Effects of in ovo vaccination time on broiler performance parameters under field conditions

Abstract

Hatchery performance is often evaluated based on descriptors such as hatchability, 7-d mortality, and cost. In addition to these descriptors, it is useful to include in this analysis aspects of chick quality through post-hatch performance. Realizing the bird's complete genetic potential necessitates meeting various criteria, with effective support for the chick's immune system being among the pivotal factors. To be effective, in ovo vaccination systems must deliver the vaccines to specific sites in the egg, a circumstance that directly depends on when the injection is made. We examined production data to evaluate the impact of in ovo vaccination time on performance parameters of male Ross308AP chicks. A comprehensive survey was conducted examining records from 3,722 broiler flocks produced and raised by the same company under standard nutrition and management conditions. The selected data specifically pertained to flocks that underwent slaughter between 41 and 45 d. In our analysis, 4 different linear models were built, one for each response variable: mean weight (MW), body weight gain (BWG), corrected feeding conversion rate (cFCR), and total mortality (TM). The linear models used in the analyses included as main predictor the timing of in ovo vaccination (440, 444, 448, 452, 456, 458, and 460 h of incubation), and as additional predictors: age of the breeding flock (26–35, 36–55 and 56–66 wks old), slaughter age, identity of the hatchery, and the season at which the data was collected. Our results showed that the timing of in ovo vaccination significantly affected BWG and cFCR, with procedures performed at 460 h of incubation showing the best outcomes. Breeding flock age affected all response variables, with older breeding flocks delivering increased MW, BWG and TM, and middle-aged flocks increased cFCR. Increasing slaughter age reduced BWG while MW, cFCR and TM were all increased. These data emphasize the benefits of performing in ovo vaccination as close as possible to 460 h of incubation to extract the best BWG and cFCR from Ross308AP male broiler.

INTRODUCTION

Genetic improvement in broiler production has reached a level where for fast-growing lines more than a third of the time elapsed between the start of incubation and slaughter weight is spent in the hatchery, making the hatchery a very strategic link in the production chain (Kroetz Neto et al., 2023). Industrial hatcheries play a crucial role in large-scale vaccination, ensuring standardization and effective process control through well-trained personnel and modern equipment, in addition to the biological target of earlier stimulation of active immunity (Franzo et al., 2020). Therefore, in ovo vaccination has become increasingly widespread in the broiler industry due to its advantages over conventional methods of post-hatch vaccination. Advantages include earlier immunity, reduced chick stress, fast and uniform delivery, and lower labor costs (Ricks et al., 1999; Williams, 2007; Williams and Zedek, 2010; Peebles, 2018). Egg injections must be performed at the correct embryonic age to ensure results, otherwise they may adversely affect hatchability, immune status and chick performance (Ricks et al., 1999; Williams, 2011; Fernandes et al., 2016).

In commercial hatcheries, in ovo vaccinations are delivered by automated systems, often without constant checking of the embryonic age or site of injection to confirm their accuracy (Williams, 2011). Yet during the last days of incubation embryos undergo continuous changes in their positioning and in the proportions of extraembryonic membranes. The delivery of vaccines outside the desired compartments, such as amniotic fluid and the embryo's muscles, may impair the embryo's immune response (Wakenell et al., 2002; Avakian, 2006; Williams, 2007; Williams and Hopkins, 2011; Sokale et al., 2017; Manders et al., 2021). Some studies suggest that timing of injections can vary between 422 and 466 h of incubation without interfering with the vaccine efficacy (Sharma and Burmester, 1982; Ricks et al., 1999; Wakenell et al., 2002; Williams and Hopkins, 2011; Fernandes et al., 2016). Nevertheless, more recent data indicate that embryos vaccinated outside the window of 444 to 460 h of incubation are at greater risk of receiving the vaccine outside the ideal location, making the vaccination ineffective (Peebles, 2018). While some studies have addressed the effects of the timing of egg injection on hatchability, very few examined its influence on posthatch performance (Ricks et al., 1999; Williams and Zedek, 2010; Williams, 2011; Fernandes et al., 2016; Sokale et al., 2017). In most cases, authors focused on the impact of a particular vaccine or the vaccination system itself, rather than on the implications of the timing of vaccination on posthatch performance. Thus, we decided to perform an analytical survey of performance data from 3,722 Ross308AP male broiler flocks produced and housed by a single company to explore how different in ovo vaccination times (at 440, 444, 448, 452, 456, 458, and 460 h of incubation) affect the posthatch performance in breeding flocks of different ages (26–35, 36–55 and 56–66 wks old).

MATERIAL AND METHODS

Experimental Design

This investigation was carried out utilizing data collected from 3,722 Ross308AP male broiler flocks produced and housed by the same Brazilian company between 2019 and 2021. For the analysis, broiler flocks were separated according to the age of the breeder flock from which they were produced: young, middle, and old breeders (26–35, 36–55, and 56–66-wk old, respectively). These flocks were housed in the same geographical region, received feed processed in the same factory (i.e., formulation, raw materials, etc.), and were managed using standard company procedures (e.g., feeding program, housing conditions, lightning, ambience and welfare, bedding quality, ventilation, etc.). Only flocks vaccinated using the Embrex Inovoject System (Pfizer Poultry Health, Research Triangle Park, NC) were included, in order to reduce possible variability related to the use of different injection equipment. The data were also screened to include only eggs vaccinated with (0.05 mL/egg) of Marek – Poulvac Ovoline HVT/ Zoetis Animal Health, and Gumboro – Transmune/ Ceva Saúde Animal. The timing of the in ovo vaccination was estimated as the interval (in hours) between the start of egg incubation (eggs placed into the setter) and the start of egg transfer (which took place between 440 and 460 h). Then, the influence this factor on chick performance was estimated using data for mean weight (MW), body weight gain (BWG), corrected feed conversion ratio (cFCR), and total mortality (TM) (see below).

All procedures complied with the current regulations establish by the Ethics Committee of the College of Veterinary Medicine and Animal Sciences at the São Paulo University (CEUAVET No 6556290520).

Egg Storage, Processing, and Incubation

Similar to broiler flock feeding and management, hatchery and egg handling procedures also followed company standards. Eggs were packed onto setter trays at the farm, and transported to the hatchery on the same day or the day after oviposition, with storage temperature at the poultry farms set at 22°C to 24°C. Eggs were transported to the hatchery in air-conditioned trucks running at temperatures 1°C to 2°C above those programmed for the hatchery storage rooms. Shortly after arrival at the hatchery, egg trollies were placed in the egg storage room (17°C–19°C), where they remained stored for up to 7 d. For preheating, egg trollies were kept in front of the incubators inside the incubation room for 4 to 8 h. Multistage setters with capacities for 124,416 or 115,200 eggs (models CASP MG 125 and COOPERMAQ 1152, respectively) were used. Incubation temperature and relative humidity remained between 99.3°F to 99.5°F and 80°F to 85°F (dry and wet bulb, respectively), depending on the breeder age and flock, throughout the entire incubation period. Egg were turned hourly until it was time to remove them from the setters for in ovo vaccination and transferring the eggs to the hatchers. Temperature and relative humidity in the hatchers ranged from 98.5°F to 97.5°F and 88°F to 84°F (dry and wet bulb, respectively). At 507 to 510 h of incubation, chicks were removed from the hatchers, sexed and sent to the company's farms to be housed.

Posthatch Performance

The chicks were housed in broiler farms on the day of hatch (within 4–8 h of removal from the hatchers). Although the company recorded mean weight, body weight gain, feed conversion ratio, and mortality data on a weekly basis, only the results at the time of slaughter were considered. Broilers’ weekly weight was measured using an automatic scale hanging from the ceiling with a box where animals were placed in groups. Around 1% of the flock was weighed weekly. The weight sampling system required the collection of individuals from 4 different areas of the poultry barn and weighing took place at the same time every week. In the first week, around 30 birds were weighed at a time (in the same box), in the second and third weeks 20 birds were weighed at a time, in the fourth and fifth weeks 15 birds were weighed at a time, and the total weights were added up and divided by the number of birds to obtain the average weight. These data were later used to calculate mean weight (MW of 10% of the housed broilers), body weight gain (BWG = average bird weight/ age of the bird) (Tavares and Schiassi, 2016); corrected feed conversion ratio (cFCR = [(2.8–average flock weight)/4] + feed conversion ratio) (Aviagen, 2022), and total mortality (TM = (number of dead animals /numbers of housed animals) x 100).

Statistical Analysis

All statistical analyses were performed using the R environment programming language (v4.0.0, R core team). For the statistical analysis, 4 different linear models were built, one for each response variable. The response variables used were mean weight (MW), body weight gain (BWG), corrected feeding conversion rate (cFCR) and total mortality (TM). All the linear models included as main predictor the treatments where the timing of in ovo vaccination was different. Additionally, the linear models included as predictors the following factors: age of the breeding flock, slaughter age, identity of the hatchery that provided the data, and the season at which the data was collected. The inclusion of these additional factors made it possible to control for the potential confounding effects of other unmeasured and/or source of errors on the 4 response variables. We first explored linear models that included the interaction between the age of the breeder flock and times of vaccination, as maternal effects from the breeder flock could boost or mitigate the effects of the vaccine. However, since this interaction was not significant for all response variables, we used the more parsimonious models, that is, without this interaction term.

The statistical significance of each predictor was assessed using Wald's type II chi-square tests and confidence intervals of the estimates of the coefficients (effects) of predictors. For Wald's type II tests, predictors are statistically significant when the corresponding P-value is smaller than 0.05 (i.e., P < 0.05). For the confidence interval analysis, when confidence intervals do not overlap zero, the effects of a given predictor can be considered statistically significant. In our linear models all results from the Wald's type II tests agreed with the confidence intervals approach, suggesting that the model is robust under different statistical tests. Model diagnosis was performed by visually inspecting the residuals. All the residuals were normally distributed, and the variance was homogeneous.

RESULTS

Analysis showed that broiler MW was significantly affected by the breeding flock age and the time before slaughter (Figure 1 and Table 1). Broilers from older breeding flocks and later slaughter exhibited higher MW (–0.052 ± 0.012 for young breeders, and 0.159 ± 0.015 for the highest slaughter age). In contrast, the timing of in ovo vaccination did not alter MW, although broilers from eggs vaccinated at 460 h of incubation showed a slight increase in MW compare to the others (0.017 ± 0.018). BWG was significantly influenced by all predictor variables (Figure 2 and Table 2). Lower BWG was obtained from young and middle-aged parents and in broilers slaughtered above 41 d (–1.147 ± 0.208 for young breeders, and –2.281 ± 0.337 for the highest slaughter age). Timing of in ovo vaccination significantly increased BWG with the highest increase occurring in broilers coming from eggs vaccinated at 460 h of incubation (0.778 ± 0.404).

Figure 1.

Relationship between broiler mean body weight and (A, B) timing of in ovo vaccination, (C) breeding flock age, and (D) slaughter age. In panels (B–D) each point represent coefficient estimates from the fitted linear model, while solid lines represent 95% confidence intervals. For any given coefficient estimate, it is considered significant (P < 0.05) whenever the confidence intervals do not cross zero (dashed lines).

Table 1.

Results of the Wald's type II test for the linear model fitted with broiler mean body weight as a response variable. Season and hatchery plant were included in the model to control for potential unmeasured and/or source of errors.

Predictor variable	Sum of squares	Df	F value	Pr (>F)
Breeder flock age	1.356155121	2	57.18215	<0.001⁎
Vaccination group	0.074697028	6	1.049864	0.39
Time before slaughter	6.722551631	4	141.7279	<0.001⁎
Season	3.140139014	3	88.26911	<0.001⁎
Hatchery plant	2.365568132	19	10.49936	<0.001⁎
Residuals	43.72119377	3687	-	-

^⁎P < 0.05.

Figure 2.

Relationship between broiler body weight gain and (A, B) timing of in ovo vaccination, (C) breeding flock age, and (D) slaughter age. In panels (B–D) each point represent coefficient estimates from the fitted linear model, while solid lines represent 95% confidence intervals. For any given coefficient estimate, it is considered significant (P < 0.05) whenever the confidence intervals do not cross zero (dashed lines).

Table 2.

Results of the Wald's type II test for the linear model fitted with broiler body weight gain as a response variable. Season and hatchery plant were included in the model to control for potential unmeasured and/or source of errors.

Predictor variable	Sum of squares	Df	F value	Pr (>F)
Breeder flock age	814.2788309	2	62.58519	<0.001⁎
Vaccination group	197.1275219	6	5.050384	<0.001⁎
Time before slaughter	1653.546919	4	63.54552	<0.001⁎
Season	1645.063293	3	84.29266	<0.001⁎
Hatchery plant	1186.875344	19	9.602403	<0.001⁎
Residuals	23985.27591	3687		-

^⁎P < 0.05.

The results of the analysis showed that all predictor variables significantly impacted cFCR (Figure 3 and Table 3). Broilers from middle-aged breeding flocks and slaughtered after 41 d increased cFCR (0.010 ± 0.008 and 0.078 ± 0.005, respectively), whereas individuals from eggs vaccinated at 460 h of incubation reduced it. Meanwhile, total mortality was significantly affected by breeding flock age and slaughter age, but not by timing of in ovo vaccination (Figure 4 and Table 4). Broilers produced by young and middle-aged breeding flocks had lower total mortality when compared to those from old breeding flocks (–0.570 ± 0.127 for middle-aged breeding flocks), while birds slaughtered after 42 d showed higher total mortality (0.498 ± 0.206).

Figure 3.

Relationship between broiler corrected feeding conversion rate and (A, B) timing of in ovo vaccination, (C) breeding flock age, and (D) slaughter age. In panels (B–D) each point represent coefficient estimates from the fitted linear model, while solid lines represent 95% confidence intervals. For any given coefficient estimate, it is considered significant (P < 0.05) whenever the confidence intervals do not cross zero (dashed lines).

Table 3.

Results of the Wald's type II test for the linear model fitted with broiler corrected feeding conversion rate as a response variable. Season and hatchery plant were included in the model to control for potential unmeasured and/or source of errors.

Predictor variable	Sum of squares	Df	F value	Pr (>F)
Breeder flock age	0.054615057	2	3.497018	<0.05⁎
Vaccination group	0.195812072	6	4.179301	<0.001⁎
Time before slaughter	1.953715631	4	62.54849	<0.001⁎
Season	3.246599283	3	21.88218	<0.001⁎
Hatchery plant	0.8764959	19	37.41486	<0.001⁎
Residuals	28.79106114	3687	-	-

^⁎P < 0.05.

Figure 4.

Relationship between broiler total mortality and (A, B) timing of in ovo vaccination, (C) breeding flock age, and (D) slaughter age. In panels (B–D) each point represent coefficient estimates from the fitted linear model, while solid lines represent 95% confidence intervals. For any given coefficient estimate, it is considered significant (P < 0.05) whenever the confidence intervals do not cross zero (dashed lines).

Table 4.

Results of the Wald's type II test for the linear model fitted with broiler total mortality as a response variable. Season and hatchery plant were included in the model to control for potential unmeasured and/or source of errors.

Predictor variable	Sum of squares	Df	F value	Pr (>F)
Breeder flock age	199.8556417	2	41.0954	<0.05⁎
Vaccination group	19.81221549	6	1.357965	0.23
Time before slaughter	71.69747022	4	7.371412	<0.001⁎
Season	68.49648634	3	9.389746	<0.001⁎
Hatchery	1567.974636	19	33.93847	<0.001⁎
Residuals	8965.330688	3687		-

^⁎P < 0.05.

DISCUSSION

The aim of this study was to improve understanding of how the timing of in ovo vaccination affects post-hatch performance of Ross308AP male broilers. The data from 3,723 commercial broiler flocks analyzed, showed that vaccinations delivered at 460 h of incubation rather than between 440 and 458 h improved performance indicators such as BWG and cFCR. Reasonable evidence already exists that the optimal time to safely inject eggs is between 420 and 460 h of incubation (i.e., 17.5 and 19.2 d of incubation), otherwise risks of damaging embryos or other supporting structures can increase leading to a decrease in hatchability (Ricks et al., 1999; Williams, 2007, 2011). However, despite the full automation of in ovo vaccination, in practice, its timing can be advanced or delayed due to logistical issues stemming from limited staff availability, such as during weekends, holidays, and similar situations (Fernandes et al., 2016). An overall perspective of the pros and cons of these adjustments in vaccination timing, considering not only hatchability but also chick performance, can assist hatcheries’ decision-making on which course to take. Previously, there has only been one study addressing the performance of broilers vaccinated at different embryonic ages, in which no difference was found among embryos vaccinated at 16, 17, 18, and 19 d of incubation (Fernandes et al., 2016). Unfortunately, in addition to dealing with very small sample sizes, the authors also commented that the hygienic and environmental conditions in their experimental poultry barn may not mimic those observed in commercial facilities (i.e., they are not exposed to the same challenges observed in the field).

In an attempt to explain how the timing of in ovo vaccination could interfere with broiler performance, 3 possibilities were considered. Firstly, delaying vaccination to 460 h is associated with a greater homogeneity of vaccine administration and faster development of immune response. Previous studies have shown that injections delivered between 19 and 19.2 d (456-460 h) showed a higher frequency of intraembryonic deposition (Aviakan, 2006; Lozano, 2016). In addition, Islam et al. (2001) reported that intra-embryonic vaccination induced greater protection than extra-embryonic deposition against challenge with a virulent strain of Marek's disease virus. It seems possible that the higher homogeneity and immunity provided by vaccination at 460 h of incubation improved control of diseases in the field, reflecting in improved productivity.

The second possible explanation relates to the fact that the needle puncture in the shell represents a 25% to 30% increase in the relative pore volume of the egg (Williams, 2011), which may accelerate water loss and consequently accelerate the hatching process. If earlier vaccination advances hatch time, and the pull time is not adjusted to match the change, then it could lead to chick dehydration, longer fasting time, poor flock uniformity, and yield loss. In addition, early transfer from the setter to the hatcher in itself can cause deleterious effects unrelated to the in ovo vaccination process (Williams, 2011; Fernandes et al., 2016). All these explanations may also justify why vaccination at 460 h of incubation positively impacted the performance of the analyzed flocks.

A third important aspect to be considered in explaining the survey results concerns the use of multi-stage incubators, which tend to have much more variable temperatures and a longer hatch window than single stage as a consequence. In this situation, the developmental stage of the embryos will also be more variable. Increased variability in development at the point of transfer could lead to a situation where vaccine is deposited in different (and sometimes incorrect) embryonic compartments conferring low vaccine protection, which in turn impairs flock performance. Perhaps in these cases proceeding with vaccination as close as possible to internal and external pipping would be helpful in ensuring greater uniformity of injections into the amnion or embryo body, as described by Avakian (2006) and Lozano (2016). This approach could explain our findings of better weight gain and feed conversion in flocks vaccinated at 460 h of incubation. Under ideal conditions, later transfer in itself can also lead to shorter hatch windows and, consequently, to a shorter stay of chicks inside the hatchers. This would improve hydration and functionality of the digestive tract of newly hatched chicks (Maiorka et al., 2003; Careghi et al., 2005; Deines et al., 2021). For this reason, postponing vaccination up to the latest time maximum recommended could be a good alternative, especially for hatcheries that still base their operations on multistage incubators.

Another factor that stood out in our survey as a determinant of broiler performance was the age of the breeding flocks. Our and other studies noted that chicks from older flocks achieved higher BWG than those from young or middle flocks (Machado et al., 2020; Nangsuay et al., 2021; Santos et al., 2022). This improvement in BWG may be the result of a greater nutrient uptake as previous work suggests that newly-hatched chicks from older breeders have longer gastroinstestinal tracts which appear to be better adapted to exogenous feeding (Machado et al., 2020; Santos et al., 2022). Analyzing the development of the immune system of chicks, Santos et al. (2022) concluded that older breeders have a more effective transfer of maternal immunity, protecting the chicks up to 14 d of age, an advantage that possibly has a positive impact on BWG. Regarding the role of breeding flock age on feed conversion of broilers, no clear pattern was observed in our study since broilers from old flocks only had lower cFCR when compared to broilers from middle-aged flocks, but not in relation to those from young flocks. So far, existing data on this topic revealed similar FCRs between broilers produced by young and old breeding flocks (Ipek and Sozcu, 2015; Machado et al., 2020; Nangsuay et al., 2021). As for total mortality, we detected a higher incidence in broilers from old breeding flocks, an outcome similar to that obtained by Ipek and Sozcu (2015). However, the effect of breeder age on broiler mortality was not observed by Sinclair et al. (1990). According to Yassin et al. (2009), poorly healed navels and yolk sac infections are 2 common conditions found in chicks from older breeders which are apparently associated with higher mortality during the first week of age. There are also authors who believe that the higher mortality of chicks from older breeders originates from early hatching that may increase the risks of dehydration and poor intestinal development (Yassin et al., 2009).

The inclusion of slaughter age as one of the predictors in our analyses was due to its known influence on the response variables assessed in our study. With this in mind, we only used data from flocks slaughtered at ages between 41 and 45 d (42 d being the slaughter age sought by the partner company as this is when the target average weight of 2,800 g is reached). Data from the company providing the genetics show that between 38 to 45 d broilers of this line reach a plateau in their daily weight gain (Aviagen, 2022). Thus, not surprisingly, our analysis confirmed that the gradual increase in slaughter age led to a progressive reduction in weight gain and, at the same time, an increase in weight and feed conversion. The increase in mortality observed with the extension of the slaughter time seems equally logical to us since each day the flock remains in the field increases the chances of more individuals dying from various causes.

In summary, our data showed that postponing vaccination to nearly 460 h of incubation improves broiler performance, at least with regard to body weight gain and feed conversion. This measure possibly ensures delivery of the vaccines at the right compartments of embryonated eggs, in addition to offering greater homogeneity of the process. These aspects may enhance immune response and avoid acceleration of hatching which, in turn, promote adequate health, hydration and development of the digestive system. In parallel, we found that broiler performance is also influenced by the age of the breeding flocks with chicks produced by older breeders presenting better mean weight, body gain but higher mortality. This information may assist commercial hatcheries to better adjust their in ovo vaccination programs to their logistical staffing challenges in order to achieve the best productive potential of broilers.