Effects of in ovo injection of different doses of coccidiosis vaccine and turn-out times on broiler performance,,

Effects of in ovo injection of different doses of coccidiosis vaccine and turn-out times on... ABSTRACT Inovocox EM1 vaccine (EM1) is hatchery-applied via in ovo injection for the control of coccidiosis in broiler chickens. Effects of 3 in ovo injection treatments (INT) and 2 turn-out times (TOT) on the hatching chick quality variables and 35 d posthatch performance of Ross × Ross 708 broilers were investigated. In a single-stage incubator, 1,440 hatching eggs were randomly distributed among 3 INT groups on each of 8 tray levels. At 19 d of incubation (doi), embryonated eggs were subjected to one of the following INT by in ovo injection: noninjected control; 1 × dose of EM1; 10 × dose of EM1. On 21 doi, hatchability of injected eggs (HI), hatching body weight (HBW), and hatching chick quality variables were determined. Additionally, for the grow-out phase, birds belonging to each INT were randomly subjected to a 7 or 10 d TOT. Twenty chicks were initially placed in each of 48 floor pens (6 INT × TOT combination groups × 8 replications) for growth performance evaluation from 0 to 35 d posthatch. The main effect of INT on hatching chick quality variables, as well as the main and interactive effects of INT and TOT on various grow-out performance variables were determined. Although there was no significant INT effect on HI or HBW, significant INT effects on chick total BW, yolk-free BW, and yolk sac weight were observed. There were significant INT effects on BWG and FCR in the 21- to 28-d posthatch interval, as well as on BWG and FCR in the 0- to 35-d posthatch interval. There was no main effect of TOT or interactive effect of INT and TOT on BW and other performance variables from 0 to 35 d posthatch. There was a significant main effect of INT on relative intestine weight at 28 d posthatch. In conclusion, the injection of EM1 vaccine at a 10 × dose may affect hatching chick quality variables and growth performance up to 35 d posthatch. INTRODUCTION The protozoan parasite of the genus Eimeria, which causes avian coccidiosis, develops in the intestinal tract of birds, causing morbidity, mortality, poor feed efficiency, and poor weight gain, and predisposes birds to secondary diseases such as necrotic enteritis (Williams, 2005). Coccidia oocysts are ubiquitous to commercial chicken houses, and therefore, only control of coccidiosis, rather than complete eradication, is attainable under commercial conditions (Chapman, 2000; Tewari and Maharana, 2011). The control of coccidiosis is achieved by use of in-feed anticoccidials and coccidiosis vaccines in different types of grow-out programs, such as rotation or bio-shuttle programs (Chapman et al., 2002; Montoya and Quiroz, 2013). When used in rotation programs, coccidiosis vaccines help to restore the precocious strains (drug-resistant) of coccidia oocysts that have sensitivity to in-feed anticoccidials (Chapman and Jeffers, 2014). In a bio-shuttle program, coccidiosis vaccines are used at day of hatch, and are followed by low levels of ionophore anticoccidials in the grower and finisher feeds (Montoya and Quiroz, 2013). In addition, due to consumer demand and changing regulatory landscapes, poultry producers are not only reducing the use of antibiotic growth promoters, but also ionophore anticoccidials in poultry feed. The result of this is that producers have fewer options to help control coccidiosis, and therefore rely on either non-ionophore (synthetic) anticoccidials or almost exclusively on vaccines for the control of coccidiosis. Several disease models that evaluate the bird's response to test substances like feed additives have utilized coccidia vaccines up to 10× normal levels to challenge the bird's intestine, and increase the likelihood of overgrowth of Clostridium perfringens that produces dysbacteriosis, necrotic enteritis, or sub-optimal growth conditions in the birds. In order to effectively simulate these conditions under research conditions similar to commercial conditions, it is important to consider the life cycle of coccidia and the environmental and management factors that can help to maximize the outcome of the challenge, especially those that affect sporulation and re-cycling of the coccidia oocysts. The most recognized predisposing factor for necrotic enteritis is damage to the intestine caused by a coccidiosis infection. Damage to intestinal villus and crypt morphology leads to a subsequent loss in performance and an increase in mortality in affected birds (Timbermont et al., 2011). From a practical standpoint, ensuring the efficacy of an in ovo coccidiosis vaccine is an interplay of several factors, which begins at the hatchery and continues into the chicken house. At the hatchery, it is important to ensure the precise stage of embryo development, as this will ensure that the vaccine is delivered to the correct injection site (Sokale et al., 2017a). Previous work has shown that in ovo vaccination of embryos at 18 d of incubation (doi) for Marek's Disease (Williams and Zedek, 2010) or 18.5 doi for coccidiosis (Sokale et al., 2017a) resulted in a high delivery accuracy. However, the vaccination of embryos later than these times (e.g., at 19 doi) is a common practice under commercial conditions. In the chicken house, exposure of birds to multiple coccidia life cycles (oocyst cycling) can potentially initiate an immune response necessary for the control of coccidiosis (Chapman et al., 2002; Tewari and Maharana, 2011). In order to achieve this oocyst cycling from coccidiosis vaccination, partial house brooding management has been recommended by vaccine manufacturers. Partial house brooding during the first few d of placement allows the birds to have a repetitive fecal-oral exposure to high numbers of coccidia oocysts, which initiates their development of immune competence against coccidiosis (Mathis, 2001; Schering-Plough Animal Health, 2007). However, the appropriate duration of repeated exposures of birds to coccidia oocysts in the Inovocox EM1 vaccine (EM1; Zoetis, Kalamazoo, MI) under varying application conditions, as well as the effects on growth performance, have not been documented in the scientific literature. Therefore, the objective of this study was to determine the effects of EM1vaccine injected into 19-day-old broiler embryos at a high or low dose, with 2 partial house brooding time-points, on grow-out performance. For this study, we chose 19 doi in order to mimic industry conditions of late-stage embryo vaccination. In addition, 7- and 10-d posthatch were selected as the 2 partial house brooding time-points to evaluate the effects early and late turn-out times have on coccidia oocyst cycling when EM1 is injected at a high or low dose. We evaluated growth performance and intestinal morphometrics as an indication of how the broiler chickens responded under these given conditions. MATERIALS AND METHODS General This study was conducted according to a protocol that was approved by the Institutional Animal Care and Use Committee of Mississippi State University. A total of 2,160 Ross × Ross 708 broiler hatching eggs were obtained from a 45-week-old commercial breeder flock, and held for 2 d under standard storage conditions prior to setting. On 0 doi, eggs were weighed and labeled according to methods previously described by Sokale et al. (2017a). A total of 1,440 eggs were randomly assigned to 3 injection treatment (INT) groups, each containing 60 eggs, on each of 8 replicate trays (blocks). After candling on 18 doi, a total of 1,310 embryonated eggs were retained across the 3 INT and their 8 replicate groups (each replicate group contained approximately 54 embryonated eggs). All eggs were incubated in a Jamesway model PS 500 single-stage incubator (Jamesway Incubator Co. Inc., Cambridge, Ontario, Canada), under standard conditions (Peebles and Brake, 1987). On 19 doi, eggs were subjected to one of the 3 INT: noninjected control (NIC), 1 × dose of EM1 (1 × EM1), 10 × dose of EM1 (10 × EM1). The dosages for the EM1 were attained as described below. Injection and Experimental Layout Injection of eggs was performed on 19 doi using an Embrex Inovoject injector system (Zoetis Animal Health, Research Triangle Park, NC), as described by Sokale et al. (2017a). According to recommendations by the vaccine manufacturer (Zoetis Animal Health), 3 vials (8,000 doses each) of EM1 were reconstituted in 1,200 mL of sterile commercial MD vaccine diluent (Merial Co., Duluth, GA) for the 1 × EM1, and 30 vials (8,000 doses each) of EM1 were reconstituted in 1,200 mL for the 10 × EM1. At the time of immunization, the EM1 vaccine still had approximately 7 months before its expiration date. The vaccine containing oocysts of E. acervulina, E. maxima, and E. tenella were administered at the rate of 50 μL per egg. Eggs belonging to each INT group were injected separately with an intervening machine cleaning cycle to avoid cross-contamination. Once the entire injection process was completed, eggs were transferred to the hatcher unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada) on their corresponding replicate tray levels. Eggs in the hatcher baskets were arranged in a way to prevent cross-contamination between chicks belonging to the vaccine injected and non-injected groups. The site of injection was confirmed according to the method previously described by Sokale et al. (2017a). At 21 doi, the effects of INT on the various hatching chick quality variables described below were determined. In the grow-out phase of the study, the turn-out time (TOT), which indicates the duration of repetitive exposure to coccidia oocysts, was included along with INT in the performance evaluation. Thus, a 3 INT × 2 TOT factorial design was employed. A group of 6 miniature floor pens, each representing a specific INT × TOT combination were randomly assigned to a block, with 8 total replicate blocks in the grow-out facility. This resulted in a total of 48 INT × TOT treatment replicate pens. On 21 doi, a total of 20 straight-run chicks randomly selected from each INT replicate group were allocated to 1 of 2 TOT, and were wing-banded, weighed, and placed in each of the 48 pens. Each pen measured 1.1 m2 within a temperature-controlled research facility. The birds (20 birds × 48 pens = 960 total birds) were placed on litter that had been previously used for 2 grow-out cycles. Birds were fed ad libitum with a crumbled starter diet from 0 to 14 d posthatch, pelletized grower diet from 15 to 28 d posthatch, and pelletized finisher diet from 29 to 35 d posthatch. Diets were formulated to meet or exceed NRC (1994) recommendations. House temperature conditions were monitored and recorded 2 times daily from 0 to 35 d posthatch. In order to achieve the specified TOT, pens were divided using plastic wire mesh into three-fourths (0.83 m2) and one-fourth (0.28 m2) portions. The plastic wire mesh prevented birds from crossing over to the unused side of each pen without interfering with air flow. All 20 chicks were initially placed in the three-fourths portion of each pen, at a stocking density of 0.04 m2/bird, and on either 7 or 10 d posthatch, birds within each of the 3 INT in each of the 8 replicate blocks were turned out (8 × 3 = 24 pens at each of the 2 TOT). Turning-out involved the removal of the plastic wire mesh used to divide each pen, so that birds were allowed the entire 1.1 m2 space in each pen, at a stocking density of 0.06 m2/bird, up to 35 d posthatch. Data Collection Set egg weight (SEW) was recorded on 0 doi. On d of injection (19 doi), both the site of injection and embryo stage score were determined to evaluate vaccine delivery accuracy as described by Sokale et al. (2017a). On 21 doi, the hatchability of injected fertilized eggs (HI) and mean hatching body weight (HBW) of chicks in each INT replicate group were determined. Additionally, 16 chicks from each INT group were individually selected, wing-banded, euthanized, weighed, and necropsied to determine the following hatching (21 doi) chick quality parameters: total body weight (BW), and absolute yolk sac (YSW), liver (LW), whole intestine (IW), and heart (HW) weight. In addition, the following were determined: yolk-free BW (YFBW), YSW relative to total BW (RYBW), IW relative to total (RIBW) and yolk-free (RIYFW) BW, LW relative to total (RLBW) and yolk-free (RLYFW) BW, HW relative to total (RHBW) and yolk-free (RHYFW) BW, total BW relative to SEW (RBSW), YFBW relative to SEW (RYFWSW), and yolk-free body mass (YFBM). On 28 d posthatch, 2 chicks from each pen were individually selected, euthanized, weighed, and necropsied for determination of total BW, IW, and RIBW. For evaluation of bird performance, BW gain (BWG), feed intake (FI), and mortality-adjusted feed conversion ratio (FCR) were determined for the weekly and entire 0- to 35-d posthatch intervals. Percentage cumulative mortality (CPM) for the entire 0- to 35-d posthatch interval was also determined. Additionally, on 35 d posthatch, 3 birds taken from 3 replicate groups within each INT × TOT treatment combination were randomly selected, euthanized, weighed, and necropsied for intestinal histomorphometric analysis. The small intestinal samples were collected and preserved in 10% buffered neutral formalin. The tissue samples were trimmed, and transverse sections of approximately 5 μm were stained with hematoxylin and eosin. All slides were evaluated and digitalized for histomorphometry using an Amscope 5 MP camera and image J software (AmScope, Irvine, CA). Images were analyzed for villus height and crypt depth, and the villus-to-crypt depth (VCD) ratio was calculated. Five villi and crypts were measured per sample, and the mean was calculated. In addition to the morphometric analysis, the enumeration of absolute count of coccidia in each sample was accomplished by histological method. The absolute count of coccidia stages (sporocysts) was obtained by counting a total of 10 fields in each treatment sample (at 400× magnification) and the mean for each treatment was calculated and reported. Statistical Description A randomized complete block design was used in both the incubational and grow-out periods. In the incubational phase, each tray level represented a block, and all treatments were equally and randomly represented in each block. The chick quality data on 21 doi were analyzed using the 3 INT groups (NIC, 1 × EM1, and 10 × EM1). A 1-way analysis of variance (ANOVA) was used to analyze the main effect of INT on the hatching chick quality variables. The performance data were arranged in a 3 INT × 2 TOT factorial design to evaluate the main and interactive effects of INT and TOT on performance. A 2-way ANOVA was used to analyze the main and interactive effects of INT and TOT on 28 d posthatch IW and RIBW, CPM in the 0- to 35-d posthatch interval, and weekly BWG, FI, and FCR. All individual data within each replicate group was averaged and all percentage data was arcsine transformed prior to analysis. A split-plot analysis was performed for weekly absolute BW with INT and TOT as whole plot factors split over posthatch d. In the aforementioned analysis, INT and TOT were considered as fixed effects and block as a random effect. For histomorphometric variable evaluation, a 1-way ANOVA was used to analyze the main effects of INT or TOT on coccidia count, and a 2-way ANOVA was used to analyze the main and interactive effects of INT and TOT on villus height, crypt depth, and VCD ratio. For this analysis, INT and TOT were considered as fixed effects and each individual bird as a random effect. Least-square means were compared in the event of significant global effects (Steel and Torrie, 1980). All variables were analyzed using the MIXED procedure of SAS software 9.3 (SAS Institute, 2012). Global and least-square means differences were considered significant at P ≤ 0.05. RESULTS Using approximately 7% of the in ovo-injected embryonated eggs, the site of injection and embryo stage score at 19 doi were evaluated. Mean embryo stage score was 4.60 ± 0.99, and site of injection evaluation indicated that 6.8 and 93.2% of the eggs received vaccine in the amnion and embryo body, respectively, with embryo body injections being 81.5% intramuscular and 11.7% subcutaneous. There was no significant INT effect on SEW at 0 doi or on HI, HBW, YFBW, YFBM, RYFWSW, IW, RIBW, RIYFW, LW, RLBW, RLYFW, HW, RHBW, and RHYFW at 21 doi. However, there was a significant INT effect on chick BW, RBSW, and YSW at 21 doi. The BW, RBSW and YSW values were higher in the NIC group compared to the 10 × EM1 group, with the 1 × EM1 group being intermediate. The INT means for those 3 variables and all of the other hatching chick quality variables evaluated at 21 doi are provided in Table 1. Table 1. Main effect of injection treatment (INT): noninjected control group (NIC), 1× dose EM1 (1× EM1), and 10× dose EM1 (10× EM1) vaccine on the somatic and yolk variable means of selected chicks at 21 d of incubation.1,2 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 a,bMeans within a variable with no common superscript differ (P ≤ 0.05). 1Set egg weight (SEW), hatchability of injected fertilized egg (HI), hatching body weight (HBW), body weight (BW), yolk-free BW (YFBW), BW as a percentage of set egg weight (RBSW), yolk-free BW as a percentage of set egg weight (RYFWSW), intestine weight (IW), intestine weight as a percentage of BW (RIBW), intestine weight as a percentage of yolk-free BW (RIYFW), liver weight (LW), liver weight as a percentage of BW (RLBW), liver weight as a percentage of yolk-free BW (RLYFW), heart weight (HW), heart weight as a percentage of BW (RHBW), heart weight as a percentage of yolk-free BW (RHYFW), yolk sac weight (YSW), yolk sac weight as a percentage of BW (RYBW), and yolk-free body mass (YFBM). 2Two birds in each of 8 replicate units per treatment (16 birds per treatment group) were used to calculate each treatment mean. 3Sixty eggs in each of 8 replicate units per treatment (480 eggs per treatment group) were used to calculate mean SEW. 4Data from 8 replicate units was used for calculation of HI and HBW means for each treatment group. View Large Table 1. Main effect of injection treatment (INT): noninjected control group (NIC), 1× dose EM1 (1× EM1), and 10× dose EM1 (10× EM1) vaccine on the somatic and yolk variable means of selected chicks at 21 d of incubation.1,2 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 a,bMeans within a variable with no common superscript differ (P ≤ 0.05). 1Set egg weight (SEW), hatchability of injected fertilized egg (HI), hatching body weight (HBW), body weight (BW), yolk-free BW (YFBW), BW as a percentage of set egg weight (RBSW), yolk-free BW as a percentage of set egg weight (RYFWSW), intestine weight (IW), intestine weight as a percentage of BW (RIBW), intestine weight as a percentage of yolk-free BW (RIYFW), liver weight (LW), liver weight as a percentage of BW (RLBW), liver weight as a percentage of yolk-free BW (RLYFW), heart weight (HW), heart weight as a percentage of BW (RHBW), heart weight as a percentage of yolk-free BW (RHYFW), yolk sac weight (YSW), yolk sac weight as a percentage of BW (RYBW), and yolk-free body mass (YFBM). 2Two birds in each of 8 replicate units per treatment (16 birds per treatment group) were used to calculate each treatment mean. 3Sixty eggs in each of 8 replicate units per treatment (480 eggs per treatment group) were used to calculate mean SEW. 4Data from 8 replicate units was used for calculation of HI and HBW means for each treatment group. View Large There were no significant main effects or interactions involving INT or TOT on weekly BW; BWG, FI, and FCR in the 0- to 7-d, 7- to 14-d, 14- to 21-d, or 28- to 35-d posthatch intervals; or on FI in the 21- to 28-d and 0- to 35-d posthatch intervals. There was also no significant TOT main effect or INT × TOT interaction for BWG and FCR in the 21- to 28-d or 0- to 35-d posthatch intervals. However, there was a significant main effect of INT on BWG and FCR in the 21- to 28-d posthatch interval, and of INT on BWG and FCR in the 0- to 35-d posthatch interval. In both posthatch intervals, BWG was significantly higher in the NIC treatment group compared to both the 1 × EM1 and 10 × EM1 treatment groups, with no statistical difference between the 1 × EM1and 10 × EM1 treatment groups (Table 3). Furthermore, FCR was significantly lower in the NIC treatment group compared to both the 1 × EM1 and 10 × EM1 groups in both time intervals (Table 3). However, birds in the 1 × EM1 group displayed a better feed efficiency compared to birds in the 10 × EM1 group in the 0- to 35-d posthatch period (Table 3). The main effect means of INT for BWG, FI, and FCR for the 0- to 7-d, 7- to 14-d, and 14- to 28-d posthatch intervals are provided in Table 2, and for the 21- to 28-d, 28- to 35-d, and 0- to 35-d posthatch intervals are provided in Table 3. Table 2. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 0 to 7, d 7 to 14, and d 14 to 21 posthatch.1 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 120 birds in each of 8 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG1 = d 0 to 7 BW gain; FI1 = d 0 to 7 feed intake; FCR1 = d 0 to 7 feed conversion ratio; BWG2 = d 7 to 14 BW gain; FI2 = d 7 to 14 feed intake; FCR2 = d 7 to 14 feed conversion ratio; BWG3 = d 14 to 21 BW gain; FI3 = d 14 to 21 feed intake; FCR3 = d 14 to 21 feed conversion ratio. View Large Table 2. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 0 to 7, d 7 to 14, and d 14 to 21 posthatch.1 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 120 birds in each of 8 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG1 = d 0 to 7 BW gain; FI1 = d 0 to 7 feed intake; FCR1 = d 0 to 7 feed conversion ratio; BWG2 = d 7 to 14 BW gain; FI2 = d 7 to 14 feed intake; FCR2 = d 7 to 14 feed conversion ratio; BWG3 = d 14 to 21 BW gain; FI3 = d 14 to 21 feed intake; FCR3 = d 14 to 21 feed conversion ratio. View Large Table 3. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 21 to 28, d 28 to 35, and d 0 to 35 posthatch.1 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 a–cMeans within a column with no common superscript differ (P ≤ 0.05). 120 birds in each of 16 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG4 = d 21 to 28 BW gain; FI4 = d 21 to 28 feed intake; FCR4 = d 21 to 28 feed conversion ratio; BWG5 = d 28 to 35 BW gain; FI5 = d 28 to 35 feed intake; FCR5 = d 28 to 35 feed conversion ratio; BWG6 = d 0 to 35 BW gain; FI6 = d 0 to 35 feed intake; FCR6 = d 0 to 35 feed conversion ratio. View Large Table 3. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 21 to 28, d 28 to 35, and d 0 to 35 posthatch.1 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 a–cMeans within a column with no common superscript differ (P ≤ 0.05). 120 birds in each of 16 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG4 = d 21 to 28 BW gain; FI4 = d 21 to 28 feed intake; FCR4 = d 21 to 28 feed conversion ratio; BWG5 = d 28 to 35 BW gain; FI5 = d 28 to 35 feed intake; FCR5 = d 28 to 35 feed conversion ratio; BWG6 = d 0 to 35 BW gain; FI6 = d 0 to 35 feed intake; FCR6 = d 0 to 35 feed conversion ratio. View Large There were no significant main effects or interactions involving INT or TOT on IW at 28 d posthatch, although the main effect of TOT on IW approached significance (P = 0.08). Mean IW in the d 7 and 10 TOT treatment groups were 73.3 g and 69.8 g, respectively. Similarly, there was no significant main effect of TOT or interactive effect of INT × TOT on RIBW at 28 d posthatch, although the main effect of TOT on RIBW approached significance (P = 0.07). Mean RIBW in the d 7 and 10 TOT were 4.80% and 4.60%, respectively. There was, however, a significant main effect of INT on RIBW at 28 d posthatch. The RIBW of birds in the 1 × EM1 and 10 × EM1 groups was significantly higher compared to that in birds belonging to the NIC group, but RIBW in the 1 × EM1 group was not significantly different from that of birds in the 10 × EM1 group. The INT main effect means for RIBW at 28 d posthatch are provided in Table 4. Table 4. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1× EM1), and 10 × dose EM1 (10 × EM1) vaccine on relative intestinal weight (RIBW) of birds at d 28 posthatch, and villus height and villus to crypt depth (VCD) ratio of the small intestine at d 35 posthatch. Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 a,bMeans with no common superscript differ (P ≤ 0.05). 1n = 16 replicate units for each RIBW mean. 2n = 18 replicate units for each villus height and VCD ratio mean. View Large Table 4. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1× EM1), and 10 × dose EM1 (10 × EM1) vaccine on relative intestinal weight (RIBW) of birds at d 28 posthatch, and villus height and villus to crypt depth (VCD) ratio of the small intestine at d 35 posthatch. Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 a,bMeans with no common superscript differ (P ≤ 0.05). 1n = 16 replicate units for each RIBW mean. 2n = 18 replicate units for each villus height and VCD ratio mean. View Large There was no significant main effect of INT or TOT on villus height or VCD ratio at 35 d posthatch. There was also no significant INT × TOT interaction on villus height (P = 0.24) or VCD ratio (P = 0.11). For observational purposes, the INT means for villus height and VCD ratio are provided with those of RIBW in Table 4. There was a significant (P = 0.01) INT × TOT interaction for crypt depth. Small intestine crypt depth was higher in the NIC-d10 TOT treatment combination group in comparison to the NIC-d7 TOT and 10 × EM1-d10 TOT treatment groups, with all other treatment combination groups intermediate (Table 5). There was no significant main effect of INT or TOT on intestinal coccidia count at 35 d posthatch. There were, however, numerical differences in mean coccidia counts between the 3 INT groups and between the 2 TOT groups. The 35 d posthatch mean coccidia counts for the 3 INT groups and the 2 TOT are presented in Table 6. There was no significant main effect of INT (P = 0.35) or TOT (P = 0.30), and no significant (P = 0.34) INT × TOT interaction for the CPM of birds in the 0- to 35-d posthatch interval. However, there were numerical differences in the CPM of birds between the treatment groups in the 0- to 35-d posthatch interval. Birds belonging to the NIC-d7 TOT and NIC-d10 TOT had a CPM of 1.88% and 5.63%, respectively. Birds belonging to the 1 × EM-d7 TOT and 1 × EM-d10 TOT treatment combination groups had a CPM of 4.37% and 3.75%, respectively, and birds belonging to the 10 × EM-d7 and 10 × EM-d10 treatment groups had a CPM of 4.60% and 5.00%, respectively. Table 5. Interaction effect involving injection treatment [noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine] and turn-out time on d 7 and 10 posthatch, on small intestinal crypt depth at d 35 posthatch.1,2 TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b a,bMeans with no common superscript differ (P = 0.01). 1n = 18 replicate units for each mean. 2Pooled SEM = 48.5. View Large Table 5. Interaction effect involving injection treatment [noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine] and turn-out time on d 7 and 10 posthatch, on small intestinal crypt depth at d 35 posthatch.1,2 TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b a,bMeans with no common superscript differ (P = 0.01). 1n = 18 replicate units for each mean. 2Pooled SEM = 48.5. View Large Table 6. Mean absolute intestinal coccidia counts for injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine and turn-out times (TOT) at d 35 posthatch. Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Histological enumeration of absolute coccidia count of 10 fields per sample. 1n = 18 replicate units for each mean. View Large Table 6. Mean absolute intestinal coccidia counts for injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine and turn-out times (TOT) at d 35 posthatch. Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Histological enumeration of absolute coccidia count of 10 fields per sample. 1n = 18 replicate units for each mean. View Large DISCUSSION In previous studies in which 50 μL of the EM1 vaccine was injected into broiler hatching eggs on 18.5 doi, it was shown that EM1 did not have a negative effect on embryogenesis through 21 doi (Sokale et al., 2017a) or performance through 14 d posthatch (Sokale et al., 2017b). In this current study, the effects of 2 doses of EM1 injected at d 19.0 doi, on embryogenesis, and 2 EM1 doses and 2 TOT on performance from 0 to 35 d posthatch were evaluated. Effects of the physiological age of embryos and the site of injection on the efficacy of an in ovo vaccination has been previously described by Sokale et al. (2017a). Based on the embryo stage and site of injection scores in the current study, it is suggested that the physiological age at which the embryos were in ovo-injected corresponded to 19 doi time point, which resulted in EM1 vaccine deposition occurring mostly in the right breast muscle. The EM1 vaccine is recommended for injection at 18 to 19 doi, and although the amnion is the preferred site of injection, studies have shown that in late-stage embryos in which the volume of amniotic fluid is reduced, the possibility of depositing vaccine directly into the amnion is decreased, and the probability of it being injected in the body of the embryo is increased (Jochemsen and Jeurissen, 2002). A report by William (2007) stated that the optimum time for in ovo injection during embryo development, is from 17.5 doi up to 19 doi + 4 h. These times correspond to the stage of physiological development when the yolk sac stalk begins to ascend into the abdomen and the head is tucked under the wing up until pipping is initiated. This was also supported in studies by Williams and Zedek (2010) and by Sokale et al. (2017a) which showed that when vaccinating late-stage embryos, the Inovoject system will deliver the EM1 vaccine into the intramuscular (i.m.) and subcutaneous (s.c.) regions of the embryo and to lesser extent in the amnion. Based on these reports, the site of injection evaluation indicates that the in ovo injection of EM1 in late-stage embryos in the current study was optimum (amnion: 6.8%; i.m.: 81.5%; and s.c.:11.7%). However, there is no published literature on the development of coccidiosis vaccines administered by the in ovo i.m. or s.c route. Nonetheless, it is important to note that in ovo injection of late-stage embryos at 19.0 is a common practice in the commercial hatchery. The non-detrimental effect of the 10 × EM1 treatment on embryogenesis may be due to the high proportion of the vaccine being deposited i.m. at 19.0 doi, which could occur in late-stage embryos, hence vaccines intended for deposition in the amnion are deposited in the embryo body (i.m. or s.c.) and therefore can affect the birds’ response to the vaccine. In comparison to the NIC group, the injection of the 10 × EM1, similar to the injection of the 1 × EM1, resulted in a decrease in chick BW, RBSW, and YSW. In addition, there was a numerical decrease in HI and HBW in the vaccine injected (1 × EM1 and 10 × EM1) groups in comparison to the NIC group at 21 doi. The observed decreases in these hatchling quality variables examined suggests a common factor between the 1 × EM1 and 10 × EM1 that is different from the NIC group. This factor, being the creation of injection holes during the late embryonic stage, might have introduced a stressor to the embryos, thereby resulting in a reduction in the various chick quality variables. This is consistent with previous results reported by Sokale et al., (2017a) and Bello et al. (2013), who showed a decrease in hatchability in groups that were subjected to the injection process. Furthermore, decreases in chick BW, RBSW, and YSW in the 10 × EM1 in comparison to both the 1 × EM1 and NIC groups may also suggest that a higher dose of the EM1 has some negative effect on embryogenesis. The current and past results corroborate in indicating that specific hatching chick quality variables may be negatively affected during the in ovo injection of embryos at the late embryonic phase. Repeated exposures to coccidia oocysts (generally referred to as coccidia cycling) allows birds to develop immunity to the disease early in their life, thereby reducing the later impact of diseases. The duration of repeated exposures of birds to coccidia oocysts in the broiler house is necessary for the development of optimal immunity against coccidiosis. In practice, the recycling of oocysts is achieved through partial house brooding, in which birds are confined to a section of the house (usually a one-third to one-half portion of the house) for a limited period of time (usually between 7 and 14 d). Thereafter, birds are released (turned-out) to the entire house for the remainder of the grow-out period (Merck Animal Health Management Guidelines, 2016). In a study conducted by Weber and Evans (2003) in which Eimeria tenella sporozoites, sporocysts, or oocysts were injected in ovo, the peak of fecal oocyst shedding was observed at 7 d posthatch. Similarly, Sokale et al., (2017a) showed peaks in oocyst shedding on d 7 and 10 posthatch in broiler chickens raised in cages following the injection of in ovo injection of EM1. Based on these, in this study we selected 7 and 10 d posthatch to mimic early, and potentially ideal TOT, respectively. For in ovo coccidiosis vaccines, it has been suggested that oocysts may remain dormant in the embryo intestine so that chicks may become infected around hatching, allowing an early completion of their life cycle in comparison to hatchery sprayed vaccines (Weber et al., 2004). Under this circumstance it may be possible that certain Eimeria spp., such as E. acervulina, will complete their life cycle prior to d 7 posthatch. Furthermore, birds are already re-ingesting oocysts at this time. Therefore, choosing 7 d posthatch for early TOT is important in order to evaluate this effect. During partial house brooding, birds are exposed to several cycles of the passage and ingestion of coccidia oocysts (cycling). However, a balance between the number of ingested oocyst necessary to establish immunity against coccidiosis and that which can lead to disease is important. Based on this, the 10 d posthatch was chosen as a second TOT because it coincides with the period of moderate cycling, allowing for fecal oocyst passage and sporulation, but prior to the 2 major coccidia cycles at 14 and 21 d posthatch. The objective of evaluating these 2 TOT with the EM1 vaccine was to better define an ideal TOT when the EM1 vaccine is administered by in ovo injection. In this study, TOT alone did not have any significant effect on any of the parameters evaluated from 0 to 35 d posthatch. However, there was an INT × TOT interaction effect on 35 d posthatch intestinal morphology. The small intestine crypt depth was higher in birds belonging to the NIC-d10 TOT treatment group, which may indicate a higher damage index to the intestinal structure as a result of the birds ingesting oocysts from the environment late in the cycle, since this group was not vaccinated with EM1. However, there was no significant differences in intestinal villus height and VCD among the treatment groups. Long intestinal villi and shallow crypts are indicative of an optimal intestinal condition, whereas a shortening of the villi and a larger crypt depth indicate a faster turn-over of epithelial cells that reflects an increased challenge pressure on the intestine (Timbermont et al., 2011). The main measurable effects during the 0 to 35 d posthatch performance came from the INT (NIC, 1 × EM1 and 10 × EM1). Performance evaluation revealed a significant INT effect on BWG and FCR during the 21- to 28-d posthatch interval. The lower BWG and feed efficiency in both 1 × EM1 and 10 × EM1 groups in comparison to the NIC group may suggests an on-going coccidia cycling around this period of 21- to 28-d posthatch interval. The peak of oocyst cycling typically occur between 21 and 28 d posthatch and may predispose birds to stress, with a subsequent reduction in BW gain and poor feed efficiency (Broomhead, 2012). The effect of INT on the RIBW at 28 d posthatch further supports the thought that an increase in oocyst production and coccidia cycling may have resulted in the observed vaccine effect on performance. The RIBW was higher in birds belonging to the 1 × EM1 and 10 × EM1 groups in comparison to the NIC group, suggesting that coccidia development significantly increased in the intestine by at 28 d posthatch. This is also consistent with the results obtained in previous studies. Küçükyilmaz et al. (2012) showed a significantly higher cecal weight and intestine length in coccidia-infected birds in comparison to uninfected birds. Further, Baurhoo et al. (2007) showed no significant increase in intestinal weight during low pathogen challenge in feed supplemented with additives, compared to the control group. Increase in intestinal weight has been used as an indicator of intestinal health and is thought to be associated with inflammation following enteric challenge, although several factors can influence intestinal physiology and environment (Walton, 1988). In the overall 0- to 35-d posthatch interval, birds belonging to the NIC group had better BWG and FCR than the vaccine-injected birds. Although there was evidence of on-going coccidia cycling at age 35 d posthatch in the NIC group (shown by the mean intestinal coccidia count) the level may not have negatively influenced performance. It is noteworthy, however, that the numerical reduction in the coccidia count in birds belonging to the vaccinated groups may indicate that early vaccination with EM1 may help to reduce oocyst production by 35 d posthatch, possibly due to the development of early immunity to the coccidiosis infection. On the other hand, the low BWG observed in birds belonging to the vaccinated groups at 35 d posthatch may indicate a carryover effect from coccidia cycling between 21 and 28 d posthatch, in which vaccinated birds are less feed efficient and have reduced BW and therefore require some time to recover from the cycling of coccidia in the intestine. The interpretation of this outcome, however, is confounded by the fact that the EM1 was injected in the i.m. site in a majority of the embryos. In conclusion, the in ovo injection of the EM1 vaccine at either a 1 × or 10 × dose in this study, showed that there is a higher chance of vaccine deposition intramuscularly rather than in the amnion when embryos are injected at 19 doi. However, no detrimental effect was observed on hatchability and embryo survivability. Nevertheless, the Ross × Ross 708 broiler hatchling quality variables such as BW, RBSW, and YSW may be affected by in ovo injection. The overall growth performance of birds belonging to the vaccine-injected groups was significantly reduced at 35 d posthatch, although TOT (at 7 or 10 d posthatch) alone or in combination with INT did not significantly affect performance. The RIBW of birds belonging to the vaccine-injected groups was significantly higher at 28 d posthatch, and may have influenced the performance of these groups at 35 d posthatch. At 35 d posthatch, no significant difference in the coccidia counts and small intestine morphology were found among the NIC, 1 × EM1 and 10 × EM1 groups, which indicates that the vaccine challenge was not detrimental. The performance evaluation and intestinal weight at d 28 posthatch suggests that vaccination by EM1 injection was effective with the necessary cycling of coccidia. Therefore, it may be recommended that under commercial conditions, a 1 × EM1 vaccine in conjunction with partial house brooding up to 10 d posthatch, may be employed to ensure adequate oocyst cycling, without subsequently causing any negative effects on Ross × Ross 708 broiler growth performance. ACKNOWLEDGMENTS We express our appreciation for the financial support of Zoetis Animal Health, the expert technical assistance of Sharon K. Womack and Eric Nixon, and for the assistance of the interns, and graduate and undergraduate students 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 Baurhoo B. , Phillip L. , Ruiz-Feria C. A. . 2007 . Effects of purified lignin and mannan oligosaccharides on intestinal integrity and microbial populations in the ceca and litter of broiler chickens . Poult. Sci. 86 : 1070 – 1078 . Google Scholar CrossRef Search ADS PubMed Bello A. , Zhai W. , Gerard P. D. , Peebles E. D. . 2013 . Effects of the commercial in ovo injection of 25-hydroxycholecalciferol on the hatchability and hatching chick quality of broilers . Poult. Sci. 92 : 2551 – 2559 . 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Poult. Prod . 15 : 7 – 8 . Williams C. J. , Zedek A. S. . 2010 . Comparative field evaluations of in ovo applied technology . Poult. Sci. 89 : 189 – 193 . Google Scholar CrossRef Search ADS PubMed © 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 injection of different doses of coccidiosis vaccine and turn-out times on broiler performance,,

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT Inovocox EM1 vaccine (EM1) is hatchery-applied via in ovo injection for the control of coccidiosis in broiler chickens. Effects of 3 in ovo injection treatments (INT) and 2 turn-out times (TOT) on the hatching chick quality variables and 35 d posthatch performance of Ross × Ross 708 broilers were investigated. In a single-stage incubator, 1,440 hatching eggs were randomly distributed among 3 INT groups on each of 8 tray levels. At 19 d of incubation (doi), embryonated eggs were subjected to one of the following INT by in ovo injection: noninjected control; 1 × dose of EM1; 10 × dose of EM1. On 21 doi, hatchability of injected eggs (HI), hatching body weight (HBW), and hatching chick quality variables were determined. Additionally, for the grow-out phase, birds belonging to each INT were randomly subjected to a 7 or 10 d TOT. Twenty chicks were initially placed in each of 48 floor pens (6 INT × TOT combination groups × 8 replications) for growth performance evaluation from 0 to 35 d posthatch. The main effect of INT on hatching chick quality variables, as well as the main and interactive effects of INT and TOT on various grow-out performance variables were determined. Although there was no significant INT effect on HI or HBW, significant INT effects on chick total BW, yolk-free BW, and yolk sac weight were observed. There were significant INT effects on BWG and FCR in the 21- to 28-d posthatch interval, as well as on BWG and FCR in the 0- to 35-d posthatch interval. There was no main effect of TOT or interactive effect of INT and TOT on BW and other performance variables from 0 to 35 d posthatch. There was a significant main effect of INT on relative intestine weight at 28 d posthatch. In conclusion, the injection of EM1 vaccine at a 10 × dose may affect hatching chick quality variables and growth performance up to 35 d posthatch. INTRODUCTION The protozoan parasite of the genus Eimeria, which causes avian coccidiosis, develops in the intestinal tract of birds, causing morbidity, mortality, poor feed efficiency, and poor weight gain, and predisposes birds to secondary diseases such as necrotic enteritis (Williams, 2005). Coccidia oocysts are ubiquitous to commercial chicken houses, and therefore, only control of coccidiosis, rather than complete eradication, is attainable under commercial conditions (Chapman, 2000; Tewari and Maharana, 2011). The control of coccidiosis is achieved by use of in-feed anticoccidials and coccidiosis vaccines in different types of grow-out programs, such as rotation or bio-shuttle programs (Chapman et al., 2002; Montoya and Quiroz, 2013). When used in rotation programs, coccidiosis vaccines help to restore the precocious strains (drug-resistant) of coccidia oocysts that have sensitivity to in-feed anticoccidials (Chapman and Jeffers, 2014). In a bio-shuttle program, coccidiosis vaccines are used at day of hatch, and are followed by low levels of ionophore anticoccidials in the grower and finisher feeds (Montoya and Quiroz, 2013). In addition, due to consumer demand and changing regulatory landscapes, poultry producers are not only reducing the use of antibiotic growth promoters, but also ionophore anticoccidials in poultry feed. The result of this is that producers have fewer options to help control coccidiosis, and therefore rely on either non-ionophore (synthetic) anticoccidials or almost exclusively on vaccines for the control of coccidiosis. Several disease models that evaluate the bird's response to test substances like feed additives have utilized coccidia vaccines up to 10× normal levels to challenge the bird's intestine, and increase the likelihood of overgrowth of Clostridium perfringens that produces dysbacteriosis, necrotic enteritis, or sub-optimal growth conditions in the birds. In order to effectively simulate these conditions under research conditions similar to commercial conditions, it is important to consider the life cycle of coccidia and the environmental and management factors that can help to maximize the outcome of the challenge, especially those that affect sporulation and re-cycling of the coccidia oocysts. The most recognized predisposing factor for necrotic enteritis is damage to the intestine caused by a coccidiosis infection. Damage to intestinal villus and crypt morphology leads to a subsequent loss in performance and an increase in mortality in affected birds (Timbermont et al., 2011). From a practical standpoint, ensuring the efficacy of an in ovo coccidiosis vaccine is an interplay of several factors, which begins at the hatchery and continues into the chicken house. At the hatchery, it is important to ensure the precise stage of embryo development, as this will ensure that the vaccine is delivered to the correct injection site (Sokale et al., 2017a). Previous work has shown that in ovo vaccination of embryos at 18 d of incubation (doi) for Marek's Disease (Williams and Zedek, 2010) or 18.5 doi for coccidiosis (Sokale et al., 2017a) resulted in a high delivery accuracy. However, the vaccination of embryos later than these times (e.g., at 19 doi) is a common practice under commercial conditions. In the chicken house, exposure of birds to multiple coccidia life cycles (oocyst cycling) can potentially initiate an immune response necessary for the control of coccidiosis (Chapman et al., 2002; Tewari and Maharana, 2011). In order to achieve this oocyst cycling from coccidiosis vaccination, partial house brooding management has been recommended by vaccine manufacturers. Partial house brooding during the first few d of placement allows the birds to have a repetitive fecal-oral exposure to high numbers of coccidia oocysts, which initiates their development of immune competence against coccidiosis (Mathis, 2001; Schering-Plough Animal Health, 2007). However, the appropriate duration of repeated exposures of birds to coccidia oocysts in the Inovocox EM1 vaccine (EM1; Zoetis, Kalamazoo, MI) under varying application conditions, as well as the effects on growth performance, have not been documented in the scientific literature. Therefore, the objective of this study was to determine the effects of EM1vaccine injected into 19-day-old broiler embryos at a high or low dose, with 2 partial house brooding time-points, on grow-out performance. For this study, we chose 19 doi in order to mimic industry conditions of late-stage embryo vaccination. In addition, 7- and 10-d posthatch were selected as the 2 partial house brooding time-points to evaluate the effects early and late turn-out times have on coccidia oocyst cycling when EM1 is injected at a high or low dose. We evaluated growth performance and intestinal morphometrics as an indication of how the broiler chickens responded under these given conditions. MATERIALS AND METHODS General This study was conducted according to a protocol that was approved by the Institutional Animal Care and Use Committee of Mississippi State University. A total of 2,160 Ross × Ross 708 broiler hatching eggs were obtained from a 45-week-old commercial breeder flock, and held for 2 d under standard storage conditions prior to setting. On 0 doi, eggs were weighed and labeled according to methods previously described by Sokale et al. (2017a). A total of 1,440 eggs were randomly assigned to 3 injection treatment (INT) groups, each containing 60 eggs, on each of 8 replicate trays (blocks). After candling on 18 doi, a total of 1,310 embryonated eggs were retained across the 3 INT and their 8 replicate groups (each replicate group contained approximately 54 embryonated eggs). All eggs were incubated in a Jamesway model PS 500 single-stage incubator (Jamesway Incubator Co. Inc., Cambridge, Ontario, Canada), under standard conditions (Peebles and Brake, 1987). On 19 doi, eggs were subjected to one of the 3 INT: noninjected control (NIC), 1 × dose of EM1 (1 × EM1), 10 × dose of EM1 (10 × EM1). The dosages for the EM1 were attained as described below. Injection and Experimental Layout Injection of eggs was performed on 19 doi using an Embrex Inovoject injector system (Zoetis Animal Health, Research Triangle Park, NC), as described by Sokale et al. (2017a). According to recommendations by the vaccine manufacturer (Zoetis Animal Health), 3 vials (8,000 doses each) of EM1 were reconstituted in 1,200 mL of sterile commercial MD vaccine diluent (Merial Co., Duluth, GA) for the 1 × EM1, and 30 vials (8,000 doses each) of EM1 were reconstituted in 1,200 mL for the 10 × EM1. At the time of immunization, the EM1 vaccine still had approximately 7 months before its expiration date. The vaccine containing oocysts of E. acervulina, E. maxima, and E. tenella were administered at the rate of 50 μL per egg. Eggs belonging to each INT group were injected separately with an intervening machine cleaning cycle to avoid cross-contamination. Once the entire injection process was completed, eggs were transferred to the hatcher unit (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada) on their corresponding replicate tray levels. Eggs in the hatcher baskets were arranged in a way to prevent cross-contamination between chicks belonging to the vaccine injected and non-injected groups. The site of injection was confirmed according to the method previously described by Sokale et al. (2017a). At 21 doi, the effects of INT on the various hatching chick quality variables described below were determined. In the grow-out phase of the study, the turn-out time (TOT), which indicates the duration of repetitive exposure to coccidia oocysts, was included along with INT in the performance evaluation. Thus, a 3 INT × 2 TOT factorial design was employed. A group of 6 miniature floor pens, each representing a specific INT × TOT combination were randomly assigned to a block, with 8 total replicate blocks in the grow-out facility. This resulted in a total of 48 INT × TOT treatment replicate pens. On 21 doi, a total of 20 straight-run chicks randomly selected from each INT replicate group were allocated to 1 of 2 TOT, and were wing-banded, weighed, and placed in each of the 48 pens. Each pen measured 1.1 m2 within a temperature-controlled research facility. The birds (20 birds × 48 pens = 960 total birds) were placed on litter that had been previously used for 2 grow-out cycles. Birds were fed ad libitum with a crumbled starter diet from 0 to 14 d posthatch, pelletized grower diet from 15 to 28 d posthatch, and pelletized finisher diet from 29 to 35 d posthatch. Diets were formulated to meet or exceed NRC (1994) recommendations. House temperature conditions were monitored and recorded 2 times daily from 0 to 35 d posthatch. In order to achieve the specified TOT, pens were divided using plastic wire mesh into three-fourths (0.83 m2) and one-fourth (0.28 m2) portions. The plastic wire mesh prevented birds from crossing over to the unused side of each pen without interfering with air flow. All 20 chicks were initially placed in the three-fourths portion of each pen, at a stocking density of 0.04 m2/bird, and on either 7 or 10 d posthatch, birds within each of the 3 INT in each of the 8 replicate blocks were turned out (8 × 3 = 24 pens at each of the 2 TOT). Turning-out involved the removal of the plastic wire mesh used to divide each pen, so that birds were allowed the entire 1.1 m2 space in each pen, at a stocking density of 0.06 m2/bird, up to 35 d posthatch. Data Collection Set egg weight (SEW) was recorded on 0 doi. On d of injection (19 doi), both the site of injection and embryo stage score were determined to evaluate vaccine delivery accuracy as described by Sokale et al. (2017a). On 21 doi, the hatchability of injected fertilized eggs (HI) and mean hatching body weight (HBW) of chicks in each INT replicate group were determined. Additionally, 16 chicks from each INT group were individually selected, wing-banded, euthanized, weighed, and necropsied to determine the following hatching (21 doi) chick quality parameters: total body weight (BW), and absolute yolk sac (YSW), liver (LW), whole intestine (IW), and heart (HW) weight. In addition, the following were determined: yolk-free BW (YFBW), YSW relative to total BW (RYBW), IW relative to total (RIBW) and yolk-free (RIYFW) BW, LW relative to total (RLBW) and yolk-free (RLYFW) BW, HW relative to total (RHBW) and yolk-free (RHYFW) BW, total BW relative to SEW (RBSW), YFBW relative to SEW (RYFWSW), and yolk-free body mass (YFBM). On 28 d posthatch, 2 chicks from each pen were individually selected, euthanized, weighed, and necropsied for determination of total BW, IW, and RIBW. For evaluation of bird performance, BW gain (BWG), feed intake (FI), and mortality-adjusted feed conversion ratio (FCR) were determined for the weekly and entire 0- to 35-d posthatch intervals. Percentage cumulative mortality (CPM) for the entire 0- to 35-d posthatch interval was also determined. Additionally, on 35 d posthatch, 3 birds taken from 3 replicate groups within each INT × TOT treatment combination were randomly selected, euthanized, weighed, and necropsied for intestinal histomorphometric analysis. The small intestinal samples were collected and preserved in 10% buffered neutral formalin. The tissue samples were trimmed, and transverse sections of approximately 5 μm were stained with hematoxylin and eosin. All slides were evaluated and digitalized for histomorphometry using an Amscope 5 MP camera and image J software (AmScope, Irvine, CA). Images were analyzed for villus height and crypt depth, and the villus-to-crypt depth (VCD) ratio was calculated. Five villi and crypts were measured per sample, and the mean was calculated. In addition to the morphometric analysis, the enumeration of absolute count of coccidia in each sample was accomplished by histological method. The absolute count of coccidia stages (sporocysts) was obtained by counting a total of 10 fields in each treatment sample (at 400× magnification) and the mean for each treatment was calculated and reported. Statistical Description A randomized complete block design was used in both the incubational and grow-out periods. In the incubational phase, each tray level represented a block, and all treatments were equally and randomly represented in each block. The chick quality data on 21 doi were analyzed using the 3 INT groups (NIC, 1 × EM1, and 10 × EM1). A 1-way analysis of variance (ANOVA) was used to analyze the main effect of INT on the hatching chick quality variables. The performance data were arranged in a 3 INT × 2 TOT factorial design to evaluate the main and interactive effects of INT and TOT on performance. A 2-way ANOVA was used to analyze the main and interactive effects of INT and TOT on 28 d posthatch IW and RIBW, CPM in the 0- to 35-d posthatch interval, and weekly BWG, FI, and FCR. All individual data within each replicate group was averaged and all percentage data was arcsine transformed prior to analysis. A split-plot analysis was performed for weekly absolute BW with INT and TOT as whole plot factors split over posthatch d. In the aforementioned analysis, INT and TOT were considered as fixed effects and block as a random effect. For histomorphometric variable evaluation, a 1-way ANOVA was used to analyze the main effects of INT or TOT on coccidia count, and a 2-way ANOVA was used to analyze the main and interactive effects of INT and TOT on villus height, crypt depth, and VCD ratio. For this analysis, INT and TOT were considered as fixed effects and each individual bird as a random effect. Least-square means were compared in the event of significant global effects (Steel and Torrie, 1980). All variables were analyzed using the MIXED procedure of SAS software 9.3 (SAS Institute, 2012). Global and least-square means differences were considered significant at P ≤ 0.05. RESULTS Using approximately 7% of the in ovo-injected embryonated eggs, the site of injection and embryo stage score at 19 doi were evaluated. Mean embryo stage score was 4.60 ± 0.99, and site of injection evaluation indicated that 6.8 and 93.2% of the eggs received vaccine in the amnion and embryo body, respectively, with embryo body injections being 81.5% intramuscular and 11.7% subcutaneous. There was no significant INT effect on SEW at 0 doi or on HI, HBW, YFBW, YFBM, RYFWSW, IW, RIBW, RIYFW, LW, RLBW, RLYFW, HW, RHBW, and RHYFW at 21 doi. However, there was a significant INT effect on chick BW, RBSW, and YSW at 21 doi. The BW, RBSW and YSW values were higher in the NIC group compared to the 10 × EM1 group, with the 1 × EM1 group being intermediate. The INT means for those 3 variables and all of the other hatching chick quality variables evaluated at 21 doi are provided in Table 1. Table 1. Main effect of injection treatment (INT): noninjected control group (NIC), 1× dose EM1 (1× EM1), and 10× dose EM1 (10× EM1) vaccine on the somatic and yolk variable means of selected chicks at 21 d of incubation.1,2 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 a,bMeans within a variable with no common superscript differ (P ≤ 0.05). 1Set egg weight (SEW), hatchability of injected fertilized egg (HI), hatching body weight (HBW), body weight (BW), yolk-free BW (YFBW), BW as a percentage of set egg weight (RBSW), yolk-free BW as a percentage of set egg weight (RYFWSW), intestine weight (IW), intestine weight as a percentage of BW (RIBW), intestine weight as a percentage of yolk-free BW (RIYFW), liver weight (LW), liver weight as a percentage of BW (RLBW), liver weight as a percentage of yolk-free BW (RLYFW), heart weight (HW), heart weight as a percentage of BW (RHBW), heart weight as a percentage of yolk-free BW (RHYFW), yolk sac weight (YSW), yolk sac weight as a percentage of BW (RYBW), and yolk-free body mass (YFBM). 2Two birds in each of 8 replicate units per treatment (16 birds per treatment group) were used to calculate each treatment mean. 3Sixty eggs in each of 8 replicate units per treatment (480 eggs per treatment group) were used to calculate mean SEW. 4Data from 8 replicate units was used for calculation of HI and HBW means for each treatment group. View Large Table 1. Main effect of injection treatment (INT): noninjected control group (NIC), 1× dose EM1 (1× EM1), and 10× dose EM1 (10× EM1) vaccine on the somatic and yolk variable means of selected chicks at 21 d of incubation.1,2 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 SEW3 HI4 HBW4 BW YFBW RBSW RYFWSW IW RIBW RIYFW LW RLBW RLYFW HW RHBW RHYFW YSW RYBW YFBM INT (g) (%) (g) (g) (g) (%) (%) (g) (g) (%) (g) (%) (%) (g) (%) (%) (g) (%) (%) NIC 64.8 96.1 45.2 45.9a 40.9 70.8a 63.1 2.19 4.76 5.33 1.22 2.66 2.98 0.354 0.761 0.850 4.94a 10.8 89.2 1× EM1 64.6 93.1 44.8 44.2a,b 39.5 68.5a,b 61.2 2.20 4.98 5.57 1.16 2.63 2.94 0.341 0.773 0.862 4.70a,b 10.6 89.4 10× EM1 64.5 95.1 44.7 43.0b 39.1 66.7b 60.6 2.21 5.14 5.66 1.21 2.81 3.09 0.333 0.781 0.861 3.95b 9.15 90.9 SEM 0.18 1.17 0.22 0.61 0.56 0.92 0.83 0.06 0.13 0.14 0.03 0.06 0.07 0.01 0.02 0.02 0.28 0.60 0.60 P-value 0.39 0.22 0.32 0.01 0.06 0.01 0.09 0.96 0.15 0.24 0.42 0.12 0.26 0.49 0.91 0.97 0.04 0.13 0.13 a,bMeans within a variable with no common superscript differ (P ≤ 0.05). 1Set egg weight (SEW), hatchability of injected fertilized egg (HI), hatching body weight (HBW), body weight (BW), yolk-free BW (YFBW), BW as a percentage of set egg weight (RBSW), yolk-free BW as a percentage of set egg weight (RYFWSW), intestine weight (IW), intestine weight as a percentage of BW (RIBW), intestine weight as a percentage of yolk-free BW (RIYFW), liver weight (LW), liver weight as a percentage of BW (RLBW), liver weight as a percentage of yolk-free BW (RLYFW), heart weight (HW), heart weight as a percentage of BW (RHBW), heart weight as a percentage of yolk-free BW (RHYFW), yolk sac weight (YSW), yolk sac weight as a percentage of BW (RYBW), and yolk-free body mass (YFBM). 2Two birds in each of 8 replicate units per treatment (16 birds per treatment group) were used to calculate each treatment mean. 3Sixty eggs in each of 8 replicate units per treatment (480 eggs per treatment group) were used to calculate mean SEW. 4Data from 8 replicate units was used for calculation of HI and HBW means for each treatment group. View Large There were no significant main effects or interactions involving INT or TOT on weekly BW; BWG, FI, and FCR in the 0- to 7-d, 7- to 14-d, 14- to 21-d, or 28- to 35-d posthatch intervals; or on FI in the 21- to 28-d and 0- to 35-d posthatch intervals. There was also no significant TOT main effect or INT × TOT interaction for BWG and FCR in the 21- to 28-d or 0- to 35-d posthatch intervals. However, there was a significant main effect of INT on BWG and FCR in the 21- to 28-d posthatch interval, and of INT on BWG and FCR in the 0- to 35-d posthatch interval. In both posthatch intervals, BWG was significantly higher in the NIC treatment group compared to both the 1 × EM1 and 10 × EM1 treatment groups, with no statistical difference between the 1 × EM1and 10 × EM1 treatment groups (Table 3). Furthermore, FCR was significantly lower in the NIC treatment group compared to both the 1 × EM1 and 10 × EM1 groups in both time intervals (Table 3). However, birds in the 1 × EM1 group displayed a better feed efficiency compared to birds in the 10 × EM1 group in the 0- to 35-d posthatch period (Table 3). The main effect means of INT for BWG, FI, and FCR for the 0- to 7-d, 7- to 14-d, and 14- to 28-d posthatch intervals are provided in Table 2, and for the 21- to 28-d, 28- to 35-d, and 0- to 35-d posthatch intervals are provided in Table 3. Table 2. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 0 to 7, d 7 to 14, and d 14 to 21 posthatch.1 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 120 birds in each of 8 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG1 = d 0 to 7 BW gain; FI1 = d 0 to 7 feed intake; FCR1 = d 0 to 7 feed conversion ratio; BWG2 = d 7 to 14 BW gain; FI2 = d 7 to 14 feed intake; FCR2 = d 7 to 14 feed conversion ratio; BWG3 = d 14 to 21 BW gain; FI3 = d 14 to 21 feed intake; FCR3 = d 14 to 21 feed conversion ratio. View Large Table 2. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 0 to 7, d 7 to 14, and d 14 to 21 posthatch.1 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 INT BWG1 (kg) FI1 (kg) FCR1 BWG2 (kg) FI2 (kg) FCR2 BWG3 (kg) FI3 (kg) FCR3 NIC 0.120 0.158 1.32 0.286 0.367 1.29 0.419 0.599 1.43 1 × EM1 0.119 0.157 1.32 0.274 0.349 1.28 0.409 0.591 1.45 10 × EM1 0.116 0.154 1.33 0.275 0.355 1.29 0.403 0.586 1.46 SEM 0.002 0.003 0.024 0.005 0.005 0.010 0.006 0.007 0.015 P-value 0.447 0.612 0.954 0.172 0.071 0.515 0.211 0.426 0.579 120 birds in each of 8 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG1 = d 0 to 7 BW gain; FI1 = d 0 to 7 feed intake; FCR1 = d 0 to 7 feed conversion ratio; BWG2 = d 7 to 14 BW gain; FI2 = d 7 to 14 feed intake; FCR2 = d 7 to 14 feed conversion ratio; BWG3 = d 14 to 21 BW gain; FI3 = d 14 to 21 feed intake; FCR3 = d 14 to 21 feed conversion ratio. View Large Table 3. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 21 to 28, d 28 to 35, and d 0 to 35 posthatch.1 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 a–cMeans within a column with no common superscript differ (P ≤ 0.05). 120 birds in each of 16 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG4 = d 21 to 28 BW gain; FI4 = d 21 to 28 feed intake; FCR4 = d 21 to 28 feed conversion ratio; BWG5 = d 28 to 35 BW gain; FI5 = d 28 to 35 feed intake; FCR5 = d 28 to 35 feed conversion ratio; BWG6 = d 0 to 35 BW gain; FI6 = d 0 to 35 feed intake; FCR6 = d 0 to 35 feed conversion ratio. View Large Table 3. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine on performance variable means from d 21 to 28, d 28 to 35, and d 0 to 35 posthatch.1 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 BWG4 (kg) FI4(kg) FCR4 BWG5 (kg) FI5(kg) FCR5 BWG6 (kg) FI6(kg) FCR6 NIC 0.691a 1.08 1.57b 0.555 1.19 2.16 2.07a 3.40 1.54c 1 × EM1 0.642b 1.05 1.64a 0.544 1.18 2.18 1.99b 3.32 1.57b 10 × EM1 0.640b 1.06 1.66a 0.543 1.20 2.20 1.98b 3.35 1.59a SEM 0.011 0.010 0.022 0.012 0.019 0.043 0.022 0.034 0.006 P-value 0.003 0.056 0.018 0.726 0.768 0.747 0.014 0.276 <0.001 a–cMeans within a column with no common superscript differ (P ≤ 0.05). 120 birds in each of 16 replicate units per treatment was used to calculate each treatment mean. Feed conversion ratio was adjusted for mortality. BWG4 = d 21 to 28 BW gain; FI4 = d 21 to 28 feed intake; FCR4 = d 21 to 28 feed conversion ratio; BWG5 = d 28 to 35 BW gain; FI5 = d 28 to 35 feed intake; FCR5 = d 28 to 35 feed conversion ratio; BWG6 = d 0 to 35 BW gain; FI6 = d 0 to 35 feed intake; FCR6 = d 0 to 35 feed conversion ratio. View Large There were no significant main effects or interactions involving INT or TOT on IW at 28 d posthatch, although the main effect of TOT on IW approached significance (P = 0.08). Mean IW in the d 7 and 10 TOT treatment groups were 73.3 g and 69.8 g, respectively. Similarly, there was no significant main effect of TOT or interactive effect of INT × TOT on RIBW at 28 d posthatch, although the main effect of TOT on RIBW approached significance (P = 0.07). Mean RIBW in the d 7 and 10 TOT were 4.80% and 4.60%, respectively. There was, however, a significant main effect of INT on RIBW at 28 d posthatch. The RIBW of birds in the 1 × EM1 and 10 × EM1 groups was significantly higher compared to that in birds belonging to the NIC group, but RIBW in the 1 × EM1 group was not significantly different from that of birds in the 10 × EM1 group. The INT main effect means for RIBW at 28 d posthatch are provided in Table 4. Table 4. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1× EM1), and 10 × dose EM1 (10 × EM1) vaccine on relative intestinal weight (RIBW) of birds at d 28 posthatch, and villus height and villus to crypt depth (VCD) ratio of the small intestine at d 35 posthatch. Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 a,bMeans with no common superscript differ (P ≤ 0.05). 1n = 16 replicate units for each RIBW mean. 2n = 18 replicate units for each villus height and VCD ratio mean. View Large Table 4. Main effect of injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1× EM1), and 10 × dose EM1 (10 × EM1) vaccine on relative intestinal weight (RIBW) of birds at d 28 posthatch, and villus height and villus to crypt depth (VCD) ratio of the small intestine at d 35 posthatch. Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 Injection-treatment RIBW1(%) Villus Height2 (μm) VCD2 NIC 4.42b 1172 4.14 1 × EM1 4.91a 1131 4.33 10 × EM1 4.78a 1217 4.64 SEM 0.09 172 0.66 P-value 0.03 0.94 0.87 a,bMeans with no common superscript differ (P ≤ 0.05). 1n = 16 replicate units for each RIBW mean. 2n = 18 replicate units for each villus height and VCD ratio mean. View Large There was no significant main effect of INT or TOT on villus height or VCD ratio at 35 d posthatch. There was also no significant INT × TOT interaction on villus height (P = 0.24) or VCD ratio (P = 0.11). For observational purposes, the INT means for villus height and VCD ratio are provided with those of RIBW in Table 4. There was a significant (P = 0.01) INT × TOT interaction for crypt depth. Small intestine crypt depth was higher in the NIC-d10 TOT treatment combination group in comparison to the NIC-d7 TOT and 10 × EM1-d10 TOT treatment groups, with all other treatment combination groups intermediate (Table 5). There was no significant main effect of INT or TOT on intestinal coccidia count at 35 d posthatch. There were, however, numerical differences in mean coccidia counts between the 3 INT groups and between the 2 TOT groups. The 35 d posthatch mean coccidia counts for the 3 INT groups and the 2 TOT are presented in Table 6. There was no significant main effect of INT (P = 0.35) or TOT (P = 0.30), and no significant (P = 0.34) INT × TOT interaction for the CPM of birds in the 0- to 35-d posthatch interval. However, there were numerical differences in the CPM of birds between the treatment groups in the 0- to 35-d posthatch interval. Birds belonging to the NIC-d7 TOT and NIC-d10 TOT had a CPM of 1.88% and 5.63%, respectively. Birds belonging to the 1 × EM-d7 TOT and 1 × EM-d10 TOT treatment combination groups had a CPM of 4.37% and 3.75%, respectively, and birds belonging to the 10 × EM-d7 and 10 × EM-d10 treatment groups had a CPM of 4.60% and 5.00%, respectively. Table 5. Interaction effect involving injection treatment [noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine] and turn-out time on d 7 and 10 posthatch, on small intestinal crypt depth at d 35 posthatch.1,2 TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b a,bMeans with no common superscript differ (P = 0.01). 1n = 18 replicate units for each mean. 2Pooled SEM = 48.5. View Large Table 5. Interaction effect involving injection treatment [noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine] and turn-out time on d 7 and 10 posthatch, on small intestinal crypt depth at d 35 posthatch.1,2 TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b TOT d 7 d 10 Injection treatment ——————(μm)—————– NIC 234b 412a 1 × EM1 289a,b 276a,b 10 × EM1 329a,b 219b a,bMeans with no common superscript differ (P = 0.01). 1n = 18 replicate units for each mean. 2Pooled SEM = 48.5. View Large Table 6. Mean absolute intestinal coccidia counts for injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine and turn-out times (TOT) at d 35 posthatch. Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Histological enumeration of absolute coccidia count of 10 fields per sample. 1n = 18 replicate units for each mean. View Large Table 6. Mean absolute intestinal coccidia counts for injection treatment (INT): noninjected control group (NIC), 1 × dose EM1 (1 × EM1), and 10 × dose EM1 (10 × EM1) vaccine and turn-out times (TOT) at d 35 posthatch. Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Treatment1 Absolute coccidia counts NIC 216 1 × EM1 92 10 × EM1 7.00 SEM 79 P-value 0.31 TOT d7 167 TOT d10 43 SEM 70 P-value 0.28 Histological enumeration of absolute coccidia count of 10 fields per sample. 1n = 18 replicate units for each mean. View Large DISCUSSION In previous studies in which 50 μL of the EM1 vaccine was injected into broiler hatching eggs on 18.5 doi, it was shown that EM1 did not have a negative effect on embryogenesis through 21 doi (Sokale et al., 2017a) or performance through 14 d posthatch (Sokale et al., 2017b). In this current study, the effects of 2 doses of EM1 injected at d 19.0 doi, on embryogenesis, and 2 EM1 doses and 2 TOT on performance from 0 to 35 d posthatch were evaluated. Effects of the physiological age of embryos and the site of injection on the efficacy of an in ovo vaccination has been previously described by Sokale et al. (2017a). Based on the embryo stage and site of injection scores in the current study, it is suggested that the physiological age at which the embryos were in ovo-injected corresponded to 19 doi time point, which resulted in EM1 vaccine deposition occurring mostly in the right breast muscle. The EM1 vaccine is recommended for injection at 18 to 19 doi, and although the amnion is the preferred site of injection, studies have shown that in late-stage embryos in which the volume of amniotic fluid is reduced, the possibility of depositing vaccine directly into the amnion is decreased, and the probability of it being injected in the body of the embryo is increased (Jochemsen and Jeurissen, 2002). A report by William (2007) stated that the optimum time for in ovo injection during embryo development, is from 17.5 doi up to 19 doi + 4 h. These times correspond to the stage of physiological development when the yolk sac stalk begins to ascend into the abdomen and the head is tucked under the wing up until pipping is initiated. This was also supported in studies by Williams and Zedek (2010) and by Sokale et al. (2017a) which showed that when vaccinating late-stage embryos, the Inovoject system will deliver the EM1 vaccine into the intramuscular (i.m.) and subcutaneous (s.c.) regions of the embryo and to lesser extent in the amnion. Based on these reports, the site of injection evaluation indicates that the in ovo injection of EM1 in late-stage embryos in the current study was optimum (amnion: 6.8%; i.m.: 81.5%; and s.c.:11.7%). However, there is no published literature on the development of coccidiosis vaccines administered by the in ovo i.m. or s.c route. Nonetheless, it is important to note that in ovo injection of late-stage embryos at 19.0 is a common practice in the commercial hatchery. The non-detrimental effect of the 10 × EM1 treatment on embryogenesis may be due to the high proportion of the vaccine being deposited i.m. at 19.0 doi, which could occur in late-stage embryos, hence vaccines intended for deposition in the amnion are deposited in the embryo body (i.m. or s.c.) and therefore can affect the birds’ response to the vaccine. In comparison to the NIC group, the injection of the 10 × EM1, similar to the injection of the 1 × EM1, resulted in a decrease in chick BW, RBSW, and YSW. In addition, there was a numerical decrease in HI and HBW in the vaccine injected (1 × EM1 and 10 × EM1) groups in comparison to the NIC group at 21 doi. The observed decreases in these hatchling quality variables examined suggests a common factor between the 1 × EM1 and 10 × EM1 that is different from the NIC group. This factor, being the creation of injection holes during the late embryonic stage, might have introduced a stressor to the embryos, thereby resulting in a reduction in the various chick quality variables. This is consistent with previous results reported by Sokale et al., (2017a) and Bello et al. (2013), who showed a decrease in hatchability in groups that were subjected to the injection process. Furthermore, decreases in chick BW, RBSW, and YSW in the 10 × EM1 in comparison to both the 1 × EM1 and NIC groups may also suggest that a higher dose of the EM1 has some negative effect on embryogenesis. The current and past results corroborate in indicating that specific hatching chick quality variables may be negatively affected during the in ovo injection of embryos at the late embryonic phase. Repeated exposures to coccidia oocysts (generally referred to as coccidia cycling) allows birds to develop immunity to the disease early in their life, thereby reducing the later impact of diseases. The duration of repeated exposures of birds to coccidia oocysts in the broiler house is necessary for the development of optimal immunity against coccidiosis. In practice, the recycling of oocysts is achieved through partial house brooding, in which birds are confined to a section of the house (usually a one-third to one-half portion of the house) for a limited period of time (usually between 7 and 14 d). Thereafter, birds are released (turned-out) to the entire house for the remainder of the grow-out period (Merck Animal Health Management Guidelines, 2016). In a study conducted by Weber and Evans (2003) in which Eimeria tenella sporozoites, sporocysts, or oocysts were injected in ovo, the peak of fecal oocyst shedding was observed at 7 d posthatch. Similarly, Sokale et al., (2017a) showed peaks in oocyst shedding on d 7 and 10 posthatch in broiler chickens raised in cages following the injection of in ovo injection of EM1. Based on these, in this study we selected 7 and 10 d posthatch to mimic early, and potentially ideal TOT, respectively. For in ovo coccidiosis vaccines, it has been suggested that oocysts may remain dormant in the embryo intestine so that chicks may become infected around hatching, allowing an early completion of their life cycle in comparison to hatchery sprayed vaccines (Weber et al., 2004). Under this circumstance it may be possible that certain Eimeria spp., such as E. acervulina, will complete their life cycle prior to d 7 posthatch. Furthermore, birds are already re-ingesting oocysts at this time. Therefore, choosing 7 d posthatch for early TOT is important in order to evaluate this effect. During partial house brooding, birds are exposed to several cycles of the passage and ingestion of coccidia oocysts (cycling). However, a balance between the number of ingested oocyst necessary to establish immunity against coccidiosis and that which can lead to disease is important. Based on this, the 10 d posthatch was chosen as a second TOT because it coincides with the period of moderate cycling, allowing for fecal oocyst passage and sporulation, but prior to the 2 major coccidia cycles at 14 and 21 d posthatch. The objective of evaluating these 2 TOT with the EM1 vaccine was to better define an ideal TOT when the EM1 vaccine is administered by in ovo injection. In this study, TOT alone did not have any significant effect on any of the parameters evaluated from 0 to 35 d posthatch. However, there was an INT × TOT interaction effect on 35 d posthatch intestinal morphology. The small intestine crypt depth was higher in birds belonging to the NIC-d10 TOT treatment group, which may indicate a higher damage index to the intestinal structure as a result of the birds ingesting oocysts from the environment late in the cycle, since this group was not vaccinated with EM1. However, there was no significant differences in intestinal villus height and VCD among the treatment groups. Long intestinal villi and shallow crypts are indicative of an optimal intestinal condition, whereas a shortening of the villi and a larger crypt depth indicate a faster turn-over of epithelial cells that reflects an increased challenge pressure on the intestine (Timbermont et al., 2011). The main measurable effects during the 0 to 35 d posthatch performance came from the INT (NIC, 1 × EM1 and 10 × EM1). Performance evaluation revealed a significant INT effect on BWG and FCR during the 21- to 28-d posthatch interval. The lower BWG and feed efficiency in both 1 × EM1 and 10 × EM1 groups in comparison to the NIC group may suggests an on-going coccidia cycling around this period of 21- to 28-d posthatch interval. The peak of oocyst cycling typically occur between 21 and 28 d posthatch and may predispose birds to stress, with a subsequent reduction in BW gain and poor feed efficiency (Broomhead, 2012). The effect of INT on the RIBW at 28 d posthatch further supports the thought that an increase in oocyst production and coccidia cycling may have resulted in the observed vaccine effect on performance. The RIBW was higher in birds belonging to the 1 × EM1 and 10 × EM1 groups in comparison to the NIC group, suggesting that coccidia development significantly increased in the intestine by at 28 d posthatch. This is also consistent with the results obtained in previous studies. Küçükyilmaz et al. (2012) showed a significantly higher cecal weight and intestine length in coccidia-infected birds in comparison to uninfected birds. Further, Baurhoo et al. (2007) showed no significant increase in intestinal weight during low pathogen challenge in feed supplemented with additives, compared to the control group. Increase in intestinal weight has been used as an indicator of intestinal health and is thought to be associated with inflammation following enteric challenge, although several factors can influence intestinal physiology and environment (Walton, 1988). In the overall 0- to 35-d posthatch interval, birds belonging to the NIC group had better BWG and FCR than the vaccine-injected birds. Although there was evidence of on-going coccidia cycling at age 35 d posthatch in the NIC group (shown by the mean intestinal coccidia count) the level may not have negatively influenced performance. It is noteworthy, however, that the numerical reduction in the coccidia count in birds belonging to the vaccinated groups may indicate that early vaccination with EM1 may help to reduce oocyst production by 35 d posthatch, possibly due to the development of early immunity to the coccidiosis infection. On the other hand, the low BWG observed in birds belonging to the vaccinated groups at 35 d posthatch may indicate a carryover effect from coccidia cycling between 21 and 28 d posthatch, in which vaccinated birds are less feed efficient and have reduced BW and therefore require some time to recover from the cycling of coccidia in the intestine. The interpretation of this outcome, however, is confounded by the fact that the EM1 was injected in the i.m. site in a majority of the embryos. In conclusion, the in ovo injection of the EM1 vaccine at either a 1 × or 10 × dose in this study, showed that there is a higher chance of vaccine deposition intramuscularly rather than in the amnion when embryos are injected at 19 doi. However, no detrimental effect was observed on hatchability and embryo survivability. Nevertheless, the Ross × Ross 708 broiler hatchling quality variables such as BW, RBSW, and YSW may be affected by in ovo injection. The overall growth performance of birds belonging to the vaccine-injected groups was significantly reduced at 35 d posthatch, although TOT (at 7 or 10 d posthatch) alone or in combination with INT did not significantly affect performance. The RIBW of birds belonging to the vaccine-injected groups was significantly higher at 28 d posthatch, and may have influenced the performance of these groups at 35 d posthatch. At 35 d posthatch, no significant difference in the coccidia counts and small intestine morphology were found among the NIC, 1 × EM1 and 10 × EM1 groups, which indicates that the vaccine challenge was not detrimental. The performance evaluation and intestinal weight at d 28 posthatch suggests that vaccination by EM1 injection was effective with the necessary cycling of coccidia. Therefore, it may be recommended that under commercial conditions, a 1 × EM1 vaccine in conjunction with partial house brooding up to 10 d posthatch, may be employed to ensure adequate oocyst cycling, without subsequently causing any negative effects on Ross × Ross 708 broiler growth performance. ACKNOWLEDGMENTS We express our appreciation for the financial support of Zoetis Animal Health, the expert technical assistance of Sharon K. Womack and Eric Nixon, and for the assistance of the interns, and graduate and undergraduate students of the Mississippi State University Poultry Science Department. 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Poultry ScienceOxford University Press

Published: Feb 15, 2018

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