Influence of in-ovo and after hatching treatment with inorganic selenium on productive performance, some hormones and antioxidant status of broiler

: The current study aimed to evaluate the effects of inorganic selenium (sodium selenite) added to the drinking water of hatched chicks from eggs injected with it on productive performance, antioxidant status, and some hormonal parameters. 216 unsexed one-day-old broiler chicks were distributed into 6 groups (3 replicates, 12 birds/replicate) till 42 days of age, as follows: The first (1 st ) and second (2 nd ) groups: hatched chicks came from non-injected eggs without any substance. The third (3 rd ) and fourth (4 th ) groups: hatched chicks came from eggs injected with deionized water (0.2 ml/egg) in the chorioallantoic sac, while the fifth (5 th ) and sixth (6 th ) groups: hatched chicks came from eggs injected with 0.02 mg sodium selenite/egg in the chorioallantoic sac. Chicks of the 1 st , 3 rd , and 5 th groups were given tap water, while chicks of the other groups were given drinking water supplemented with 0.3 mg sodium selenite/L. Inorganic selenium significantly improved the initial and final body weight, weight gain, feed conversion ratio, and significantly reduced the feed consumption at 35 and 42 days of age. It also enhanced vitality percentage, productive index, and reduced mortality percentage, and it was significant (P≤0.05) at 42 days of age. On the other hand, in-ovo injection with sodium selenite reduced malondialdehyde and corticosterone values, while total antioxidant capacity and leptin increased, and continuing adding it with drinking water led to a significant decrease in malondialdehyde while leptin and thyroxine values significantly increased. In conclusion, the in-ovo injection with sodium selenite and continuous treatment with it after hatching improve the antioxidant status and productive performance of the broiler, and it can be recommended to use sodium selenite in early egg nutrition during incubation period and its supplementation with drinking water of chicks after hatching.


INTRODUCTION.
The poultry industry faced great challenges in the world in terms of reproduction and production improvement (Zia et al., 2017). Therefore, the search for natural ingredients that can be added to feed and water to achieve high production efficiency in poultry has become necessary, especially after the European Union banned the use of growth-stimulating antibiotics. Despite the positive and significant role of antibiotics in the development of the poultry industry, they are causing health damage to animals and humans due to consuming their products (Dibne and Richard, 2005). The research was focused on improving poultry production in ways that depended on adding antibiotic alternatives such as probiotics, plant extracts, and organic acids in addition to enzymes (Ahmed et al., 2017;Pirgozliev et al., 2015). In recent years, interest in the poultry industry has increased due to the wide development in its production, whether meat or eggs, compared to other animal products (Windhorst, 2006). The development of breeding programs for broilers also increased in order to increase the speed of growth in modern breeds, and because of genetic improvement, produced flocks were characterized by rapid growth and a high conversion coefficient. However, these flocks were characterized by rapid metabolism, which made them more susceptible to diseases and more susceptible to oxidative stress resulting from the increased generation of free radicals, which will affect the performance of poultry and therefore will need antioxidants for production (Surai, 2016). As a result, recent reports have indicated that adding antioxidants to poultry feed can improve the antioxidant status, health, and performance of birds (Karadas et al., 2014;Pirgozliev et al., 2019).
Selenium is one of the antioxidants added to broiler diets that can reduce the risks of oxidative stress. Selenium is one of the mineral elements that researchers have paid attention to because it enters all cells and tissues of the body at various distribution rates. Selenium has many effects on the vital functions of both humans and animals (Zhang et al., 2018;Edens and Sefton, 2016). Selenium plays an important role in the immune system function and growth of poultry (Liao et al., 2012). It is also the main component of many antioxidant enzymes including glutathione peroxidase GSH-Px (Pilarczyk et al., 2012), which protects the cell from free radical damage (Ighodaro and Akinloye, 2018). Selenium has an important role in the synthesis and metabolism of thyroid hormones (Brown and Arthur, 2001), also plays as cofactor of the enzyme (5 deiodinase), which is important in the synthesis of Triiodothyronine (T 3 ) hormone. The T 3 is important in controlling the growth of animals, especially poultry, by controlling energy and protein assimilation. Also selenium increases protein digestibility and energy utilization (Chen et al., 2014;Saleh et al., 2014). There are two types of selenium that can be added to poultry rations, organic selenium (selenomethionine and selenium-rich yeast, etc.), and inorganic selenium in the form of salts such as sodium selenite, which is the most widely used source of selenium (Surai and Fisinin, 2014;Yuan et al., 2011).
Inorganic selenium has been used as a source of selenium to be added to poultry rations, as it is less expensive than the organic source and is absorbed from the intestinal lining by simple diffusion (Surai and Fisinin, 2014). Recommended concentrations for adding selenium to broiler diets are 0.15 ppm throughout the growth period, and toxic concentrations are 10-20 mg/kg feed (N.R.C., 1994). However, it was recommended (European Commission, 2014) not to allow selenium to be given more than 0.5 mg/kg feed. Accordingly, the study aimed to determine the effect of inorganic selenium on the antioxidant status and productive performance of broilers.

MATERIALS AND METHODS
This experiment was conducted in the livestock fields/College of Agriculture and Forestry/ University of Mosul. The current study aimed to evaluate the effects of sodium selenite (SS) addition to the drinking water of chick's strain Ross-308 hatched from in-ovo injected eggs with SS on productive performance, antioxidant status, and some hormonal parameters. The treatments were as follows: -The 1 st group: chicks hatched from non-injected eggs and reared on a standard ration and tap water. -The 2 nd group: as in the 1 st group except that the chicks were reared on drinking water supplemented with SS 0.3 mg/L.
-The 3 rd group: chicks hatched from in-ovo injected eggs with D.W. (0.2 ml/egg) in the chorioallantoic sac and reared on a standard ration and tap water. -The 4 th group was as in the 3 rd group except that the chicks were reared on drinking water supplemented with SS 0.3 mg/L.
Rahawi, Abdul-Majeed, Abdul-Rhaman -The 5 th group: chicks hatched from in-ovo injected eggs with SS 0.02 mg/egg inthe chorioallantoic sac and reared on a standard ration and tap water.
-The 6 th group: as in the 3 rd group except that the chicks were reared on drinking water supplemented with SS 0.3 mg/L. The treatments continued to 42 days of age, bird were raised under ideal environmental conditions of ventilation and lighting and fed standard ration according to (N.R.C., 1994) containing 20.16% crude protein and 2841.6 kcal/kg metabolizable energy. At the end of the experiment, the parameters of body weight (B.wt), feed consumption (FC), feed conversion ratio (FCR), weight gain rate, productive index, mortality rate, and vitality of birds at the ages of 35 and 42 days were studied. Blood samples were collected at days of age42, then the serum was isolated and stored at -20°C and used for the determination of total antioxidant capacity (TAC), glutathione (GSH), malondialdehyde (MDA), Leptin, corticosterone, insulin like growth factor-1 (IGF-1), thyroxine (T 4 ) and triiodothyronine (T 3 ) were determined by the use of a kit made by Elabscience, USA.
Statistical analysis was conducted as a factorial experiment with two factors, and to determine the differences between the groups were determined using the Duncan's test (Gupta et al., 2016) for all the measurements covered by the study, at least significant differences (P≤0.05) and using the ready-made SAS program (SAS, 2009).

RESULTS AND DISCUSSION.
Tables 1 and 2 revealed that in-ovo injection with sodium selenite (SS) caused a significant (P≤0.05) increase in the initial B.wt of the hatched chicks, final body weight and weight gain 35 and 42 days of age compared with the negative and positive control. There was, also a significant decrease in FC in the SS injected group compared with the negative control group, and significantly improved FCR compared with both negative and positive control. In regard to the continuity of treatments with SS in drinking water after hatching, the addition of SS to the drinking water significantly reduces (P≤0.05) FC and FCR for the two periods (35 and 42 days) as compared with the discontinuous treatment. For the interaction effects, the in-ovo injection with SS regardless of the after-hatching treatment showed the highest Rahawi, Abdul-Majeed, Abdul-Rhaman -**Sodium selenite is injected on the tenth day of incubation at a dose of 0.02 mg/egg and 0.2 ml/egg in the chorioallantoic sac. -Continuous treatments: Sodium selenite was added to the drinking water of the hatched chicks at a dose of 0.3 mg/L until 42 days of age.
hatched chick B.wt (Table 1 and 2). Whereas the chicks hatched from the in-ovo injection with SS and those hatched from in-ovo injection eggs with SS recorded a significant increase in B.wt and W.G at 35 days of age, and those hatched from in-ovo injection eggs and treated after hatching with SS showed a significant increase in B.wt and GW at 42 days of age at (P≤0.05). They also showed a significant improvement in FCR at 35 and 42 days of age. On the other hand, the chicks hatched from the noninjected eggs recorded the highest FC values at 35 and 42 days of age (P≤0.05). These results agreed with the results of Wang et al. (2021) as it was noted that the addition of inorganic selenium at two different levels led to a significant increase in live body weight and a significant improvement in the feed conversion factor, as well as a decrease in the total mortality rate. The results of the current study agreed with the results of Ghazalah et al. (2020) as it was reported that the Final body weight, rate of weight gain and the improvement of the food conversion ratio when adding inorganic selenium compared with the control treatment. The reason for the improvement in product performance may be due to the fact that selenium is a necessary complement and activator of key enzymes in the manufacture and activity of T 3 hormone such as deiodinase, and that T 3 is an important regulator of growth in poultry by regulating energy production and protein metabolism, so selenium can increase growth by improving the digestion and assimilation of protein and energy consumption (Chen et al., 2014: Saleh et al., 2014. Our results did not agree with the results of Prasoon et al. (2018) as no significant differences were observed in body weight, feed consumption, or feed conversion among all treatments. Table 3 revealed that in-ovo injection with SS enhance significantly (P≤0.05) the productive index at 35 and 42 days of age. In addition, the addition of SS to drinking water after hatching (continuous treatment) reduced mortality percentage and increased vitality percentage and the productive index significantly at 42 days of age. Concerning the effects of interaction, the addition of SS to the drinking water of chicks hatched from in-ovo injected or non-injected eggs, and chicks hatched from in-ovo injected eggs with SS. Whether treated with SS with drinking water or not, reduced significantly mortality percentage at 35 and 42 days of age, and enhance significantly vitality% and productive index at 35 and 42 days of age (P≤0.05). Table 4 showed that the in-ovo injection with sodium selenite SS improved the antioxidant status as represented by the significant reduction in MDA and the significant increase in TAC as compared with other treatments. Whereas the addition of SS to the drinking water of hatched chicks reduced the MDA significantly at (P≤0.05). For the effects of interaction, the in-ovo injection with SS and the addition of SS to the drinking water of non-injected eggs (negative control) and to chicks hatched from D.W. in-ovo    Table 5 showed that in-ovo injection with SS significantly increased leptin hormone values as compared with the negative and positive control groups, whereas the in-ovo injection with D.W. significantly increased corticosterone values as compared with the negative control and SS injection (P≤0.05). Also, the addition of SS to drinking water after hatching (continuous treatment) significantly increased leptin and T 4 values as compared with the discontinued group. For interaction effects, the in-ovo injection with SS and the after hatching treatment with SS with drinking water showed the highest values in leptin and T 3 , whereas the chicks hatched from in-ovo injected eggs with D.W. showed the highest corticosterone values regardless of the after hatching treatment with SS. On the other hand, the chicks that hatched from SS in-ovo injected eggs and the chicks that hatched from non-injected eggs (negative control) and those that hatched from D.W. in-ovo injected eggs (positive control) and treated with SS after hatching recorded the highest T 4 values at (P≤0.05).
The results agreed with Ghazalah et al. (2020) if it was noticed that the addition of selenium led to a significant increase in the concentration of thyroxine hormone in the treatment of adding sodium selenite. The results did not agree with Choupani et al. (2014) if it was noticed that the addition of selenium led to a significant increase in the concentration of T3 hormone in the treatment of adding selenium to the broiler diet.
Type 1 of iodothyronine deiodinase, which catalyzes the conversion of T 4 to active T 3 , requires selenium for production (Jianhua et al., 2000). Plasma T 3 concentration is produced by 5¯deiodination of thyroxin in non-thyroidal tissue, particularly the liver and kidney (Beckett et al.,1987). Burk. (2002) Rahawi, Abdul-Majeed, Abdul-Rhaman thyroid hormones and enhancing the assimilation of fat, sugar and proteins which is essential to improve body growth.