment, based on 0.5% saline solution prepared with 0.5 g of NaCl dissolved in 100 mL of sterile distilled water Research, Society and Development, v. 9, n. 8, e43985178, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5178 6 (Saline group). Honey from commercial polyfloral origins was included as a nutritional component in the saline solution at 20% (Honey group). Both solutions were autoclaved at 120 ºC for 15 minutes to ensure the sterility of the material to be inoculated.
After sterilization, the honey solution was analyzed for the presence of hydroxymethylfurfural, because this component is considered toxic if present above 60 mg/kg; both saline and honey solutions were analyzed for the presence or absence of microorganisms (IAL, 2008). After these tests, the solutions were considered suitable for in ovo nutrition.
At 12 days of incubation, ovoscopy was performed to identify fertile eggs and determine the mortality rate using embryo diagnosis, determining fertile eggs, infertile eggs, early embryonic mortality (1 to 7 days) and intermediate embryonic mortality (8 to 14 days Because amniotic fluid ingestion starts from the 17th day of incubation and ends on the 19th day (Bohorquez, 2010), we decided to perform inoculations on the 17th day of incubation.
Innoculations were carried out at the Microbiology and Immunology Laboratory (Labmin) of UDESC -CEO, in a room previously heated at 30 ºC. Before in ovo nutrition, the equipment used was placed in a sterile chamber with ultraviolet light to ensure sterility. The eggs were cleaned with 70% alcohol and subjected to ovoscopy for identification and delimitation of the inoculation site (Foye et al., 2006). The inoculation process was carried out in a sterile continuous flow hood. After ovoscopy, the shell was perforated in the region of the air chamber using a portable micro-artificial with a 2-mm diameter drill, avoiding the perforation of the inner membrane of the egg shell. The inoculum was delivered directly into the amniotic cavity with a 7 x 2.5 mm syringe and, immediately after inoculation, the hole was sealed with PVA glue. After the procedure, the eggs were individually packed in filo bags and returned to the incubator.
After 20 days of incubation, the temperature and humidity of the incubators were adjusted to 36°C and 62%, respectively, and the turning feature was turned off. Hatching was attended without interference; during this process we checked the activity and vigor of the chicks. At the end of the 22 days of incubation, the eggs that did not hatch were opened for analysis using embryo diagnosis, determining mortality after in ovo nutrition. Research, Society and Development, v. 9, n. 8, e43985178, 2020 (CC BY 4 To minimize the variation between the experimental units and ensure the same condition of equal weight for the treatments, at the time of accommodation, all birds were weighed individually and divided into five weight ranges (<37.5 g, 37.5 g at 40 g, 40 g to 42.5 g, 42.5 g to 45 g and > 45 g). The number of available birds per weight range was divided by the number of experimental units, depending on the treatments. The birds were then distributed according to the weight ranges, such that the experimental units had the same initial average weight (Sakomura and Rostagno, 2007).
The feed provided throughout the experimental period had 22.4% crude protein, based on soybean meal and corn meal (Table 1). Feed consumption was recorded weekly with the weight of the feeder using analytical balance.
At 28 days of age, two birds per experimental unit were sacrificed (one male and one female), weighing equal to or close to the average weight of the brood. The birds were kept under an 8-hour water fast for later sacrifice. We weighed and sacrificed them using cervical dislocation, a method that kills bird by cutting the spinal cord and stretching the main blood vessels (Ludtke et al., 2010). According to Concea (2015) this method can be used to sacrifice birds up to 3 kg. After this procedure, bleeding was carried out and the birds were weighed again. Then they were scalded and plucked.
The carcasses were eviscerated, discarding non-edible viscera. The weights of the cold carcasses and their cuts (chest, wing, thigh and drumstick, and back) and edible viscera (heart, liver, and gizzard) were obtained using an analytical scale. The small and large intestines were separated, separating the duodenum, jejunum and ileum (small intestine), and the cecum, colon, and rectum (large intestine). Each intestinal portion was weighed on an analytical scale and measured using a millimeter ruler.
The data obtained were analyzed for normality; subsequently, the analysis of variance was performed using the PROC GLM of the SAS. When there was a significant difference, values were compared using the Tukey test at 5% significance level.

Results and Discussion
The parameters related to incubation were displayed in for the Honey group and 9.4% for the Saline group). According to Plano et al. (2005), the increase of the internal humidity of the egg during the final days of incubation causes increased embryonic mortality, with the embryos adhering to the internal membrane, making hatching difficult. Pedroso et al. (2006a) attributed this increase to what they considered an excessive volume of the inoculation (0.5 mL). Pedroso et al. (2006b) inoculated 0.2 mL volumes and found no differences between the control group and the group that received the inoculum, differently from the results observed in the present study. Research, Society and Development, v. 9, n. 8, e43985178, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5178 without in ovo injection). The post-inoculation mortality rate reported by Neves et al. (2016) was also high, ranging from 32.3% in the 37.5 nmol glycerol/mL treatment to 48.5% in the 25 and 50 nmol glycerol/mL treatments. Campos et al. (2011) found hatchability of 93.57% for eggs without inoculation, 84.64% for eggs that received saline and 82.5% for eggs that received glucose + sucrose; they also observed higher embryonic mortality in the post-peck phase, different from what we observed in the present study, that is, the highest mortality occurred after the inoculation performed on the 17 th day. Late mortality was observed only for the chicks that received some type of inoculation. When comparing the results obtained from the Saline and Honey groups to Control group, we noted that the increased post-inoculation mortality may have been related to the inoculation process, which was done manually, and may not have been performed with precision; factors such as the speed of injection, location and volume injected, even the needle used can interfere with the results of in ovo injection (Neves et al, 2016).
There was no significant difference (p>0.05) for live weight at 28 days (Table 3) and for carcass cuts (Table 4). There was an effect of the treatment, that is, those that passed through the inoculation with honey had less feed conversion; as well as greater heart weight (Table 3 and 4).   (Campos et al., 2011) or maltose and dextrin  found better breast yield in chickens. Most honey contains glucose and fructose (Alvarez-Suarez, 2013); for this reason, it is possible that the presence of lower amounts of sucrose did not allow an increase in the carcass yield of broilers in the honey group compared to the control group.
Authors reported increased weight and yield of breast to bone weight (5.07%) and breast fillet (5.47%) in the group that received 2.5% glucose + 3% sucrose, compared to the control group (Campos et al. 2011). Uni et al. (2005) also reported an increase of 8.7% and 8.3% in breast yield at 25 days in broilers of the Cobb and Ross lineage by inoculating carbohydrate-based solution at 17.5 days of incubation.
It is possible that the composition of the honey-based nutrient solution was insufficient (i.e., 20%) to cause the expected effect of increased chick performance. Ipek et al. (2004) inoculated low glucose levels (5, 10 and 15 mg) and did not observe an increase in embryonic mortality or improvement in broiler performance, concluding that low glucose levels do not contribute as an energy supplement for a bird during embryonic development. Tako et al. (2004) pointed out that the use of disaccharides such as maltose and sucrose in egg supplementation provided better results due to the stimulus of the synthesis of enzymes and greater absorption of nutrients. It is noteworthy that honey contains 70% monosaccharides and between 10 to 15% disaccharides (including maltose, sucrose, maltulose, turanose, isomaltose, laminaribiose, nigerose, kojibiose, gentiobiose and ß-trehalose) and oligosaccharides (maltotriose, erlose, melezitose, centose 3-α5 isomaltosylglucose, l-kestose, isomaltotriose, panose, psopanose and theanderose) . Low glucose Development, v. 9, n. 8, e43985178, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5178 13 levels (100, 200 and 300 mg) provide 0.34, 0.69 and 1.02 kcal of metabolizable energy, respectively; this is insufficient for the process of breaking the inner and outer membranes of the eggshell (Longo et al., 2005). Honey has 82 g of carbohydrates in 100 g that provide 304 calories; the amount of honey diluted in saline may have been insufficient to provide the energy needed to improve performance and hatchability.
The hearts in the group that received honey were heavier at the end of the experimental period (28 days) (p<0.05) than those of the control group. Few studies show increased heart weights attributable to addition of nutrients in the egg; Neves et al. (2016) found greater heart weights in chickens that received glycerol in ovo and related this increase to increased blood volume. We found no significant difference (p>0.05) for liver or gizzard weights, or for small and large intestine variables. Neves et al. (2016) evaluated various levels of glycerol in in ovo nutrition and did not observe significant differences in the viscera weights, except for the spleen, heart and gizzard.

Final Considerations
In ovo nutrition is a technique used to increase hatchability and improve the viability of newborn chicks. Therefore, we hypothesize that the inoculation of honey in ovo is a viable alternative, as this is a highly energetic food.
The inoculation of the 20% bee honey solution in ovo to nutrition does not alter hatchability, as well as the weight and carcass yield of chickens at 28 days. However, it positively influenced dietary conversion and heart weight. Nevertheless, some issues remain to be clarified, including the level of honey content to be used, the efficiency of the nutrition method, and its benefits for the production indexes in a complete production cycle.