Research Note: In ovo and in-feed probiotic supplementation improves layer embryo and pullet growth

Abstract

Probiotics are widely used as feed supplements in the poultry industry to promote growth and performance in chickens. Specifically, this supplementation starts around the time of lay and continues through the production cycle in laying hens. However, the embryonic period is critical to the growth and development of metabolically active organs thereby influencing subsequent health and productivity in adult birds. Therefore, the present study investigated the potential use of probiotics to promote embryonic growth in layers. Further, a pilot grow-out study was conducted to evaluate the effect of in ovo and in-feed probiotic application on pullet growth. For the study, fertile White Leghorn eggs were sprayed with phosphate buffered saline (control, CON) or probiotic cocktail (in ovo only, IO; Lactobacillus paracasei DUP 13076 and L. rhamnosus NRRL B 442) prior to and during incubation. The embryos were sacrificed on d 7, 10, 14, and 18 of incubation for embryo morphometry. On d 18, remaining eggs were set in the hatcher to assess hatchability and hatchling morphometry. For the pullet trial, hatchlings were raised on feed with or without probiotics until wk 5. Pullets were sacrificed weekly, and morphometric parameters were recorded. Results of our study demonstrate that in ovo probiotic application significantly improved relative embryo weight, crown-rump length, hatchability, and hatchling weight when compared to the control (P < 0.05). Further, this enhanced embryonic development was associated with a concomitant increase in posthatch growth. Specifically, pullets raised from probiotic-sprayed eggs had significantly improved crown-rump length, tibial length, tibial bone weight, and body weight when compared to the control (P < 0.05). Moreover, among the different treatment schemes employed in this study [CON (no probiotics), in-feed only (IF), IO only, and in ovo and in-feed probiotic supplementation (IOIF)], sustained probiotic supplementation (IOIF) was found to be the most effective in promoting growth. Therefore, in ovo and in-feed probiotic supplementation could be employed to promote embryo and pullet growth to support subsequent performance in layers.

INTRODUCTION

Over the last few decades, increased consumer awareness and demand for a healthy diet have highlighted the significant role of poultry products as a source of high-quality protein for human consumption (Gautron et al., 2022; Korver, 2023). In order to meet this ever-growing demand, the poultry industry adopted intensive rearing practices which led to a tremendous increase in meat and egg production. In fact, the United States is a major producer of eggs in the world second only to mainland China (Gautron et al., 2022). Recent data indicate that in 2022, the U.S. table egg flock size consisted of 306 million layers producing 7.83 billion eggs (USDA, 2023). This tremendous growth and production has been a result of planned genetic selection of poultry breeds targeting higher feed conversion efficiency and performance.

In addition to genetic vigor, in-feed supplementation of growth promoters including probiotics played a major role in maximizing poultry production (Perelta-Sanchez et al., 2019). These are live microbial feed supplements that can improve nutrient absorption, increase productivity, reduce chick mortality, and stimulate immunity (Park et al., 2016). In this regard, few studies demonstrated the potential for dietary supplementation of probiotics in day-old chicks on improving growth in pullets and performance in adult layers (Aksu et al., 2005; Agustono et al., 2022; Hahn-Didde and Purdum, 2016). Although most studies have focused on the effect of in-feed probiotic supplementation on layer growth and performance, optimum embryonic growth is critical to posthatch growth and performance (Walstra et al., 2010; Willems et al., 2013).

In ovo administration of probiotics by spraying, air sac inoculation, and inoculation into amnion or directly to the embryo during late-term developmental stages have been studied in broilers (Das et al., 2021). However, to our knowledge similar studies in layers are limited to none. The embryonic and immediate postnatal developmental period represents a significant phase that is critical to pullet growth (Walstra et al., 2010; Willems et al., 2013). In fact, accumulating evidence indicates that the perinatal period is critical to the growth and development of metabolically active organs thereby influencing subsequent health and productivity in adult birds (Uni et al., 2005; Foye et al., 2006). Therefore, in ovo administration of probiotics could be a potential and viable alternative to promote embryonic development in layers. Further, a proof-of-concept pilot grow-out study was conducted to evaluate the effect of in ovo and in-feed probiotic application on subsequent growth in pullets.

MATERIALS AND METHODS

LAB Culture Conditions and Preparation of Probiotic Spray

Probiotic strains, L. paracasei DUP-13076, and L. rhamnosus NRRL-B-442 were obtained from Dr. Bhunia, Food Science Department, Purdue University, and the USDA NRRL culture collection, respectively. These cultures were selected based on previous in ovo trials (Muyyarikkandy et al., 2023). Each strain was cultured separately in 10 mL of de Mann, Rogosa, Sharpe broth (MRS) at 37°C for 24 h, sedimented by centrifugation (3,600 × g for 15 min), washed twice with sterile phosphate buffered saline (PBS, pH 7.0), and resuspended in 10 mL PBS. Equal portions from each of the strains were combined to make a 2-strain probiotic cocktail. The bacterial population in the 2-strain mixture was determined by plating 0.1-mL portions of appropriate dilutions on MRS agar, followed by incubation at 37°C for 24 h.

Experimental Design, Egg Treatment, Incubation, and Sampling

All trials were conducted with the approval of the UConn Institutional Animal Care and Use Committee. Fertilized White Leghorn eggs from 42- to 50-wk-old birds were obtained from the University of Connecticut poultry farm. The eggs were randomly assigned to 1 of 2 groups namely i) control (CON; eggs sprayed with PBS only) and ii) in ovo (IO; eggs sprayed with the probiotic cocktail; ∼9 log CFU/egg). Prior to incubation, all settable eggs were weighed (starting egg weight) and individually sprayed on d 0, 3, 7, 10, 14, and 18 of incubation as described previously (Muyyarikkandy et al., 2023). Briefly, eggs in the control group were individually sprayed with PBS while eggs in treatment groups were sprayed with the probiotic cocktail (∼9 log CFU/egg) using an atomizer (Amalaradjou, 2022). The treatment regimen was based on maintaining significant probiotic populations on the eggs (∼4 log CFU/egg) throughout incubation as determined in our preliminary trials. The sprayed eggs were then incubated in a thermostat incubator (2362N Hova-Bator, GQF Manufacturing Company Inc., Savannah, GA) with an automatic egg turner (1,611 egg turner with 6 universal racks, GQF Manufacturing Company Inc.), temperature and humidity control for 18 d at 37.8°C and 55% relative humidity. Two incubators (replicates) were assigned per group with a total of 180 eggs per incubator and 3 independent trails were conducted (total of 6 incubators per group).

Embryo Morphometry

On d 7, 10, 14, and 18 of incubation 10 embryos from each treatment and incubator were randomly sampled in each trial for a total of 60 embryos per group. The eggs were weighed, opened through the blunt end and embryos euthanized by cervical dislocation to assess embryo growth. Morphometric measurements including yolk sac weight, yolk-free body mass (embryo weight), crown-rump length, tibial length, radial length, and length of the third digit were measured (Pineda et al., 2013; De Oliviera et al., 2014; Alabdallach et al., 2020). Relative embryo weight and yolk sac weight were calculated as percentage of starting egg weight to account for variation in egg weights. Data from the 3 independent trials were pooled and analyzed to determine the effects of probiotic application on embryonic growth.

Hatchability and Hatchling Morphometry

Embryos from trial 3 were hatched and a pilot study was run to determine effects on pullet growth. On d 18, the remaining eggs were sprayed with the probiotic cocktail or PBS and transferred to separate hatchers and held at 37.8°C and 65% relative humidity for 3 d or until hatch. On the day of hatch (d 21), percent hatchability was recorded, and hatchlings were weighed prior to placement on floor pens. Twenty hatchlings from each treatment group and incubator were sacrificed, and body weight, length measurements and organ weights were recorded (Kalavathy et al., 2003; Alabdallach et al., 2020).

Pullet Growth—Pilot Study

Pullets from the control group were randomly split into 2 groups: CON (PBS only, no probiotic) and IF (no egg treatment, probiotic supplementation in-feed only; ∼9 log CFU/kg of feed until sacrifice). Similarly, hatchlings from the IO group were randomly assigned to 1 of 2 groups namely, IO (probiotic cocktail treatment on eggs only) and in ovo and in-feed probiotic supplementation (IOIF; in ovo probiotic application on eggs and posthatch in-feed supplementation; ∼9 log CFU/kg of feed). These different treatment modalities were performed to determine whether IO administration can exert a sustained growth promoting effect posthatch. Specifically, this will address our hypothesis that supporting embryonic development promotes posthatch growth. Further, inclusion of the IF group enabled the comparison of IO with routinely employed in-feed (IF) probiotic supplementation in the poultry industry. All birds were grouped in separate pens depending on the treatment type (75 birds/treatment type) and standard management practices were followed. They were fed ad libitum with a standard commercial pelleted diet of 2,800 kcal ME and 18.5% CP for starters as per the Lohman layer management guide. For in-feed supplementation, the probiotic cocktail was prepared as described previously. Prior to each feeding, appropriate volume of the cocktail culture was added to the feed and mixed thoroughly to obtain the desired concentration (∼9 log CFU/kg) in the feed.

Pullet Morphometry

During the posthatch rearing phase, individual body weights were monitored weekly. Feed consumed was recorded daily on per pen basis, the uneaten feed was collected once daily before morning feeding and feed conversion ratio was calculated as the proportion of live weight over the feed consumed (Kalavathy et al., 2003; Willems et al., 2013). In addition, 10 to 12 birds from each treatment group were sacrificed weekly and morphometric measurements including organ weights were recorded. Further, in wk 4 and 5, tibial bone weights were measured as a preliminary indicator of bone growth (Kalavathy et al., 2003).

Statistical Analysis

All data were analyzed using the Statistical Analysis Software package v 9.2 (SAS Institute Inc., Cary, NC). All experiments were set out as a completely randomized design. Data were sorted by day/age and analyzed using PROC MIXED procedure of SAS with treatment as the fixed effect. Treatment mean comparisons were performed using LSMEANS statement and PDIFF option. Differences were determined to be significant at P ≤ 0.05

RESULTS AND DISCUSSION

Among the different additives used to promote growth and performance in laying hens, the application of probiotics including lactic acid bacteria has demonstrated significant potential (Abdulrahim et al., 1996; Jha et al., 2020). Traditionally dietary additives to promote production and general health status have been provided to laying hens around the start of lay. However, one of the critical periods of development that influences health and performance in poultry is the embryonic and posthatch phase (d 1 to sexual maturity; Nahashon et al., 1994). Furthermore, studies have shown that major diseases of later life originate from impaired embryonic growth and development (Godfrey and Barker, 2000; Saki and Mahmoudi, 2015). Consequently, an approach that supports growth and development during these early stages is expected to have a significant effect on overall health and performance of laying hens (Foye et al., 2007; Hulet et al., 2007).

In this regard, researchers have demonstrated that thermal manipulation during embryogenesis regulated chick weight while exerting long-lasting effects on posthatch thermoregulation and stress response (Goel et al., 2022, 2023). Similarly, Saki and Mahmoudi (2015) demonstrated that in ovo injections of bovine lactoferrin in layer eggs resulted in increased egg weight and improved tibial strength in 28-wk-old laying hens. Along these lines, promoting embryonic development and subsequent pullet growth via sustained probiotic supplementation could serve as a potential means to improve production and health status of laying hens. Hence, the current study investigated the effect of in ovo probiotic supplementation on layer embryo growth. Further, a pilot grow-out study was performed to determine the effect on posthatch growth in pullets.

In ovo application of probiotics led to significant improvements in embryo growth as evidenced from the relative embryo weights (yolk-free body mass; P < 0.05). For instance, on 7th, 10th, 14th, and 18th day of incubation, relative embryo weights were significantly higher (P < 0.05) in the probiotic group (IO; 1.61 ± 0.05%, 5.42 ± 0.16%, 19.89 ± 0.43%, and 38.84 ± 0.94%) compared to the CON group (1.38 ± 0.09%, 4.23 ± 0.24%, 18.84 ± 0.35%, and 35.12 ± 1.28%; Table 1), respectively. Overall, we observed a 5 to 28% increase in relative embryo weights in the IO group throughout incubation. Similarly, in ovo probiotic application also resulted in a significant increase in crown-rump and tibial length when compared to the control (Table 1; P < 0.05). Particularly on d 14 and 18, we observed a 5 to 6.3% and 4 to 9.8% increase in crown-rump and tibial length in the IO group, respectively. Following 18 d of incubation, the eggs were transferred to the hatcher and monitored for hatch. We observed that the improvements to embryonic growth were associated with a significant increase in hatchability (P < 0.05). The hatchability of the probiotic-treated group was 83.50 ± 4.95% compared to the control which was 74.58 ± 2.95%.

Table 1.

Effects of in ovo probiotic supplementation on embryo and hatchling morphometry.

Morphometric parameters	Embryonic day1								Hatchling2
	7		10		14		18		Day of hatch
	CON	IO	CON	IO	CON	IO	CON	IO	CON	IO
Relative embryo weight (%)	1.38 ± 0.09a	1.61 ± 0.05 b	4.23 ± 0.24 a	5.42 ± 0.16b	18.84 ± 0.35 a	19.89 ± 0.43b	35.12 ± 1.28 a	38.84 ± 0.94b
Relative percent increase		+16.7%		+28.1%		+5.6%		+10.6%
Relative yolk sac weight (%)1			76.39 ± 0.77	75.33 ± 0.47	65.00 ± 0.80	66.53 ± 0.75	32.28 ± 1.29	31.17 ± 1.01
Crown-rump length (cm)			3.43 ± 0.07	3.28 ± 0.06	5.34 ± 0.12 a	5.68 ± 0.08 b	7.42 ± 0.16 a	7.80 ± 0.07 b	9.15 ± 0.09 a	10.08 ± 0.09 b
Relative percent increase						+6.37%		+5.12%		+10.16%
Tibial length (cm)			0.57 ± 0.02	0.55 ± 0.01	1.02 ± 0.03 a	1.12 ± 0.03 b	1.87 ± 0.03 a	1.95 ± 0.02 b	2.73 ± 0.03 a	2.82 ± 0.03 b
Relative percent increase						+9.80%		+4.28%		+3.30%
Radial length (cm)					0.98 ± 0.03	0.92 ± 0.03	1.32 ± 0.02	1.60 ± 0.02	1.63 ± 0.03 a	1.75 ± 0.03 b
Third digit length (cm)					1.15 ± 0.06	1.11 ± 0.07	2.35 ± 0.06	2.43 ± 0.06	2.20 ± 0.04 a	2.50 ± 0.04 b
Live weight (g)									39.75 ± 0.33 a	41.0 ± 0.40 b
Relative percent increase										+3.14%

Data are represented as mean ± SEM.

¹Embryonic morphometry data represent combined data from 3 independent trials.

²Hatchling morphometry data represent combined data from 2 replicate hatchers per group.

^a,bDifferent superscripts indicate significant difference in embryo morphometry between treatments within each day at P < 0.05.

CON: control (no probiotic application); IO: in ovo probiotic application.

Relative embryo weight: embryo weight (yolk-free body mass) as a percentage of starting egg weight.

Relative yolk sac weight: yolk sac weight as a percentage of starting egg weight.

Crown-rump length: measured along the curvature of the back from the top of the head to the tip of the tail.

Tibial length: measured from kneecap to hock joint. Radial length: measured from elbow to carpal joint.

As seen with the embryo weights, in ovo probiotic supplementation also resulted in significantly higher hatchling weights by 3.14% when compared to the control (CON: 39.75 ± 0.33 g; IO: 41.01 ± 0.4 g; P < 0.05; Table 1). In addition, length measurements including crown-rump, tibial, radial, and third digit length were significantly longer in the IO group by 10.16, 3.30, 7.36, and 13.63%, respectively (Table 1). Contrary to our findings, Pender et al. (2016) did not observe any significant effect of in ovo probiotic inoculation (via air sac route) on hatchability and morphometry in broilers. The difference in the observed results could be attributed to probiotic application as early as d 1 of incubation, difference in the method of application, route of administration and strain of probiotic bacteria employed. Further, it is critical to recognize that similar studies in layers examining effects on embryo development and hatchability are limited to none. Overall, chicks hatched from probiotic-sprayed eggs were 10% longer and 3% heavier than the control. This is noteworthy since longer chicks have been associated with better use of egg nutrients and higher posthatch growth (Molenaar et al., 2008; De Oliveira et al., 2014). As part of the study, we also measured relative organ weights (heart, liver, spleen, gizzard, intestine) and did not observe any significant difference between the treatments (P > 0.05)

As observed with the embryonic phase, supplementation with probiotics was also associated with significantly higher live weights when compared to the control throughout the grow-out period (Table 2). For instance, the mean live weight of the pullets on wk 1, 3, and 5 wk in IOIF group were 4.44% (79.02 ± 1.34 g), 9.45% (257.36 ± 5.55 g), and 14.22% (498.11 ± 13.41 g) higher when compared to the control (75.66 ± 1.30 g, 235.14 ± 5.26 g, and 436.07 ± 14.48 g), respectively (Table 2). Similarly, in the IO group, pullets were heavier than the control throughout the study although significantly different only on wk 1. In the IF group, although not significantly different, live weights were comparable to IO on wk 4 and wk 5 and heavier than the control (Table 2). This is in line with previous findings demonstrating improved pullet growth following in-feed supplementation of commercial probiotics (Bactocell and Levucell, Lallemand Animal Nutrition, Milwaukee, WI). They observed that Bactocell improved the weight gain of the pullet during the first 4 wk after hatch, while Levucell did not have any effects (Hahn-Didde and Purdum, 2015). Similarly, Nahashon et al. (1996) used Lactobacillus and condensed cane molasses as a growth promoter in pullets, and it significantly improved the body weight gain. Besides body weight, we also measured relative organ weights and did not observe any significant difference between the treatments throughout the study (P > 0.05).

Table 2.

Effect of probiotic supplementation on pullet morphometry.

Pullet age (wk)	Treatments	Morphometric measurements
		Live weight (g)	Crown-rump length (cm)	Tibial length (cm)	Tibial bone weight (g)	Radial length (cm)	Third digit length (cm)
Wk 1	CON	75.66 ± 1.30a	11.99 ± 0.22a	3.44 ± 0.06a		2.79 ± 0.14a	2.74 ± 0.11a
	IF	74.6 ± 1.74a	12.36 ± 0.10a	2.87 ± 0.04b		2.55 ± 0.04a	2.51 ± 0.04b
	IO	79.84 ± 1.02b	12.85 ± 0.13b	3.47 ± 0.05a		3.17 ± 0.04b	3.11 ± 0.05c
	IOIF	79.02 ± 1.34ab	12.41 ± 0.20a	3.35 ± 0.03a		3.09 ± 0.05b	3.06 ± 0.03c
Wk 2	CON	147.84 ± 4.22a	15.74 ± 0.16a	4.00 ± 0.07a		3.57 ± 0.04a	3.65 ± 0.06
	IF	149.3 ± 4.01a	15.58 ± 0.27a	3.83 ± 0.07a		3.48 ± 0.07a	3.59 ± 0.04
	IO	149.13 ± 3.67a	16.06 ± 0.20ab	4.38 ± 0.07b		4.28 ± 0.06b	3.79 ± 0.09
	IOIF	162.31 ± 4.14b	16.42 ± 0.17b	4.57 ± 0.06c		4.33 ± 0.06b	3.79 ± 0.05
Wk 3	CON	235.14 ± 5.26a	18.12 ± 0.11a	4.72 ± 0.06a		4.21 ± 0.08a	4.34 ± 0.05
	IF	234.95 ± 5.05a	18.03 ± 0.15a	4.51 ± 0.05a		4.16 ± 0.07a	4.37 ± 0.08
	IO	244.69 ± 6.98ab	18.62 ± 0.18b	5.08 ± 0.1b		4.70 ± 0.10b	4.40 ± 0.08
	IOIF	257.36 ± 5.55b	18.75 ± 0.19b	5.31 ± 0.05b		4.79 ± 0.05b	4.30 ± 0.08
Wk 4	CON	287.20 ± 7.46a	19.77 ± 0.28a	5.15 ± 0.07a	3.05 ± 0.17a	4.63 ± 0.07a	4.80 ± 0.08a
	IF	295.10 ± 8.63a	20.10 ± 0.15a	4.99 ± 0.13a	3.12 ± 0.12a	4.55 ± 0.06a	4.72 ± 0.11a
	IO	315.10 ± 12.65a	21.14 ± 0.23b	5.88 ± 0.10b	3.62 ± 0.22b	5.49 ± 0.08b	4.96 ± 0.11a
	IOIF	342.00 ± 8.02b	20.84 ± 0.29b	5.82 ± 0.08b	3.70 ± 0.19b	5.61 ± 0.04b	5.72 ± 0.11b
Wk 5	CON	436.07 ± 14.48a	23.55 ± 0.32a	6.35 ± 0.28a	4.41 ± 0.12a	5.65 ± 0.11a	5.53 ± 0.11
	IF	461.39 ± 13.52a	22.95 ± 0.24b	6.29 ± 0.11a	4.57 ± 0.20a	5.64 ± 0.07a	5.42 ± 0.20
	IO	461.66 ± 15.03a	24.75 ± 0.46c	7.12 ± 0.15b	4.52 ± 0.25a	6.52 ± 0.11b	5.49 ± 0.80
	IOIF	498.11 ± 13.41b	24.65 ± 0.40c	7.10 ± 0.16b	5.12 ± 0.15b	6.54 ± 0.09b	5.54 ± 0.20

Data are represented as mean ± SEM.

^a,b,cDifferent superscripts indicate significant difference in pullet morphometry between treatments within each time point at P < 0.05.

CON: control (no probiotic treatment); IF: in-feed probiotics only; IO: in ovo probiotics only; IOIF: in ovo and in-feed probiotics.

As seen with the embryos from the IO group, continued posthatch probiotic supplementation (IOIF) resulted in a significant increase in crown-rump, tibial, and radial lengths throughout the study. For instance, on wk 4 and 5, crown-rump length and tibial length were 4.6 to 5.4% and 11 to 13% longer in the IOIF group when compared to the control pullets. Furthermore, the tibial bone was significantly heavier (P < 0.05) in IOIF pullets by 16 to 21% on wk 4 and 5 when compared to the control (Table 2). These results provide preliminary indication for a positive effect for in ovo and in-feed probiotic supplementation on bone growth and development posthatch. Besides these morphometric measurements, we also recorded pullet feed consumption. We observed a reduction in FCR in the IOIF group when compared to IO, IF and CON. FCR was lower when probiotic treatment was applied either on egg surface or in-feed compared to the control group. FCR at wk 5 was lowest in IOIF (4.46), followed by IO (4.83) and IF (4.85) and was highest in the CON (5.33). Hahn-Didde et al. (2015) observed a similar trend in improved feed efficiency in pullets between 5 and 10 wk of age following in-feed probiotic supplementation with Bactocell. Our study is in agreement with Hahn-Didde et al. (2015) since both in ovo and in-feed probiotic supplementation had a lower FCR than the control. Studies in broilers have shown that in ovo probiotic administration affect the digestive efficacy of pancreas in the growing embryo and would affect the FCR posthatch (Pruszynska-Oszmalek et al., 2015). Further in-feed supplementation of probiotics to hatchlings and pullets has been shown to improve feed utilization and growth in poultry (Maiorano et al., 2012; Beshara et al., 2019). Nevertheless, given the pilot nature of the posthatch trial, additional replications are warranted to validate this finding.

Our results demonstrate that in ovo probiotic administration significantly enhanced the growth and development of the layer embryo. Further, data from our study also demonstrate a potential for improving hatchability, hatchling morphometry and pullet growth. Specifically, the improvement in embryo and hatchling growth in the IO group was associated with an improvement in posthatch growth. This demonstrates that improving embryonic growth can promote subsequent development in posthatch birds. Among the different posthatch treatment regimens, sustained probiotic application (IOIF) was found to be the most effective in promoting overall growth in embryo, hatchlings, and pullets. Therefore, in ovo and in-feed probiotic supplementation could be employed to promote critical pullet growth and development thereby subsequently supporting layer performance. However, additional studies in pullets and laying hens are warranted to validate these findings.