Effects of the In ovo Administration of L-Ascorbic Acid on Tissue L-Ascorbic Acid Concentrations, Systemic Inﬂammation, and Tracheal Histomorphology of Ross 708 Broilers Subjected to Elevated Levels of Atmospheric Ammonia †

U.S

L-ascorbic acid is the active form of vitamin C, which exists under physiological conditions as an ascorbate anion [19]. As a reducing agent, L-AA can donate two electrons from the double bond between the second and third carbon atoms to oxidizing species, or oxidants. Therefore, because L-AA releases electrons, it acts as an antioxidant or free radical scavenger [20]. Electrons from the ascorbate anion can also reduce metals, such as copper (Cu) and iron, leading to the formation of super oxides and hydrogen peroxide [21]. Dietary L-AA has been shown to improve the metabolism of calcium (Ca) and the binding capacity of proteins [22]. L-ascorbic acid is also required for the conversion of vitamin D into its metabolite form, which accommodates Ca regulation and the calcification process [23]. The in ovo supplementation of 4 µg of L-AA in broilers has been shown to increase the levels of ash, phosphorus, and Ca in the femur of broilers at 43 days of age (doa) [24]. Additionally, chick-embryo ciliated tracheal organ cultures demonstrated resistance to viral infections after supplementation with L-AA [25]. In humans, supplementary L-AA has resulted in a 15-fold increase in resistance to infectious bronchitis and a 50% reduction in the incidence of respiratory symptoms compared to control treatments [26,27]. These results suggest an immunomodulatory role of L-AA during an infection.
Nitrogen is excreted as uric acid (80%), ammonia (NH 3 ; 10%), and urea (5%). The conversion to NH 3 is accomplished by microbial degradation and several microbial enzymes in manure [28,29]. Uric acid is the major source of NH 3 formation in the ceca of poultry [30][31][32]. Ammonia is alkaline, corrosive, and recognized as the most abundant toxic gas in poultry houses [33]. The production of NH 3 is also affected by the ventilation, temperature, moisture, and pH of litter [34]. Numerous studies have reported the effects of NH 3 exposure on broiler performance [35][36][37]. Broilers subjected to various atmospheric NH 3 levels have experienced damage to their respiratory system [36,37], which has been characterized by a loss of tracheal cilia and histopathological changes to the tracheal epithelium [35,38]. Oyetunde et al. [39] demonstrated that exposure to 100 ppm of NH 3 for 4 weeks resulted in deciliation of the epithelium of the upper portion of the trachea in broilers. Studies by Al-Mashhadani and Beck [40] and Ritz et al. [41] have also shown deciliation and epithelial hyperplasia in the trachea of birds exposed to elevated levels of NH 3 . Broilers exposed to 50 ppm of NH 3 for 1 to 4 weeks experienced a drop in the number of cilia in their trachea and lungs and an increase in various inflammatory factors throughout their bodies [39][40][41]. Broilers exposed to 25 ppm of NH 3 for 6 weeks can experience mucosal stimulation [42]. In addition, exposing broilers to 60 to 100 ppm of aerial NH 3 for an approximate 4-week period can cause respiratory inflammatory reactions, which include tracheitis [43] and airsacculitis [44,45]. These findings indicate that long-term exposure to NH 3 has negative effects on tracheal histomorphology and causes a severe inflammatory response in the respiratory system of broilers.
The long-term exposure to NH 3 has been shown to result in severe ocular abnormalities and infection due to an increase in oxidative reaction and inflammation [46]. Other associated physiological effects include severe ocular abnormalities [46] and irritation of mucous membranes, leading to corneal ulceration and tracheitis [43,47]. Shi et al. [48] also showed that exposing broilers for 6 weeks to aerial NH 3 concentrations between 19.5 and 45.5 ppm caused abnormalities in their tracheal immune response. L-ascorbic acid is considered an antioxidant agent, and it is highly involved in the reduction in systemic inflammation. In addition, our laboratory found that male broilers have higher L-AA concentration as compared to female at hatch [7]. In ovo supplementation of L-AA showed promising but inconsistent results in the live performance of broilers exposed to elevated aerial NH 3 concentrations [49]. However, the physiological and morphological mechanisms involved in the recovery of broilers from the negative effects of atmospheric NH 3 in response to the in ovo administration of L-AA are not well understood. Therefore, the objectives of the study reported herein included a determination of the effects of in ovo injection of L-AA on the plasma concentrations of nitric oxide (NO), Ca, and the trace minerals copper Cu and zinc (Zn) in Ross 708 broilers subjected to elevated levels of atmospheric NH 3 . Other objectives included a determination of tissue L-AA concentrations and tracheal histomorphology of these same birds. Because NH 3 is associated with numerous negative impacts on bird performance, normal tracheal histology, and vision, we hypothesized that L-AA supplementation would help alleviate these detrimental effects.

Egg Incubation
Broiler hatching eggs were collected from 35-week-old commercial Ross 708 broiler breeder hens and stored at 18 • C for 24 h. A total of 65 eggs were randomly set in each of the 4 pre-assigned treatment groups on each of the 6 replicate tray levels (1560 total eggs) in a Chick Master single-stage incubator (Chick Master Incubator Company, Medina, OH, USA) set at 37.5 • C dry bulb and 29.0 • C wet bulb temperatures. Treatment group placement was randomized on each level to avoid any positional effects in the incubator. Air temperature and relative humidity were monitored according to the method described by Fatemi et al. [5]. At 12 and 17 doi, all eggs were candled to remove infertile eggs and those that did not contain live embryos.
At 17 doi, using 100 µL solution volumes, each pre-specified treatment was freshly prepared immediately prior to injection in accordance with procedures described by Zhang et al. [4]. The 4 pre-specified treatments included 1) non-injected control; 2) injection of 100 µL of physiological (0.85% NaCl) saline (sham injection control); 3) injection of 100 µL of saline containing 12 mg of L-AA (L-AA 12); or 4) injection of 100 µL of saline containing 25 mg of L-AA (L-AA 25). Approximately 350 eggs per each pre-specified treatment were injected using a Zoetis Inovoject ® m (Zoetis Animal Health, Research Triangle Park, NC, USA) multi-egg injection machine. The injection needle parameters, as described by Zhai et al. [50], allowed for an injection depth of 2.49 cm originating at the large end of the egg to target the amnion [51,52]. The pre-specified concentrations of L-AA were initially dissolved in saline in each injector infusion bag (400 mL total volume) and were prepared according to the method described by Zhang et al. [4]. Our previous laboratory findings revealed that the L-AA used was safe and resulted in a positive effect on hatching chick quality, with no negative impacts on the hatchability of injected live embryonated eggs (HI) and hatchling body weight (BW) [7]. After in ovo injection at 17 doi, all live embryonated eggs were subsequently assigned hatcher basket positions that corresponded to their arrangements in the setter.

Posthatch Experimental Design and Atmospheric Ammonia Exposure
Hatchlings belonging to the same treatment were pooled across replicate units. From the pool of chicks belonging to a common treatment, 12 male chicks were randomly assigned to individual battery cages for each treatment within each of the 12 replicate blocks. All cages were housed in a common room of a grow-out facility. To eliminate the possibility of the release of additional atmospheric NH 3 from used litter, birds were placed in suspended battery cages with feces collected in underlying pans that were cleaned daily. Moderate and constant ventilation was available in the room to further simulate an industry-like environment. Throughout the 5-week period, the broilers were exposed to atmospheric NH 3 according to procedures described in detail in previous studies [49,53]. The average NH 3 concentration in the battery room approximated the designated level of 50 ppm. The recorded NH 3 levels ranged between 42.5 and 49.7 ppm.

Eye and Liver L-Ascorbic Acid Concentrations
Our laboratory previously found that ocular concentrations of L-AA were significantly higher in male compared to female broiler hatchlings [7]. Additionally, in comparison to saline-and non-injected treatment groups, the L-AA 12 treatment increased ocular L-AA concentrations only in male broilers at 14 doa when they were raised under normal conditions. Therefore, only male broilers were used in the current study. At 0 doa, prior to the assignment of male chicks to battery cages, 2 were randomly selected from each pool for the sampling of both eyes and a liver lobe to determine their L-AA concentrations. Furthermore, at 7, 14, 21, and 28 doa, both eyes and a liver lobe of 2 birds from each replicate battery cage were sampled for determination of their L-AA concentrations. The same lobe of the liver was taken from each bird. The liver and eye L-AA concentrations were measured according to the method described by Mousstaaid et al. [6,7].

Plasma NO, Calcium, and Trace Mineral Analyses
For systemic inflammatory response evaluation, plasma NO concentrations were determined at 0, 14, 21, and 28 doa. Moreover, plasma Ca, Cu, and Zn concentrations were determined at 0 and 21 doa. For all analyses, 12 birds per treatment (1 male bird per treatment replicate cage) were randomly selected to be individually weighed and bled by venipuncture of the wing brachial vein. All blood samples were collected into K2 EDTA tubes (Fisher Scientific, Hampton, NH, USA), and plasma was extracted by centrifugation (3000× g for 15 min at 4 • C). Approximately 0.5 mL of the plasma was stored at −20 • C in plastic microcentrifuge tubes for NO and trace mineral analysis. Plasma NO concentrations were determined using the method described by Bowen et al. [54]. The concentrations of Ca, Cu, and Zn were analyzed using ICP-MS. The analysis included use of 5 mL of trace metal nitric acid in microwave digestion tubes with 2 mL of 30% hydrogen peroxide as a digestion reagent. Mineral analyses were conducted at the Mississippi State University Chemical Laboratory, as previously described by Laur et al. [55].

Statistical Analysis
The experimental design was a randomized complete block. Individual birds served as the unit of treatment replication for the eye and liver L-AA concentrations at 0 doa. Battery cages served as the unit of treatment replication for the eye and liver L-AA concentrations after 0 doa and for all other data at all time periods. A group of battery cages was the blocking factor, with all the in ovo injection treatments being randomly represented in each block. All data within each individual period were separately analyzed by ANOVA using the procedure for linear mixed models (PROC GLIMMIX) of SAS 9.4© [60]. The following model was used for analysis of the data: where µ was the population mean; Bi was the replicate or block factor (i = 1 to 12); Tj was the effect of each in ovo injection treatment (j = 1 to 4); and Eij was the residual error. Means separations were performed by Fisher's protected least significant difference [61]. Differences were considered significant at p ≤ 0.05.

Results
In a companion study where the same eggs were used [50], it was confirmed that the frequencies of the injections across treatments were 93.4% and 6.6% in the amnion and embryo body proper, respectively.

Concentrations of Eye and Liver L-AA, and Plasma NO, Ca, Cu, and Zn
No significant treatment differences for eye and liver L-AA concentrations were observed at 0, 7, 14, 21, and 28 doa (Table 2), or for NO concentrations at 0, 14, 21, and 28 doa (Table 3). Additionally, no significant treatment differences were observed for plasma concentrations of Ca, Cu, and Zn at 0 and 21 doa (Table 4). Table 2. Effects of treatment (non-injected; saline-injected (saline); saline containing 12 mg of Lascorbic acid (L-AA 12) or 25 mg of L-ascorbic acid (L-AA-25)) on Ross 708 broiler L-AA concentration in the eye and liver from 0 to 28 days of posthatch age (doa).      3 Eggs that were injected with 100 µL saline containing L-AA 12 at 17 doi. 4 Eggs that were injected with 100 µL saline containing L-AA 25 at 17 doi. N = One bird in each of the 12 replicate groups in each treatment-doa combination was used for means calculations.

Tracheal Histomorphology
Only those treatment differences that were significant are noted. At 0 doa, the L-AA 12 and 25 mg in ovo injected treatment groups had a lower mean score for the level of tracheal attenuation compared with the non-injected and saline-injected controls. Conversely, at 21 doa, the L-AA 12, L-AA 25, and saline-injected groups had a higher mean score for the level of tracheal attenuation in comparison to the non-injected group (Table 5). At 0 doa, score 1 incidence for tracheal attenuation and score 2 incidence for tracheal ulceration were significantly lower for birds in the saline-injected, L-AA 12, and L-AA 25 treatments in comparison to those in the non-injected treatment group (Table 6). At 21 doa, score 1 incidence for tracheal attenuation was observed to be lower in the saline control and L-AA 12 and L-AA 25 treatment groups in comparison to the non-injected control group. Conversely, score 2 incidences for tracheal attenuation were significantly higher in the saline control, L-AA 12, and L-AA 25 treatment groups compared to the non-injected control group (Table 7). At 28 doa, the L-AA 12 in ovo supplemented group had a higher incidence of 0 scores for tracheal glands than both control groups, with the L-AA 25 group being intermediate. Score 1 incidence for inflammation was lower in the L-AA 12 treatment in comparison to the L-AA 25 and non-injected and saline-injected control treatment groups. In addition, the saline control, L-AA 12, and L-AA 25 treatment groups had lower score 3 incidence for tracheal attenuation compared to the non-injected control group (Table 8). Table 5. Effects of treatment (non-injected; saline-injected (saline); saline containing 12 mg of Lascorbic acid (L-AA 12) or 25 mg of L-ascorbic acid (L-AA 25) administered at 17 days of incubation (doi)) on mean score 1 for various tracheal histomorphological variables (cilial erosion, glands, inflammation, attenuation, and ulceration) at 0, 21, and 28 days of posthatch age (doa).

Discussion
The aim of the current study was to determine the effects of the in ovo administration of various amounts of L-AA on eye and liver L-AA concentrations, plasma NO and mineral concentrations, and tracheal histomorphology in broilers exposed to 50 ppm of atmospheric NH 3 from 0 to 28 doa. The effects of feed additives, including various enzymes, minerals, and vitamins, on trachea histomorphology, eye and liver L-AA concentrations, and plasma NO concentrations in chickens and livestock subjected to elevated atmospheric NH 3 levels have not been previously investigated. It was hypothesized that the resistance of developing broilers to the negative physiological impacts caused by high atmospheric NH 3 levels in the posthatch period would be improved by an earlier exposure to supplemental L-AA through in ovo injection. However, no significant treatment differences were observed for plasma NO, Ca, Cu, or Zn concentrations, or eye and liver L-AA concentrations of the birds at any of the designated posthatch time periods while they were exposed to elevated atmospheric levels of NH 3 . Because the in ovo L-AA treatments employed had no effect on the liver or eye L-AA concentrations of the birds exposed to elevated aerial NH 3 levels, it is suggested that any noted effects of treatment were not mediated by increased concentrations of L-AA in those tissues.
Nitric oxide is of paramount importance as a mediator of inflammatory responses and is produced by the oxidation of L-arginine by NO synthase. The inducible NO synthase enzyme is important for immunity and inflammation [62]. The expression of cytokines, such as IL-1β, IL-12, interferon-γ, and tumor necrosis factor, increase the production of NO [63], and the production of NO, as well as other inflammatory cytokines (TNF-α, IL-6, IL-1β, and TLR-2A), has been shown to increase in the bursa of Fabricius of broilers exposed long-term to atmospheric NH 3 levels [64]. Cytokines play an important role in the course of an inflammatory response [65], and the overproduction of molecules, such as IL-1β and IL-6, which are potent proinflammatory and immunomodulatory cytokines [66], could have potentially harmful effects on feed efficiency and growth in different species [67,68]. These findings also demonstrated that the production of NO and other inflammatory cytokines increases in sites of infection, such as the bursa of Fabricius. Nevertheless, in the current study, no changes in plasma NO concentration were observed at any of the time periods investigated. This would indicate that no systemic inflammation occurred in response to the subjection of the birds to 50 ppm of atmospheric NH 3 . Further study is needed to determine whether NO production as well as other inflammatory indicators occur in the trachea, where some levels of infection were observed in broilers subjected to chronic elevations of aerial NH 3 .
Although systemic inflammation was apparently not significantly affected by treatment, the trachea histomorphology results revealed that the in ovo injection of 12 mg of L-AA appeared to be associated with reduced tracheal inflammation at 28 doa compared to the histomorphology of birds receiving control treatments. Therefore, the current results suggest that the in ovo injection of 12 mg of L-AA may attenuate an inflammatory response after chronic exposure to high atmospheric NH 3 levels. The submucosa of the tracheal wall contains mucous glands, which secrete mucus to facilitate the entrapment of particles, and cilia (tiny hairlike structures present on the surface of tracheal epithelial cells), which propel entrapped particles for disposal. Tracheal cilia, therefore, assist in the removal of potentially harmful fluids and particles from the airways and lungs [58,59]. When erosion occurs, the ciliary function is impaired, and epithelial hypoplasia occurs [69]. In affected areas, a process known as attenuation causes ciliated columnar epithelial cells to appear abnormally shortened in height with multiple defects, including ulceration [59]. Birds in the L-AA 12 and 25 in ovo injection treatment groups exhibited less epithelial cell attenuation at 0 doa compared to birds in the non-injected and saline-injected control groups and at 21 doa compared to those in the non-injected control group. Birds that received the L-AA 12 or 25 treatments also had fewer sites of ulceration in the trachea and fewer areas of open mucous membranes at 0 doa compared to the non-injected control treatment. A reduction in inflammation incidence and the presence of tracheal lymphocytes were also observed in birds belonging to the L-AA 12 treatment group alone. Therefore, the L-AA in ovo injection treatment promoted the establishment of these ciliated epithelial cells and could thereby improve the tracheal function.
In the immune system, the major role of L-AA as a physiological antioxidant is to protect host cells against oxidative stress caused by infections [70]. It is well documented that NH 3 exposure is highly associated with systematic and local oxidative stress [71]. The exposure of broilers to atmospheric NH 3 has been shown to result in an increase in the concentrations of free radical indicators, including gamma-glutamyl transferase, malondialdehyde (MDA), and hydrogen peroxide, and a decrease in the antioxidant enzymatic activities of catalase, glutathione, and glutathione peroxidase in the bursa of Fabricius [71]. In addition, aerial NH 3 is linked to impaired immune function, where the numbers of CD8+ T-lymphocytes and the activity of adenosine triphosphate are reduced in broilers subjected long-term to elevated atmospheric NH 3 concentrations [72]. Furthermore, an increase in antioxidant activity is linked to a reduction in systematic and local oxidative stress [73,74]. Therefore, an increase in antioxidant activity leading to an improvement in local immunity may be beneficial during chronic exposure to elevated atmospheric NH 3 levels.
In our laboratory, the in ovo injection of 12 and 25 mg of L-AA was previously found to improve the enzymatic (serum superoxide dismutase concentrations) and non-enzymatic (MDA) activities in broilers under normal conditions [8]. In the companion study, broilers that received 12 mg of in ovo injected L-AA exhibited lower incidence of corneal erosion and higher gain of body weight between 0 and 28 doa compared to other treatment groups while being chronically exposed to elevated atmospheric NH 3 [49]. Therefore, improved ocular and trachea histomorphological features could be partially linked to increased systemic and local antioxidant activities in broilers chronically exposed to elevated atmospheric NH 3 . However, further research is needed to determine the effects of various levels of in ovo injected L-AA on the enzymatic and non-enzymatic antioxidant capacities of birds in response to elevated aerial NH 3 concentrations.
Previous studies have reported a decrease in the concentrations of plasma trace minerals, such as sodium (Na+) and chloride (Cl-), in broilers subjected to 50 ppm of aerial NH 3 [47]. However, dietary supplementation of L-AA at 250 mg/kg has been reported to have no significant effect on the plasma levels of Ca, Cu, and Zn in broilers experiencing oxidative stress induced by Cu toxicity [75]. Furthermore, the plasma levels of Cu have not been determined in chickens provided L-AA supplementation in the diet or by in ovo injection while housed under commercial conditions. In addition to these previous reports, the plasma minerals measured in this study were not affected by the levels of L-AA that were administered by in ovo injection while the birds were being continually exposed to elevated atmospheric NH 3 levels. Hence, the treatment effects noted for the broilers in this study did not involve changes in their plasma levels of Ca, Cu, or Zn.

Conclusions
In conclusion, the results of this study showed that the in ovo injection of 12 or 25 mg of L-AA did not significantly affect the plasma NO and mineral concentrations, as well as the tissue L-AA concentrations, of the broilers between 0 and 28 doa. However, the in ovo injection of 12 mg of L-AA was associated with decreased inflammation and improved histologic changes of the trachea of the birds while they were exposed to elevated atmospheric levels of NH 3 . The promising results concerning tracheal histomorphology could be linked to the antioxidant and immunomodulatory activities of L-AA, which have been observed in previous studies. Because the beneficial results reported in earlier studies were observed in birds housed under normal conditions, further studies should continue to explore the potential modulatory influences of other levels of supplemental L-AA administered by in ovo injection on tracheal histomorphology, local antioxidant and immunomodulatory activities, and the systemic and local inflammatory responses of broilers chronically subjected to high levels of aerial NH 3 .