Effects of in ovo administration of L-ascorbic acid on broiler hatchability and its influence on the effects of pre-placement holding time on broiler quality characteristics,,

Effects of in ovo administration of L-ascorbic acid on broiler hatchability and its influence on... ABSTRACT The current study was conducted to determine the possible effects of the in ovo administration of different dosages of L-ascorbic acid (AA) to broiler hatching eggs on hatchability and its potential for reducing the adverse effects of delayed placement. A total of 702 broiler hatching eggs was hand-injected at 17 d of incubation (DOI) with 100 μL of sterile saline (0.85%) alone or containing 0.5, 1.5, 4.5, or 13.5 mg AA. Hatchability was recorded every 5 h from 480 h to 505 hours. Results showed that AA injection did not affect embryo BW as percentage of set egg weight or yolk sac weight as percentage of embryo weight at 19.5 DOI. The hatching time of all embryos that received an AA in ovo injection was between 480 and 495 h of incubation, and significantly fewer embryos hatched before 480 h in comparison to non-injected controls. Hatchability (above 92% in all groups) was not significantly affected by injection treatment. However, fertile eggs injected with saline containing 4.5 mg AA had the highest hatchability. At 21 DOI, hatching BW as a percentage of set egg weight and yolk sac weight as a percentage of BW were numerically higher in AA injection groups. An in ovo injection of AA at a 13.5 mg/egg level resulted in a numerically higher BW as a percentage of set egg weight. The in ovo injection of AA did not reduce the adverse effects of a 48-hour posthatch pre-placement holding time on BW or on yolk sac absorption. Overall, in ovo injection of L-ascorbic acid (0.5 to 13.5 mg/egg) into fertile broiler hatching eggs at 17 DOI did not negatively affect hatchability or embryo development, and did not prevent the negative effects of a 48-hour posthatch holding time on BW and yolk sac absorption. The range of tolerance as well as the optimal dosage of in ovo-injected AA warrants future study. INTRODUCTION The in ovo injection of vitamins is a potential means by which to improve the hatchability and postnatal growth of broiler chickens (Keralapurath et al., 2010; Bello et al., 2013; Li et al., 2016). Ascorbic acid (AA), a water-soluble vitamin with multiple biochemical functions, has been shown to benefit avian embryonic development during incubation in a dose-dependent manner. Zakaria and Al-Anezi (1996) reported that injecting AA at a dose of 3 mg per egg at 11 and 15 d of incubation (DOI) improved hatchability and embryo BW, and decreased the number of culled chicks, whereas a dose of 12 mg per egg decreased embryo BW and hatchability, and increased embryonic mortality and the number of culled chicks. Ipek et al. (2004) observed that injecting 3 mg of AA into the air cell of individual eggs at 13 DOI increased hatchability (83.5 vs. 76.2%) and decreased embryo death between 18 and 21 DOI. Similarly, Elibol et al. (2001) reported that the injection of 3 mg of AA in large broiler eggs (laid by 64-week-old breeder hens) at 13 DOI reduced late term embryo mortality. Furthermore, it was reported that dipping eggs into a solution containing 10 g of AA per liter for a period of 2 min increased the hatchability of eggs from hens that were 29 wk of age (Shafey, 2002). These studies suggested that the in ovo administration of AA by injection into the air cell in the early or middle phase of incubation (before 17 DOI) may be beneficial to embryos. Research has shown that the amnion and embryo body are optimal sites for vaccine delivery (Avakian, 2006). Similarly, the amnion is an ideal site for the in ovo administration of nutrients for late pre-hatch birds (Kadam et al., 2013), and it has the potential for industrial application using commercial multi-egg injection systems in broiler hatcheries. However, little is known concerning the effects of the in ovo administration of AA via the amnion during late incubation. The use of AA has been shown to reduce heat stress in broilers exposed to high ambient temperatures (Mckee et al., 1997; Mahmoud et al., 2004; Rafiee et al., 2016). Chick embryos may be subjected to heat stress by the excessive production of metabolic and environmental heat during the later hatching phase (Tullett, 1990). Thus, the injection of AA during incubation may be beneficial in combating embryonic stress. A hot environmental incubation temperature may result in an antioxidant-oxidant imbalance during the late phase of incubation; therefore, it is hypothesized that exogenous supplementation of nutrients with antioxidant activities to embryos may be of benefit to their development. After hatching, modern broiler chicks are commonly transported to different grow-out locations, and may undergo fasting for the first 36 to 72 h post hatch. The capability of the in ovo injection of AA during the later incubation phase to reduce the adverse effects of early fasting is unknown. Thus, the current study was performed with the goal to investigate the possible influences of AA injection into the amnion during incubation on hatchability and on the effects of early fasting on broiler BW. MATERIALS AND METHODS Incubation The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee of Mississippi State University (IACUC-17–406). Four cases of fertile broiler eggs from Ross 708 breeder hens of 35 wk of age were obtained from a commercial source and were stored in an egg storage facility under commercial conditions (12.8°C and 10.4°C dry and wet bulb temperatures, respectively) for 48 hours. The eggs were warmed to room temperature (23.9°C dry bulb) for 4 h before being set. The average weight of the eggs was 60.08 g, with a range of 56 to 64 g. A total of 756 eggs was set and incubated under standard conditions (37.5°C and 29.4°C dry and wet bulb temperatures, respectively) in a Jamesway model PS 500 setter unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada). Forty-two eggs were randomly assigned to each of 6 treatments on each of 3 middle tray levels (replicate units) in the setter. To avoid a positional effect on treatment group placement in the setter, the arrangements of the treatments on each tray level were randomized with respect to each other. The eggs were weighed on a batch tray weight basis at set, and at 10 and 17 DOI. In addition, 7 eggs on each tray were marked and weighed individually on d of set. Embryos from marked eggs were sampled at 19.5 DOI. All eggs were candled at 10 and 17 DOI and any clear eggs were broken open to identify eggs as being either infertile or containing embryos that died at the early or middle stages of incubation. Treatment Solutions L-ascorbic acid (A92902, Sigma-Aldrich Inc., St. Louis, MO) solutions were freshly prepared and sterilized in a dark environment (due to photosensitivity) immediately before injection. Solution sterilization was accomplished by filtration through a 0.2 μm syringe filter (PTFE, 25 mm, Scientific Strategies, Yukon, OK). Injection Procedure After eggs were candled and weighed at 17 DOI, a total of 585 fertile eggs taken from 15 setter trays (except for 117 eggs on 3 trays in the non-injected control treatment) was in ovo-injected with one of the 5 solutions: 0.1 mL saline (0.85% NaCl)-injected control, and 0.1 mL saline containing 0.5, 1.5, 4.5, or 13.5 mg AA. The saline and vitamin solutions were injected into the large end of the egg using a 2.54 cm, 22-gauge needle, possessing a short beveled tip. The entire length of the needle was inserted into the large end of the hatching egg to ensure that the needle adequately penetrated the amnion. In accordance with commercial application, the injection site remained open and was not sealed. After in ovo injection at 17 DOI, all live embryonated eggs were subsequently transferred to a Jamesway model PS 500 hatcher unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada). Eggs in their respective treatment replicate groups were assigned hatcher basket positions that corresponded to their arrangements in the setter. Data Collection At 19.5 DOI, 5 embryos from each of the 6 treatment groups on each of 3 replicate tray levels were selected, weighed, and then sexed by gonaded observation. Individual fertile egg weight, embryo BW (yolk included), and yolk sac weight (YSW) were recorded, and their weights as percentages of set egg weight (SEW) (BW/SEW × 100, YSW/SEW × 100, respectively) were calculated. Every 5 h, beginning at 480 h (20 DOI), and ending at 500 h, the cumulative number of hatched chicks was recorded. On d of hatch (500 h of incubation), all chicks were counted, and all residue eggs were opened to record stages of embryonic mortality. Chicks were feather sexed, and 6 male and 6 female chicks from each treatment (2 males and 2 females from each original tray) were sacrificed by cervical dislocation, weighed individually, and their yolk sacs were removed and weighed. Body weight as a percentage of SEW, and YSW as a percentage of BW were calculated. After sex determination and sampling on d of hatch, chicks were kept in their hatching baskets (males and females separated) for an additional 48 h at 33.0°C and 26.5°C dry and wet bulb temperatures, respectively. After the 48 h of holding period without access to feed and water, mean BW of the male and female chicks in each treatment replicate tray was determined. Subsequently, 2 male and 2 female chicks from each treatment replicate tray were randomly selected, and their BW and yolk sac weights were determined. Subsequently, BW loss during the 48-hour holding period, and BW as a percentage of SEW, and YSW as a percentage of BW were calculated. Statistical Analysis Data were analyzed using one-way ANOVA in a completely randomized block design using the mixed model of the SAS system (version 9.4; SAS Institute, 2013). The in ovo injection treatment was considered as a fixed effect, and incubator tray level (block) was considered as a random effect. Percentage data greater than 80 or less than 20 were arcsine transformed before analysis. The dose-related effect of supplemental AA was computed by GLM in the absence of the non-injected control group using contrast command for linear and quadratic effects. Because no sex effects existed, the data from each sex were pooled within treatment replicate unit. The results are presented as mean ± SEM. Differences were considered statistically significant at P ≤ 0.05. RESULTS Embryonic Characteristics at 19.5 Doi Mean SEW and the various embryonic characteristics at 19.5 DOI are shown in Table 1. There were no significant treatment effects on SEW or on egg weight, embryo weight, yolk sac free embryo BW, YSW, or embryo and YSW as a percentage of SEW within each of the 6 treatment groups. Table 1. Effects of in ovo injection of L–ascorbic acid on embryonic characteristics at 19.5 d of incubation.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78  1P-value of all the groups. 2P-value of all the injection groups. N = 3; 5 observations were pooled within each replicated tray; values are presented as means ± SE. View Large Table 1. Effects of in ovo injection of L–ascorbic acid on embryonic characteristics at 19.5 d of incubation.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78  1P-value of all the groups. 2P-value of all the injection groups. N = 3; 5 observations were pooled within each replicated tray; values are presented as means ± SE. View Large Hatching Time of Broiler Chicks Hatchability of fertile eggs in each treatment group every 5 h between 480 and 500 h of incubation is shown in Figure 1. Before 480 h (20 DOI), numerically more chicks hatched out in the non-injected control group than in the injection treatment groups, and numerically more broilers hatched out in the 485- to 495-hour period in the injection treatment groups in comparison to the non-injected control group. Nevertheless, hatchability was not significantly affected by injection treatment, with hatchability among all treatment groups ranging from 92 to 96%, which is in agreement with the production guidelines for Ross 708 broiler breeder females (Aviagen, 2016). Figure 1. View largeDownload slide Effects of in ovo injection of L-ascorbic acid (0.5 to 13.5 mg/egg) on hatching time characteristics. Figure 1. View largeDownload slide Effects of in ovo injection of L-ascorbic acid (0.5 to 13.5 mg/egg) on hatching time characteristics. Hatching Characteristics and Chick Quality at 21 Doi Effects of the in ovo injection of AA on hatchability and embryonic mortality, and chick quality at 21 DOI are shown in Table 2. There were no statistical differences between treatment groups for hatchability, dead chicks, dead embryos, or for percentages of live pipped (chicks that may have potential to hatch given a longer hatching time) and dead pipped chicks after the 500-hour incubation period. The results showed that embryonic mortality during the 17 to 21 DOI period ranged from 0.85 to 5.32%. In addition, the highest level of AA injection (13.5 mg/chick) did not cause an increase in late mortality. Table 2. Effects of in ovo injection of L –ascorbic acid on hatching characteristics and chick quality at 21 d of incubation (hatching day).   Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40    Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40  1P-value of all the groups. 2P-value of all the injection groups. 3Hatchability: Hatched chicks as a percentage of total fertile eggs. 4Live pipped: Live embryos in the pipping phase, those that may have remained alive as chicks during continuation of the hatching phase or were assisted by manual shell removal. 5Dead pipped: Embryos that died in the pipping phase. 6Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. For hatching characteristics, calculation was based on a replicate tray basis with 39 fertile eggs per tray. View Large Table 2. Effects of in ovo injection of L –ascorbic acid on hatching characteristics and chick quality at 21 d of incubation (hatching day).   Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40    Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40  1P-value of all the groups. 2P-value of all the injection groups. 3Hatchability: Hatched chicks as a percentage of total fertile eggs. 4Live pipped: Live embryos in the pipping phase, those that may have remained alive as chicks during continuation of the hatching phase or were assisted by manual shell removal. 5Dead pipped: Embryos that died in the pipping phase. 6Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. For hatching characteristics, calculation was based on a replicate tray basis with 39 fertile eggs per tray. View Large For chick quality at 21 DOI, the in ovo injection of AA into hatching eggs at 17 DOI did not affect hatching BW across sex, or residual YSW and YSW as a percentage of BW. Chick BW relative to SEW also did not differ among treatments. Chick Bw, Yolk Sac Weight, and Bw Loss after 48-Hour Fast Effects of the in ovo injection of AA on chick BW, and YSW and BW losses after 48 h of holding time in the hatchery are provided in Table 3. There were no significant treatment differences for chick BW across sex, absolute or percentage YSW, or for absolute or percentage BW loss. Table 3. Effects of in ovo injection of L-ascorbic acid on chick body weight, yolk sac weight, and BW loss after 48-hour posthatch fast.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90  1P-value of all the groups. 2P-value of all the injection groups. 3Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. View Large Table 3. Effects of in ovo injection of L-ascorbic acid on chick body weight, yolk sac weight, and BW loss after 48-hour posthatch fast.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90  1P-value of all the groups. 2P-value of all the injection groups. 3Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. View Large DISCUSSION Background Fertile eggs do not contain detectable levels of AA. During embryonic development, AA concentration in the brain is known to rise after d 6 and increase to 5.6 nmol/mg of tissue by d 10. This level is maintained at a relatively high level between 8 and 18 DOI, and then gradually declines to 32% immediately before hatch (Wilson, 1990; Wilson and Jaworski, 1992). Plasma AA concentration is known to peak on d 12 of embryonic development, and then falls before increasing again on d 20, when pulmonary respiration begins (Wilson and Jaworski, 1992). The dynamic changes in AA levels of embryos suggest the importance of AA for optimal embryonic development in the late incubation phase. Ascorbic acid has been reported to combat heat stress in the later phases of incubation and to decrease mortality and improve hatchability (Zakaria and Al-Anezi, 1996). In this study, effects of different dosages of AA delivered by in ovo injection during the late incubation phase were investigated. However, no changes in the embryonic characteristics examined at 19.5 DOI occurred. The results suggested that the in ovo injection of AA did not negatively affect the growth of embryos 2.5 d post injection. Hatchability In the present study, no difference in the hatchability of fertile eggs was observed among treatment groups, which indicated that the injection process and the injection solution were safe for the embryos and did not interrupt the hatching process. However, an improvement in hatchability using the described treatment dosages of AA was not observed. This is in agreement with the results of earlier studies (Nowaczewski et al., 2012; Hajati et al., 2014). Nowaczewski et al. (2012) observed no increase in hatchability by the in ovo injection of 3 to 6 mg of AA in fertile eggs. However, the highest rate of hatchability of fertilized eggs was noticed in the group injected with 6 mg of AA at 15 DOI. In contrast to the current study, some studies have indicated that 3 mg AA/egg reduced late-term embryo mortality and increased hatchability. (Elibol et al., 2001; Ipek et al., 2004). The observed diverse results may be explained by the differences in injection time and the number of fertile eggs examined. The site of injection and the stage of embryonic development when injection is administered influences the effect of AA on the embryo. It has been reported that the injection of 1 μg of AA in the albumen before incubation increased hatching rate by 14.2% in comparison to a control treatment (Iglesias et al., 2015). Injection of AA at a 3 mg/egg level improved hatchability when injected on d 11 and 15 of incubation (Zakaria and Al-Anezi, 1996; Zakaria et al., 1998; Zakaria, 2001). It appears that the window that is beneficial for maximizing the effect of the in ovo injection of AA on hatchability may occur in the middle or later phases of incubation. This, however, also might be achieved after elucidating the dynamic AA status or requirement of the embryo across an entire incubation phase. Generally, fertile eggs from breeder layers during peak production exhibit a higher level of hatchability, and conversely eggs laid by old breeders have lower hatching percentages (Almeida et al., 2008). In the current study, hatchability in the non-injected group was more than 90%. Therefore, it is possible that there was little room for an improvement in hatchability. Efforts made to investigate possible differences in the AA concentrations of fertile eggs from breeder hens at different ages may help to better determine the optimal timing and dosage of injected AA that is necessary to improve hatchability. Time of Hatch In contrast to the results of Zakaria et al. (1998), AA injection did not lead to a decrease in the time of hatch in the current study. This may be due to differences in incubator type and condition as well as the type of fertile eggs used. Hatching time was recorded through 500 h of incubation in the present study, while that of Zakaria et al. (1998) started at 498 h and ended at 528 h of incubation, with an additional record made at 552 h of incubation. In addition, average chick BW in the study of Zakaria et al. (1998) was approximately 1.5 kg at 49 d of age, which was far below that of modern broilers at 42 d of age (2.5 kg). Therefore, differences in the length of time in which hatch time was recorded as well differences in the bird strains used in the 2 studies may account for these contrasting results. Whether or not the in ovo injection of AA affects hatching time remains unclear. The relevance of supplemental AA on hormone secretion also would be an important factor to consider. It has been suggested that AA administration may affect stress hormone levels (Kutlu and Forbes, 1994; Mckee and Harrison, 1995). Specifically, there is an abrupt increase in circulating triiodothyronine levels before internal pipping and the subsequent transformation to pulmonary respiration (Kuhn et al., 1993). It would be worthwhile to investigate whether or not the in ovo injection of AA may affect various circulating hormone concentrations during the later phase of incubation. Relatively more chicks hatching out earlier in the non-injected control group suggests that the injection process may delay hatching to some extent. It has been reported that fertile eggs with a low rate of water loss usually hatch later (Ar and Rahn, 1980; Ar, 1991). However, in the current study, it is suggested that a saline injection volume of 0.1 mL would not affect a change in water loss, and that a holding time of 10 min during the injection process was not long enough to alter the hatching process. Thus, the possible metabolic modulatory effect of AA on the hatching process is expected and warrants further investigation. Yolk Sac During the development of avian embryo, yolk is the sole energy supply (Romanoff, 1960). Noble and Cocchi (1990) noted that almost 94% of the total energy needs of the embryo during development are provided by means of the oxidation of fatty acids. In the current study, the in ovo injection of AA at 17 DOI did not induce a change in yolk sac weight at 19.5 DOI, which indicated that AA administration did not affect the absorption or utilization of yolk during the late phase of incubation. After hatch, as the first primary source of nutrients, the yolk sac provides the hatched chick with immediate energy (Romanoff, 1960). In the current study, more than 50% of the loss in BW during the 48-hour holding period was from YSW loss, which indicates the importance of yolk as a vital nutrient source for the newly hatched chicks. The longer the time between hatch and the start of feed and water intake, the greater is the reliance of the chick on these reserves (Vieira and Pophal, 2000). Noy and Sklan (1999) indicated that chicks deprived of food after hatch used approximately 60% of their residual yolk sac in the first 48 hours. Furthermore, an exogenous supply of feed enables or stimulates yolk sac absorption. Iglesias et al. (2015) observed a decrease in YSW at 72 h post hatch in chicks having access to feed. In the current study, chicks were deprived of food and water for 48 h, and the subsequent loss of residual yolk was between 75 and 84%. This suggested that the chicks experienced other stressors in addition to that of food deprivation. In the current study, chicks that were fasted for 48 h lost an average of 6.23 g of weight, which is similar to the 0.14 g/h average reported in the study by Warriss et al. (1992). The absence of a difference in YSW loss between treatment groups in this study indicated that the in ovo injection of AA could not counteract the severe stress induced by 48 h of feed and water deprivation that the broiler chicks experienced after hatch. Delayed Removal from the Incubator Experimental data on the anti-stress effects of in ovo-injected nutrients used to counteract the effects of delayed chick placement are lacking. Zakaria et al. (1998) held hatched chicks in the incubator for 18 to 48 h, and observed that the in ovo injection of 2 mg AA/egg elevated the BW of male chicks at 7 d of age, while no effect was observed in females. However, the current experimental period did not extend into the feeding phase. Nevertheless, the fact that body and yolk sac weight losses were unchanged indicated that the injection of AA during the last phase of incubation was not sufficient to prevent the stress of a 48-hour fast. However, investigations as to the lasting effects of the in ovo injection of AA on early growth in broiler chicks are pending. Tolerance Dosage In the current study, the in ovo injection of high concentrations of AA (13.5 mg/egg) did not lead to increases in late embryo mortality. This result is different from the results observed in the study by Zakaria and Al-Anezi (1996), which showed a dramatic decrease in hatchability occurring in a 12 mg AA/egg injection treatment group. The reason that the embryos can tolerate more than a 12 mg AA per egg exposure in the current study may be associated with the later developmental stage of the embryos. In this present study, the amnion at 17 DOI was the primary site of injection, and the air cell at the large end of the egg in the early or middle phase of incubation (before 17 DOI) has been commonly used in other studies. The embryo being of a larger size may have contributed to its higher tolerance to the dosage of the injected AA. However, high dosages of injected AA may harm the development of the embryo. This was evidenced in a study by Ingram et al. (1997). In that study, AA injections of 0.75 g/mL were toxic and resulted in embryo mortality prior to hatching. In the literature, an effective dosage of AA that was in ovo-injected was shown to range from 3 to 6 mg/egg (Ipek et al., 2004; Zakaria and Al-Anezi 1996; Elibol et al., 2001; Nowaczewski et al., 2012). To date, results of studies determining the effects of AA supplementation in chicken embryos vary considerably. For example, in a study in which an in ovo injection method was applied pre-incubation using a low level of AA (1 μg/egg), hatchability was increased (Iglesias et al., 2015). Additionally, it was observed that dipping Sinai chicken eggs in 15.0 g of AA/L at 12 DOI for 2 or 3 min maximized hatchability and minimized embryonic mortality (Awad and Abd El-Halim, 2015). Due to the diversity and inconsistency of in ovo injection sites and times used in fertile eggs from breeders of different ages, it is unclear as to ascertain the effective dosage of AA or the exact mode of in ovo injection that best supports embryonic growth. However, the current study has demonstrated that the in ovo injection of AA at a concentration of 13.5 mg/egg does not adversely affect the hatchability or quality of broiler hatchlings. In conclusion, the amniotic injection of 0.5 to 13.5 mg of AA at 17 DOI does not affect the embryonic development or hatching rate of broiler chicks, and the injection of AA does not modify the negative effects of a 48-hour posthatch fast on chick body or yolk sac weight loss. The tolerance range and optimal dosage of in ovo injected AA in broiler embryos warrant further study. ACKNOWLEDGMENTS We express our appreciation for the expert technical assistance of Jonathan Moon of the Mississippi State University Poultry Science Department. Footnotes 1 This publication is a contribution of the Mississippi Agricultural and Forestry Experiment Station. 2 This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under accession number 322290. 3 Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. REFERENCES Almeida J. G., Vieira S. L., Reis R. N., Berres J., Barros R., Ferreira A. K., Furtado F. V. F.. 2008. Hatching distribution and embryo mortality of eggs laid by broiler breeders of different ages. Braz. J. Poult. Sci.  10: 89– 96. Ar A. 1991. Egg water movements during incubation. Pages 157– 173 in Avian Incubation . Tullett S. G., ed. Butterworth-Heinemann Ltd., London, UK. Ar A., Rahn H.. 1980. Water in the avian egg: Overall budget of incubation. Am. Zool.  20: 373– 384. 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The effect of in ovo injection of grape seed extract and vitamin C on hatchability, antioxidant activity, yolk sac weight, performance and ileal micro flora of broiler chickens. Res. Opin. Anim. Vet. Sci.  4: 633– 638. Iglesias D. L. P., Sgavioli S., Malheiros E. B., Garcia R. G., Rombola L. G., Caldara F. R., Nääs I. A., Boleli I. C.. 2015. Ascorbic acid in egg injection minimizes the effects of fasting between hatching and housing of broiler chicks. Int. J. Poult. Sci.  14: 387– 393. Google Scholar CrossRef Search ADS   Ingram D. R., Deao C. E., Floyd S. A., Pittman S. T.. 1997. Effects of in-ovo injection of ascorbic acid on broiler hatchability and body weight. Poult. Sci. 86th Annual Meeting Abst.  76(Suppl. 1): 52. Ipek A., Sahan U., YIlmaz B.. 2004. The effect of in ovo ascorbic acid and glucose iniection in broiler breeder eggs on hatchability and chick weight. Archiv für Geflügelkunde  68: 132– 135. Kadam M. M., Barekatain M. R., Bhanja S. K., Iji P. A.. 2013. 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A.. 1996. Effect of ascorbic acid and cooling during egg incubation on hatchability, culling, mortality and the body weights of broiler chickens. Poult. Sci.  75: 1204– 1209. Google Scholar CrossRef Search ADS PubMed  Zakaria A. H., Al-Latif A. A., Al-Anezi M. A.. 1998. Effect of ascorbic acid on embryonic development, hatch time and growth of extended delayed placement of broiler chickens. Arch. Geflugelk.  62: 11– 15. Zakaria A. H. 2001. Effect of ascorbic acid treatment during egg incubation on pre-and post-hatching development of broiler chickens. Damascus Univ. J. Agr. Sci.  17: 118– 130. © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects of in ovo administration of L-ascorbic acid on broiler hatchability and its influence on the effects of pre-placement holding time on broiler quality characteristics,,

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0032-5791
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Abstract

ABSTRACT The current study was conducted to determine the possible effects of the in ovo administration of different dosages of L-ascorbic acid (AA) to broiler hatching eggs on hatchability and its potential for reducing the adverse effects of delayed placement. A total of 702 broiler hatching eggs was hand-injected at 17 d of incubation (DOI) with 100 μL of sterile saline (0.85%) alone or containing 0.5, 1.5, 4.5, or 13.5 mg AA. Hatchability was recorded every 5 h from 480 h to 505 hours. Results showed that AA injection did not affect embryo BW as percentage of set egg weight or yolk sac weight as percentage of embryo weight at 19.5 DOI. The hatching time of all embryos that received an AA in ovo injection was between 480 and 495 h of incubation, and significantly fewer embryos hatched before 480 h in comparison to non-injected controls. Hatchability (above 92% in all groups) was not significantly affected by injection treatment. However, fertile eggs injected with saline containing 4.5 mg AA had the highest hatchability. At 21 DOI, hatching BW as a percentage of set egg weight and yolk sac weight as a percentage of BW were numerically higher in AA injection groups. An in ovo injection of AA at a 13.5 mg/egg level resulted in a numerically higher BW as a percentage of set egg weight. The in ovo injection of AA did not reduce the adverse effects of a 48-hour posthatch pre-placement holding time on BW or on yolk sac absorption. Overall, in ovo injection of L-ascorbic acid (0.5 to 13.5 mg/egg) into fertile broiler hatching eggs at 17 DOI did not negatively affect hatchability or embryo development, and did not prevent the negative effects of a 48-hour posthatch holding time on BW and yolk sac absorption. The range of tolerance as well as the optimal dosage of in ovo-injected AA warrants future study. INTRODUCTION The in ovo injection of vitamins is a potential means by which to improve the hatchability and postnatal growth of broiler chickens (Keralapurath et al., 2010; Bello et al., 2013; Li et al., 2016). Ascorbic acid (AA), a water-soluble vitamin with multiple biochemical functions, has been shown to benefit avian embryonic development during incubation in a dose-dependent manner. Zakaria and Al-Anezi (1996) reported that injecting AA at a dose of 3 mg per egg at 11 and 15 d of incubation (DOI) improved hatchability and embryo BW, and decreased the number of culled chicks, whereas a dose of 12 mg per egg decreased embryo BW and hatchability, and increased embryonic mortality and the number of culled chicks. Ipek et al. (2004) observed that injecting 3 mg of AA into the air cell of individual eggs at 13 DOI increased hatchability (83.5 vs. 76.2%) and decreased embryo death between 18 and 21 DOI. Similarly, Elibol et al. (2001) reported that the injection of 3 mg of AA in large broiler eggs (laid by 64-week-old breeder hens) at 13 DOI reduced late term embryo mortality. Furthermore, it was reported that dipping eggs into a solution containing 10 g of AA per liter for a period of 2 min increased the hatchability of eggs from hens that were 29 wk of age (Shafey, 2002). These studies suggested that the in ovo administration of AA by injection into the air cell in the early or middle phase of incubation (before 17 DOI) may be beneficial to embryos. Research has shown that the amnion and embryo body are optimal sites for vaccine delivery (Avakian, 2006). Similarly, the amnion is an ideal site for the in ovo administration of nutrients for late pre-hatch birds (Kadam et al., 2013), and it has the potential for industrial application using commercial multi-egg injection systems in broiler hatcheries. However, little is known concerning the effects of the in ovo administration of AA via the amnion during late incubation. The use of AA has been shown to reduce heat stress in broilers exposed to high ambient temperatures (Mckee et al., 1997; Mahmoud et al., 2004; Rafiee et al., 2016). Chick embryos may be subjected to heat stress by the excessive production of metabolic and environmental heat during the later hatching phase (Tullett, 1990). Thus, the injection of AA during incubation may be beneficial in combating embryonic stress. A hot environmental incubation temperature may result in an antioxidant-oxidant imbalance during the late phase of incubation; therefore, it is hypothesized that exogenous supplementation of nutrients with antioxidant activities to embryos may be of benefit to their development. After hatching, modern broiler chicks are commonly transported to different grow-out locations, and may undergo fasting for the first 36 to 72 h post hatch. The capability of the in ovo injection of AA during the later incubation phase to reduce the adverse effects of early fasting is unknown. Thus, the current study was performed with the goal to investigate the possible influences of AA injection into the amnion during incubation on hatchability and on the effects of early fasting on broiler BW. MATERIALS AND METHODS Incubation The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee of Mississippi State University (IACUC-17–406). Four cases of fertile broiler eggs from Ross 708 breeder hens of 35 wk of age were obtained from a commercial source and were stored in an egg storage facility under commercial conditions (12.8°C and 10.4°C dry and wet bulb temperatures, respectively) for 48 hours. The eggs were warmed to room temperature (23.9°C dry bulb) for 4 h before being set. The average weight of the eggs was 60.08 g, with a range of 56 to 64 g. A total of 756 eggs was set and incubated under standard conditions (37.5°C and 29.4°C dry and wet bulb temperatures, respectively) in a Jamesway model PS 500 setter unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada). Forty-two eggs were randomly assigned to each of 6 treatments on each of 3 middle tray levels (replicate units) in the setter. To avoid a positional effect on treatment group placement in the setter, the arrangements of the treatments on each tray level were randomized with respect to each other. The eggs were weighed on a batch tray weight basis at set, and at 10 and 17 DOI. In addition, 7 eggs on each tray were marked and weighed individually on d of set. Embryos from marked eggs were sampled at 19.5 DOI. All eggs were candled at 10 and 17 DOI and any clear eggs were broken open to identify eggs as being either infertile or containing embryos that died at the early or middle stages of incubation. Treatment Solutions L-ascorbic acid (A92902, Sigma-Aldrich Inc., St. Louis, MO) solutions were freshly prepared and sterilized in a dark environment (due to photosensitivity) immediately before injection. Solution sterilization was accomplished by filtration through a 0.2 μm syringe filter (PTFE, 25 mm, Scientific Strategies, Yukon, OK). Injection Procedure After eggs were candled and weighed at 17 DOI, a total of 585 fertile eggs taken from 15 setter trays (except for 117 eggs on 3 trays in the non-injected control treatment) was in ovo-injected with one of the 5 solutions: 0.1 mL saline (0.85% NaCl)-injected control, and 0.1 mL saline containing 0.5, 1.5, 4.5, or 13.5 mg AA. The saline and vitamin solutions were injected into the large end of the egg using a 2.54 cm, 22-gauge needle, possessing a short beveled tip. The entire length of the needle was inserted into the large end of the hatching egg to ensure that the needle adequately penetrated the amnion. In accordance with commercial application, the injection site remained open and was not sealed. After in ovo injection at 17 DOI, all live embryonated eggs were subsequently transferred to a Jamesway model PS 500 hatcher unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada). Eggs in their respective treatment replicate groups were assigned hatcher basket positions that corresponded to their arrangements in the setter. Data Collection At 19.5 DOI, 5 embryos from each of the 6 treatment groups on each of 3 replicate tray levels were selected, weighed, and then sexed by gonaded observation. Individual fertile egg weight, embryo BW (yolk included), and yolk sac weight (YSW) were recorded, and their weights as percentages of set egg weight (SEW) (BW/SEW × 100, YSW/SEW × 100, respectively) were calculated. Every 5 h, beginning at 480 h (20 DOI), and ending at 500 h, the cumulative number of hatched chicks was recorded. On d of hatch (500 h of incubation), all chicks were counted, and all residue eggs were opened to record stages of embryonic mortality. Chicks were feather sexed, and 6 male and 6 female chicks from each treatment (2 males and 2 females from each original tray) were sacrificed by cervical dislocation, weighed individually, and their yolk sacs were removed and weighed. Body weight as a percentage of SEW, and YSW as a percentage of BW were calculated. After sex determination and sampling on d of hatch, chicks were kept in their hatching baskets (males and females separated) for an additional 48 h at 33.0°C and 26.5°C dry and wet bulb temperatures, respectively. After the 48 h of holding period without access to feed and water, mean BW of the male and female chicks in each treatment replicate tray was determined. Subsequently, 2 male and 2 female chicks from each treatment replicate tray were randomly selected, and their BW and yolk sac weights were determined. Subsequently, BW loss during the 48-hour holding period, and BW as a percentage of SEW, and YSW as a percentage of BW were calculated. Statistical Analysis Data were analyzed using one-way ANOVA in a completely randomized block design using the mixed model of the SAS system (version 9.4; SAS Institute, 2013). The in ovo injection treatment was considered as a fixed effect, and incubator tray level (block) was considered as a random effect. Percentage data greater than 80 or less than 20 were arcsine transformed before analysis. The dose-related effect of supplemental AA was computed by GLM in the absence of the non-injected control group using contrast command for linear and quadratic effects. Because no sex effects existed, the data from each sex were pooled within treatment replicate unit. The results are presented as mean ± SEM. Differences were considered statistically significant at P ≤ 0.05. RESULTS Embryonic Characteristics at 19.5 Doi Mean SEW and the various embryonic characteristics at 19.5 DOI are shown in Table 1. There were no significant treatment effects on SEW or on egg weight, embryo weight, yolk sac free embryo BW, YSW, or embryo and YSW as a percentage of SEW within each of the 6 treatment groups. Table 1. Effects of in ovo injection of L–ascorbic acid on embryonic characteristics at 19.5 d of incubation.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78  1P-value of all the groups. 2P-value of all the injection groups. N = 3; 5 observations were pooled within each replicated tray; values are presented as means ± SE. View Large Table 1. Effects of in ovo injection of L–ascorbic acid on embryonic characteristics at 19.5 d of incubation.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Set egg weight (SEW) (g)  59.8±1.2  60.0±0.6  61.2±0.9  59.8±1.6  60.1±0.6  60.5±1.6  0.95  0.22  0.94  Egg weight (g)  53.4±0.8  53.4±0.6  54.6±0.8  52.5±1.7  53.5±0.7  53.8±1.5  0.85  0.97  0.66  Embryo weight (g)  44.3±0.7  44.4±0.4  45.3±0.8  44.3±1.2  44.4±0.6  44.9±1.6  0.97  0.96  0.88  Yolk sac-free embryo BW (g)  35.4±0.6  36.0±0.6  36.6±0.6  35.6±0.6  35.4±0.5  35.5±0.6  0.69  0.23  0.97  Yolk sac weight (g)  8.71±0.17  8.43±0.18  8.72±0.10  8.72±0.39  9.07±0.51  9.43±0.86  0.70  0.08  0.77  Embryo weight/SEW (%)  74.1±0.5  74.1±0.4  74.1±0.4  74.1±0.5  74.0±0.5  74.2±0.5  1.0  0.87  0.82  Yolk sac weight/SEW (%)  14.6±0.4  14.1±0.8  14.3±0.4  14.6±0.6  15.0±0.8  15.5±0.6  0.58  0.07  0.75  Embryo weight/egg weight (%)  83.0±0.6  83.3±0.2  83.1±0.4  84.4±0.4  83.1±0.1  83.5±0.7  0.28  0.74  0.31  Yolk sac weight/embryo weight (%)  19.6±0.3  19.0±0.4  19.3±0.4  19.6±0.4  20.3±0.9  20.9±1.2  0.44  0.09  0.78  1P-value of all the groups. 2P-value of all the injection groups. N = 3; 5 observations were pooled within each replicated tray; values are presented as means ± SE. View Large Hatching Time of Broiler Chicks Hatchability of fertile eggs in each treatment group every 5 h between 480 and 500 h of incubation is shown in Figure 1. Before 480 h (20 DOI), numerically more chicks hatched out in the non-injected control group than in the injection treatment groups, and numerically more broilers hatched out in the 485- to 495-hour period in the injection treatment groups in comparison to the non-injected control group. Nevertheless, hatchability was not significantly affected by injection treatment, with hatchability among all treatment groups ranging from 92 to 96%, which is in agreement with the production guidelines for Ross 708 broiler breeder females (Aviagen, 2016). Figure 1. View largeDownload slide Effects of in ovo injection of L-ascorbic acid (0.5 to 13.5 mg/egg) on hatching time characteristics. Figure 1. View largeDownload slide Effects of in ovo injection of L-ascorbic acid (0.5 to 13.5 mg/egg) on hatching time characteristics. Hatching Characteristics and Chick Quality at 21 Doi Effects of the in ovo injection of AA on hatchability and embryonic mortality, and chick quality at 21 DOI are shown in Table 2. There were no statistical differences between treatment groups for hatchability, dead chicks, dead embryos, or for percentages of live pipped (chicks that may have potential to hatch given a longer hatching time) and dead pipped chicks after the 500-hour incubation period. The results showed that embryonic mortality during the 17 to 21 DOI period ranged from 0.85 to 5.32%. In addition, the highest level of AA injection (13.5 mg/chick) did not cause an increase in late mortality. Table 2. Effects of in ovo injection of L –ascorbic acid on hatching characteristics and chick quality at 21 d of incubation (hatching day).   Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40    Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40  1P-value of all the groups. 2P-value of all the injection groups. 3Hatchability: Hatched chicks as a percentage of total fertile eggs. 4Live pipped: Live embryos in the pipping phase, those that may have remained alive as chicks during continuation of the hatching phase or were assisted by manual shell removal. 5Dead pipped: Embryos that died in the pipping phase. 6Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. For hatching characteristics, calculation was based on a replicate tray basis with 39 fertile eggs per tray. View Large Table 2. Effects of in ovo injection of L –ascorbic acid on hatching characteristics and chick quality at 21 d of incubation (hatching day).   Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40    Non-injected  Saline-injected  L-Ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  Hatchability (%)  95.1±2.4  92.8±2.0  94.2±3.5  93.2±3.4  95.1±1.4  93.8±2.6  0.89  0.88  0.64  Hatchability3 (%)  95.0  92.9  94.1  93.3  95.0  94.0  –  –  –  Dead chicks (%)  0  0  0.81±0.81  0  0  0  0.46  0.50  0.62  Dead embryos (%)  1.63±1.63  5.32±0.13  1.69±0.84  2.50±1.41  3.31±0.84  0.85±0.85  0.20  0.04  0.91  Live pipped4 (%)  1.63±0.81  0.93±0.93  0.81±0.81  2.63±2.63  0  1.85±1.85  0.77  0.80  0.62  Dead pipped5 (%)  1.63±1.63  0.93±0.93  1.63±1.63  0.81±0.81  0.81±0.81  2.71±1.61  0.35  0.31  0.55  Chick BW (g/chick)  42.3±0.8  42.9±0.5  43.1±0.4  42.8±0.4  42.2±0.2  42.6±0.4  0.77  0.40  0.19  Chick BW/set egg weight exclusive  70.4±1.3  70.7±1.7  71.5±0.3  71.2±1.1  70.6±1.0  71.7±0.4  0.64  0.55  0.60  of d 19.5 sample (%)                    Residual yolk sac weight (g)6  4.52±1.08  4.07±0.41  4.55±0.36  4.76±1.21  4.67±0.42  4.54±0.57  0.89  0.75  0.42  Yolk sac weight/BW (%)6  10.4±2.2  9.5±0.8  10.6±0.9  10.9±2.7  10.9±1.1  10.6±0.9  0.91  0.70  0.40  1P-value of all the groups. 2P-value of all the injection groups. 3Hatchability: Hatched chicks as a percentage of total fertile eggs. 4Live pipped: Live embryos in the pipping phase, those that may have remained alive as chicks during continuation of the hatching phase or were assisted by manual shell removal. 5Dead pipped: Embryos that died in the pipping phase. 6Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. For hatching characteristics, calculation was based on a replicate tray basis with 39 fertile eggs per tray. View Large For chick quality at 21 DOI, the in ovo injection of AA into hatching eggs at 17 DOI did not affect hatching BW across sex, or residual YSW and YSW as a percentage of BW. Chick BW relative to SEW also did not differ among treatments. Chick Bw, Yolk Sac Weight, and Bw Loss after 48-Hour Fast Effects of the in ovo injection of AA on chick BW, and YSW and BW losses after 48 h of holding time in the hatchery are provided in Table 3. There were no significant treatment differences for chick BW across sex, absolute or percentage YSW, or for absolute or percentage BW loss. Table 3. Effects of in ovo injection of L-ascorbic acid on chick body weight, yolk sac weight, and BW loss after 48-hour posthatch fast.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90  1P-value of all the groups. 2P-value of all the injection groups. 3Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. View Large Table 3. Effects of in ovo injection of L-ascorbic acid on chick body weight, yolk sac weight, and BW loss after 48-hour posthatch fast.   Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90    Non-injected  Saline-injected  L-ascorbic acid dosage (mg/egg)  P-value    Control  Control  0.5  1.5  4.5  13.5  ANOVA1  Linear2  Quadratic2  BW (across sex, g/chick)  36.2±0.6  36.7±0.5  36.9±0.4  36.4±0.4  36.1±0.1  36.3±0.2  0.71  0.33  0.21  BW/set egg weight (%)  60.3±0.7  60.5±1.1  60.8±0.4  60.7±0.5  60.3±0.4  60.9±0.4  0.97  0.74  0.60  BW/egg weight of 17 DOI (%)  66.7±0.6  67.2±1.3  67.4±0.3  66.9±0.3  66.8±0.8  67.4±0.3  0.96  0.82  0.53  BW Loss (g/chick)  6.09±0.23  6.25±0.10  6.17±0.08  6.37±0.06  6.15±0.13  6.29±0.20  0.77  0.80  0.63  BW loss/set egg weight (%)  10.2±0.3  10.3±0.1  10.2±0.1  10.6±0.2  10.3±0.2  10.6±0.3  0.57  0.36  0.93  BW loss/hatching BW (%)  14.4±0.3  14.6±0.3  14.3±0.1  14.9±0.2  14.6±0.2  14.8±0.4  0.65  0.52  0.89  Residual yolk sac weight (g/chick)3  1.07±0.14  0.96±0.09  1.11±0.13  0.78±0.06  1.08±0.16  1.08±0.18  0.49  0.43  0.96  Yolk sac weight/BW (%)3  2.93±0.37  2.61±0.67  2.82±1.15  2.14±0.56  2.78±1.29  2.88±1.45  0.56  0.41  0.90  1P-value of all the groups. 2P-value of all the injection groups. 3Four observations were pooled within each original replicated tray. N = 3; values are presented as means ± SE. View Large DISCUSSION Background Fertile eggs do not contain detectable levels of AA. During embryonic development, AA concentration in the brain is known to rise after d 6 and increase to 5.6 nmol/mg of tissue by d 10. This level is maintained at a relatively high level between 8 and 18 DOI, and then gradually declines to 32% immediately before hatch (Wilson, 1990; Wilson and Jaworski, 1992). Plasma AA concentration is known to peak on d 12 of embryonic development, and then falls before increasing again on d 20, when pulmonary respiration begins (Wilson and Jaworski, 1992). The dynamic changes in AA levels of embryos suggest the importance of AA for optimal embryonic development in the late incubation phase. Ascorbic acid has been reported to combat heat stress in the later phases of incubation and to decrease mortality and improve hatchability (Zakaria and Al-Anezi, 1996). In this study, effects of different dosages of AA delivered by in ovo injection during the late incubation phase were investigated. However, no changes in the embryonic characteristics examined at 19.5 DOI occurred. The results suggested that the in ovo injection of AA did not negatively affect the growth of embryos 2.5 d post injection. Hatchability In the present study, no difference in the hatchability of fertile eggs was observed among treatment groups, which indicated that the injection process and the injection solution were safe for the embryos and did not interrupt the hatching process. However, an improvement in hatchability using the described treatment dosages of AA was not observed. This is in agreement with the results of earlier studies (Nowaczewski et al., 2012; Hajati et al., 2014). Nowaczewski et al. (2012) observed no increase in hatchability by the in ovo injection of 3 to 6 mg of AA in fertile eggs. However, the highest rate of hatchability of fertilized eggs was noticed in the group injected with 6 mg of AA at 15 DOI. In contrast to the current study, some studies have indicated that 3 mg AA/egg reduced late-term embryo mortality and increased hatchability. (Elibol et al., 2001; Ipek et al., 2004). The observed diverse results may be explained by the differences in injection time and the number of fertile eggs examined. The site of injection and the stage of embryonic development when injection is administered influences the effect of AA on the embryo. It has been reported that the injection of 1 μg of AA in the albumen before incubation increased hatching rate by 14.2% in comparison to a control treatment (Iglesias et al., 2015). Injection of AA at a 3 mg/egg level improved hatchability when injected on d 11 and 15 of incubation (Zakaria and Al-Anezi, 1996; Zakaria et al., 1998; Zakaria, 2001). It appears that the window that is beneficial for maximizing the effect of the in ovo injection of AA on hatchability may occur in the middle or later phases of incubation. This, however, also might be achieved after elucidating the dynamic AA status or requirement of the embryo across an entire incubation phase. Generally, fertile eggs from breeder layers during peak production exhibit a higher level of hatchability, and conversely eggs laid by old breeders have lower hatching percentages (Almeida et al., 2008). In the current study, hatchability in the non-injected group was more than 90%. Therefore, it is possible that there was little room for an improvement in hatchability. Efforts made to investigate possible differences in the AA concentrations of fertile eggs from breeder hens at different ages may help to better determine the optimal timing and dosage of injected AA that is necessary to improve hatchability. Time of Hatch In contrast to the results of Zakaria et al. (1998), AA injection did not lead to a decrease in the time of hatch in the current study. This may be due to differences in incubator type and condition as well as the type of fertile eggs used. Hatching time was recorded through 500 h of incubation in the present study, while that of Zakaria et al. (1998) started at 498 h and ended at 528 h of incubation, with an additional record made at 552 h of incubation. In addition, average chick BW in the study of Zakaria et al. (1998) was approximately 1.5 kg at 49 d of age, which was far below that of modern broilers at 42 d of age (2.5 kg). Therefore, differences in the length of time in which hatch time was recorded as well differences in the bird strains used in the 2 studies may account for these contrasting results. Whether or not the in ovo injection of AA affects hatching time remains unclear. The relevance of supplemental AA on hormone secretion also would be an important factor to consider. It has been suggested that AA administration may affect stress hormone levels (Kutlu and Forbes, 1994; Mckee and Harrison, 1995). Specifically, there is an abrupt increase in circulating triiodothyronine levels before internal pipping and the subsequent transformation to pulmonary respiration (Kuhn et al., 1993). It would be worthwhile to investigate whether or not the in ovo injection of AA may affect various circulating hormone concentrations during the later phase of incubation. Relatively more chicks hatching out earlier in the non-injected control group suggests that the injection process may delay hatching to some extent. It has been reported that fertile eggs with a low rate of water loss usually hatch later (Ar and Rahn, 1980; Ar, 1991). However, in the current study, it is suggested that a saline injection volume of 0.1 mL would not affect a change in water loss, and that a holding time of 10 min during the injection process was not long enough to alter the hatching process. Thus, the possible metabolic modulatory effect of AA on the hatching process is expected and warrants further investigation. Yolk Sac During the development of avian embryo, yolk is the sole energy supply (Romanoff, 1960). Noble and Cocchi (1990) noted that almost 94% of the total energy needs of the embryo during development are provided by means of the oxidation of fatty acids. In the current study, the in ovo injection of AA at 17 DOI did not induce a change in yolk sac weight at 19.5 DOI, which indicated that AA administration did not affect the absorption or utilization of yolk during the late phase of incubation. After hatch, as the first primary source of nutrients, the yolk sac provides the hatched chick with immediate energy (Romanoff, 1960). In the current study, more than 50% of the loss in BW during the 48-hour holding period was from YSW loss, which indicates the importance of yolk as a vital nutrient source for the newly hatched chicks. The longer the time between hatch and the start of feed and water intake, the greater is the reliance of the chick on these reserves (Vieira and Pophal, 2000). Noy and Sklan (1999) indicated that chicks deprived of food after hatch used approximately 60% of their residual yolk sac in the first 48 hours. Furthermore, an exogenous supply of feed enables or stimulates yolk sac absorption. Iglesias et al. (2015) observed a decrease in YSW at 72 h post hatch in chicks having access to feed. In the current study, chicks were deprived of food and water for 48 h, and the subsequent loss of residual yolk was between 75 and 84%. This suggested that the chicks experienced other stressors in addition to that of food deprivation. In the current study, chicks that were fasted for 48 h lost an average of 6.23 g of weight, which is similar to the 0.14 g/h average reported in the study by Warriss et al. (1992). The absence of a difference in YSW loss between treatment groups in this study indicated that the in ovo injection of AA could not counteract the severe stress induced by 48 h of feed and water deprivation that the broiler chicks experienced after hatch. Delayed Removal from the Incubator Experimental data on the anti-stress effects of in ovo-injected nutrients used to counteract the effects of delayed chick placement are lacking. Zakaria et al. (1998) held hatched chicks in the incubator for 18 to 48 h, and observed that the in ovo injection of 2 mg AA/egg elevated the BW of male chicks at 7 d of age, while no effect was observed in females. However, the current experimental period did not extend into the feeding phase. Nevertheless, the fact that body and yolk sac weight losses were unchanged indicated that the injection of AA during the last phase of incubation was not sufficient to prevent the stress of a 48-hour fast. However, investigations as to the lasting effects of the in ovo injection of AA on early growth in broiler chicks are pending. Tolerance Dosage In the current study, the in ovo injection of high concentrations of AA (13.5 mg/egg) did not lead to increases in late embryo mortality. This result is different from the results observed in the study by Zakaria and Al-Anezi (1996), which showed a dramatic decrease in hatchability occurring in a 12 mg AA/egg injection treatment group. The reason that the embryos can tolerate more than a 12 mg AA per egg exposure in the current study may be associated with the later developmental stage of the embryos. In this present study, the amnion at 17 DOI was the primary site of injection, and the air cell at the large end of the egg in the early or middle phase of incubation (before 17 DOI) has been commonly used in other studies. The embryo being of a larger size may have contributed to its higher tolerance to the dosage of the injected AA. However, high dosages of injected AA may harm the development of the embryo. This was evidenced in a study by Ingram et al. (1997). In that study, AA injections of 0.75 g/mL were toxic and resulted in embryo mortality prior to hatching. In the literature, an effective dosage of AA that was in ovo-injected was shown to range from 3 to 6 mg/egg (Ipek et al., 2004; Zakaria and Al-Anezi 1996; Elibol et al., 2001; Nowaczewski et al., 2012). To date, results of studies determining the effects of AA supplementation in chicken embryos vary considerably. For example, in a study in which an in ovo injection method was applied pre-incubation using a low level of AA (1 μg/egg), hatchability was increased (Iglesias et al., 2015). Additionally, it was observed that dipping Sinai chicken eggs in 15.0 g of AA/L at 12 DOI for 2 or 3 min maximized hatchability and minimized embryonic mortality (Awad and Abd El-Halim, 2015). Due to the diversity and inconsistency of in ovo injection sites and times used in fertile eggs from breeders of different ages, it is unclear as to ascertain the effective dosage of AA or the exact mode of in ovo injection that best supports embryonic growth. However, the current study has demonstrated that the in ovo injection of AA at a concentration of 13.5 mg/egg does not adversely affect the hatchability or quality of broiler hatchlings. In conclusion, the amniotic injection of 0.5 to 13.5 mg of AA at 17 DOI does not affect the embryonic development or hatching rate of broiler chicks, and the injection of AA does not modify the negative effects of a 48-hour posthatch fast on chick body or yolk sac weight loss. The tolerance range and optimal dosage of in ovo injected AA in broiler embryos warrant further study. ACKNOWLEDGMENTS We express our appreciation for the expert technical assistance of Jonathan Moon of the Mississippi State University Poultry Science Department. Footnotes 1 This publication is a contribution of the Mississippi Agricultural and Forestry Experiment Station. 2 This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under accession number 322290. 3 Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. REFERENCES Almeida J. G., Vieira S. L., Reis R. N., Berres J., Barros R., Ferreira A. K., Furtado F. V. F.. 2008. Hatching distribution and embryo mortality of eggs laid by broiler breeders of different ages. Braz. J. Poult. Sci.  10: 89– 96. Ar A. 1991. Egg water movements during incubation. Pages 157– 173 in Avian Incubation . Tullett S. G., ed. Butterworth-Heinemann Ltd., London, UK. Ar A., Rahn H.. 1980. Water in the avian egg: Overall budget of incubation. Am. Zool.  20: 373– 384. 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Poultry ScienceOxford University Press

Published: Feb 15, 2018

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