In ovo probiotic supplementation enhances energy status and promotes growth in developing broiler embryos and hatchlings

Abstract

As opposed to mammals, nutrient and energy availability for the embryo is limited to the nutrients deposited in the egg. This can create an energy imbalance in the perinatal period resulting in impaired perinatal embryo development, reduced hatchability, suboptimal hatchling quality and decreased post-hatch performance. Hence, it is critical to improve the energy status of the developing embryo to support the hatchling and immediate post-hatch development. Towards this, we observed that in ovo probiotic application to broiler eggs improved hatchability and hatchling quality. To understand the underlying mechanism, we investigated the effect of in ovo probiotic supplementation on glycogen reserves and embryo development in broilers. A total of 900 eggs (Ross 308) were either sprayed with phosphate buffered saline (PBS; control) or probiotics [∼9 log CFU/egg of Lactobacillus rhamnosus NRRL B-442(LR) or Lactobacillus paracasei DUP 13076 (LP)] during incubation. On day 18, eggs were transferred to the hatcher and set up for hatching. Embryos were sacrificed for morphometric measurements on day 10, 12, 14, 16, 18, 19, 20 and 21 of incubation. In addition, yolk sac membrane (YSM), yolk sac content (YSC), breast, and liver samples were collected for glycogen analysis and gene expression assays. Overall, in ovo probiotic spray significantly increased relative embryo weight, relative breast muscle and hatching muscle (P < 0.05). Additionally, glycogen content was significantly elevated in YSM, YS, liver and breast muscle throughout incubation and in the hatchling (P < 0.05). Further, we observed a significant downregulation of genes associated with the gluconeogenic pathways including PYGL, FBP1, PEPCK-C, PEPCK-M, and GK in the YSM, liver and breast muscle at day 21 (P < 0.05), thereby sparing the need for muscle protein breakdown. This in turn indicates the availability of sufficient glycogen reserves in the perinatal embryo to fuel the hatching process. In summary, in ovo spray application of probiotics improved glycogen reserves in the embryo thereby supporting optimum embryo development and improving hatchability and hatchling quality.

Introduction

As modern broiler lines are intensively selected for improved performance and higher growth rate, there is an increased requirement for energy and protein by the chicken embryos. Hence late-term embryos may experience stress from insufficient energy reserves (van der Wagt et al., 2020). Consequently, the imbalance between requirement and nutrient reserves within the eggs may restrict embryonic growth and development, potentially impacting hatchability, hatchling quality and overall broiler performance (Kocamis et al., 1999; Uni and Ferket, 2004). In fact, insufficient embryonic glycogen reserves have been associated with delayed hatching, decreased body weight at hatch and reduced performance (Uni et al., 2005; Yadgary et al., 2014).

Particularly in the perinatal period, the energy status of the embryo is determined by glycogen reserves held in the yolk, liver and muscles. This is also supported by the embryos ability to derive glucose by gluconeogenesis of amino acids, glycerol, and lactate (de Oliveira et al., 2008). The yolk sac membrane (YSM) serves as a major source of glycogen to fuel late-term embryonic growth and the energy intensive hatching process (Yadgary and Uni, 2012; Yadgary et al., 2014). In fact, while the YSM glucose content tends to be higher during initial phase of incubation, glycogen synthesis in the YSM starts during mid-incubation with highest levels reported during the last week of incubation. Besides glycogen, to meet the increasing energy demand during this period, the upregulation of gluconeogenesis in the late term embryo is critical to ensure blood glucose supply for development and for muscle and liver glycogen deposition (Hu et al., 2017). Altogether, these data demonstrate a critical role for YSM in the synthesis and storage of glycogen and its supply to the late-term embryo and hatchling (Yadgary and Uni, 2012).

Besides the YSM, the liver and muscle also play a significant role in meeting the energy demands of the perinatal embryo (de Oliviera et al., 2008). Prior to hatch, the embryonic hepatic tissue serves as a glycogen reserve and participates in gluconeogenesis, glycogenesis, and gluconeogenesis (de Oliviera et al., 2008; Sunny and Bequette, 2011). In addition to its role in energy homeostasis, the liver serves as a storage organ for lipids mobilized from the yolk (de Oliviera et al., 2008). These fat reserves help sustain the hatchling in the first few days after hatch once they have full access to oxygen (Uni et al., 2005). The pectoral muscle in the perinatal embryo accounts for the greatest quantity of total glycogen stored in the body (Christensen et al., 2001). While glycogen serves as the immediate substrate for glucose, gluconeogenesis via the breakdown of protein helps meet the energy demand once glycogen is depleted (Uni and Ferket, 2004). Towards this, the pectoral muscle is the predominant source of protein that is mobilized to supply amino acids for gluconeogenesis in the late-term embryo (Keirs et al., 2002; et al.). However, significant muscle protein catabolism can negatively affect embryonic growth and development during the last stage of incubation, leading to a decrease in hatchling weight (Uni and Ferket, 2004; Uni et al., 2005). Thus, improving the energy status in the perinatal embryo is of critical importance not only to the perinatal embryo and hatching but also for sustained overall performance.

Towards this, in ovo technologies were developed to provide additional nutrients and improve energy reserves in the perinatal embryo (Givisiez et al., 2020). Studies administering in ovo injections of maltose, sucrose, dextrin, β‑hydroxy-β-methylbutyrate (Uni et al., 2005; Kornasio et al., 2011), l-arginine (Yu et al., 2018) and l-carnitine (Shafey et al., 2010) reported an increase in hatchling weight, breast muscle weight, and glycogen storage in liver and muscle. Further, the enhancement of embryonic growth and development was found to be strongly correlated with reduced post-hatch mortality and morbidity, greater efficiency of nutrient-utilization, improved embryonic glycogen reserves (de Oliveira et al., 2008), improved intestinal development and digestive capacity (Tako et al., 2005; Smirnov et al., 2006), increased muscle development and breast meat yield (Kornasio et al., 2011; Muyyarikkandy et al., 2023b).

Although probiotics are primarily used as in-feed additives in commercial poultry production, as an in ovo supplement, they can be ideal candidates to mediate early-life programming in broiler chickens (Shehata et al., 2021). Towards this, few studies have investigated the effect of in ovo probiotic inoculations to late term embryos on intestine development and gluconeogenesis in chicks with conflicting results and negative impacts on hatchability (Das et al., 2021; Wilson et al., 2020; Rodrigues et al., 2020; Arreguin-Nava et al., 2019; El-Moneim et al., 2020; Teague et al., 2017; Yamawaki et al., 2013). As opposed to these studies, we employed non-invasive spray application to deliver probiotics in ovo (Amalaradjou, 2022; Muyyarikkandy et al., 2023a, 2023b).

Our studies demonstrate in ovo probiotic spray application of Lactobacillus rhamnosus NRRL B-442 (LR) and Lactobacillus paracasei DUP 13076 (LP) improved embryonic development and muscle growth in broilers (Muyyarikkandy et al., 2023b). Further, our data demonstrate that in ovo application of LP and LR significantly improved hatchability by ∼5 % when compared to the control (Gao et al., 2024). Moreover, hatchlings in the probiotic treated group had significantly higher yolk-free body mass and hatchling weight. Since sufficient energy reserves and optimum embryo development are critical to hatching, as a next step, in this study we determined the effect of in ovo probiotic application on embryo development and glycogen reserves (energy status) in the broiler embryo. Although the underlying mechanism by which probiotics influence the glycogen metabolism is still unclear, a few studies have demonstrated a role for the beneficial bacteria in modulating the gluconeogenic and glycolytic pathways in broiler chicks (Zheng et al., 2016). Similarly, albeit in grow-out birds, Lactobacillus plantarum, L. ingluviei, and L. salivarius supplementation was reported to enhance glucose metabolism thereby improving muscle characteristics in broilers (Cai et al., 2024). Based on these findings, we hypothesize that– in ovo probiotic application promotes hatching and hatchling quality by ii) supporting optimum embryonic development and ii) improving glycogen reserves in the yolk sac membrane, liver and pectoral muscle thereby limiting the need for muscle protein break down (gluconeogenesis) by modulating key glycogenic and gluconeogenic genes.

Materials and methods

Probiotic culture preparation

Lactobacillus rhamnosus NRRL B-442 (LR) was obtained from the USDA Agriculture Research Service NRRL culture collection (Peoria, IL, USA). Lactobacillus paracasei DUP 13076 (LP) was kindly provided by Dr. Bhunia, Molecular Food Microbiology Lab, Purdue University, West Lafayette, IN, USA. LR and LP were selected based on preliminary screening and published literature (Muyyarikkandy et al., 2023a, 2023b; Amalaradjou, 2022; Gao et al., 2024). The cultures were grown in de Mann, Rogosa, Sharpe broth (MRS; Fisher Scientific, Waltham, MA, USA) at 37°C for 16 – 18 h. Overnight cultures were centrifuged (3500 g, 10 min, 4°C), and washed twice with sterile phosphate buffered saline (PBS, pH 7.0). Probiotic counts were determined following serial dilution and plating on MRS agar and incubated at 37°C for 24 – 48 h (Muyyarikkandy and Amalaradjou, 2017; Gao et al., 2024).

Experimental design, egg incubation and hatching

This study was conducted at the UConn vivarium with approval from the UConn Institutional Animal Care and Use Committee. Fertile Ross 308 eggs from 40 to 42-week-old birds were kindly provided by Aviagen (Huntsville, AL, USA). Two independent trials were performed using a total of 900 eggs. Prior to incubation, all settable eggs were weighed (starting egg weight, SEW), numbered and randomly assigned to the three treatment groups (150 eggs/group). Group 1: Eggs sprayed with PBS (vehicle control), Group 2: Eggs sprayed with LP, Group 3: Eggs sprayed with LR. Eggs were individually sprayed with different probiotic cultures (∼ 9 log CFU/egg) or sterile PBS (Control) at embryonic day (D) 0, 3, 7, 10, 14, and 18 of incubation using a handheld atomizer as previously described (Amalaradjou, 2022; Muyyarikkandy et al., 2023a, 2023b). 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 eggs were incubated in the GQF incubator with an automatic egg turner (GQF Manufacturing Company Inc., Savannah, GA, USA) at 37.8°C and 55 % – 60 % relative humidity from D 0 to 18. On D18, eggs were transferred to a GQF hatcher and incubated at 37.8°C and 65 % – 70 % relative humidity until hatch (GQF Manufacturing Company Inc., Savannah, GA, USA; Aviagen Hatchery tips, 2020). Throughout the study, eggs in different groups were placed in separate incubators/hatchers to avoid cross contamination.

Morphometric measurements and sample collection

Morphometric measurements were collected on D10, 12, 14, 16, 18, 19, 20 and 21 in control, LP and LR groups. At each sampling day, a total of 20 – 25 embryos/hatchlings from each group were randomly selected and sacrificed across both trials. The eggs were opened from the blunt end, and embryos were euthanized by cervical dislocation. The hatchlings were euthanized by CO₂ inhalation. Firstly, yolk-free-body-mass (YFBM) and yolk sac (YS) were dissected and weighed. YFBM is the embryo weight after removal of YS. Additionally, breast and liver were dissected from YFBM and weighed. Besides, hatching muscle was dissected and weighed on D18, 19, 20 and 21 in all groups. Hatchling weight was measured at D21. To account for variation in SEW, YFBM, YS were calculated as percentage of SEW at all time points except for D21. Similarly, breast muscle, hatching muscle, and liver were represented as percentage of YFBM or as percentage of the hatchling weight at D21. Following morphometry, tissue samples including YSM, YSC, liver, and pectoral muscle samples were flash frozen in liquid nitrogen and stored at −80°C for glycogen analysis. In addition, YSM, liver, and pectoral muscle samples were collected in RNAlater (Invitrogen, Waltham, MA, USA) and stored at −80°C for subsequent gene expression analysis (Yadgary and Uni, 2012; Dayan et al., 2023a; Uni et al., 2005).

Glycogen analysis

Glycogen content in the YS, liver and breast muscle were measured on D10, 12, 14, 16, 18, 20 and 21 of incubation to determine the energy status of embryos using a colorimetric method based on the reduction of iodine (Dreiling et al., 1987; Yadgary and Uni, 2012). A total of 12 to 16 biological replicates from YSM, yolk sac content (YSC), liver, and breast samples were assayed for their glycogen content. Approximately 0.1 g YSC, YSM, liver and muscle samples were weighed, and 1 ml 8 % perchloric acid was added. Samples were homogenized on ice for 1 min using a tissue master (OMNI international, Kennesaw, GA, USA). In the case of breast muscle, samples were homogenized using the PowerLyser 24 homogenizer (Qiagen, Germantown, MD, USA). After homogenization, the homogenates were centrifuged at 15,000 × g at 4°C for 5 min. The supernatant was then then processed as previously described with absorbance measured at 450 nm (Yadgary and Uni, 2012). Glycogen concentration was calculated using a standard curve, and the total glycogen content in each tissue/organ was determined. YS glycogen amount was calculated as the sum of glycogen from YSM and YSC. Total glycogen content in the embryo was determined by adding together glycogen contents from YS, liver and breast muscle.

RNA extraction, cDNA synthesis and RT-qPCR

To determine the modulatory effect of probiotic application on glycogenic, glycogenolytic and gluconeogenic genes, we performed real time quantitative PCR (RT-qPCR). Based on the results from the morphometry and glycogen analysis, gene expression assays were limited to the control and LP groups. For liver and breast muscle samples (D14, 18 and 21), RNA was extracted using RNeasy mini kit with DNase treatment using RNase free DNase set according to the manufacturer’s protocol (Qiagen, Germantown, MD, USA). For YSM samples (D10, 14, 18, 21), RNA was extracted using RNAqueous-4PCR DNA-free RNA isolation kit with DNase treatment according to the manufacturer’s protocol (Invitrogen, Waltham, MA, USA). RNA quantity (A260/A280) and quality (A260/A230) were measured using Nanodrop One (Thermo Scientific, Waltham, MA, USA). cDNA was synthesized from 1 μg of DNA-free RNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Specific primers for candidate genes were chosen according to published literature (Yadgary and Uni, 2012; Dayan et al., 2023a), including glycogen synthesis [glycogen synthase (GYS1 for breast muscle; GYS2 for YSM and liver)], gluconeogenesis [fructose 1,6-bisphosphatase (FBP1), phosphoenolpyruvate carboxykinase (PEPCK), glycerol kinase (GK), glucose 6-phosphatase (G6PC2)] and glycogenolysis [glycogen phosphorylase (PYGL)]. β-actin (ACTB) was selected as endogenous control. RT-qPCR was performed using the PowerUp SYBR green master mix (Applied Biosystem, Waltham, MA, USA) on the QuantStudio 6 Pro system (Thermo Scientific, Waltham, MA, USA) using the following conditions: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s. A total of 8 biological replicates from control and LP group were included in this study. Data were normalized using the endogenous control, and comparative quantification (2–∆∆Ct) was performed to determine the relative change in gene expression between control and LP group (Bookout and Mangelsdorf, 2003).

Statistical analysis

This study was set up as a completely randomized design with two independent trials, and the embryo/hatchling was considered as the experimental unit. All statistical analyses were performed in R software (version 3.4.0). Shapiro-Wilk test was used to check the normality of data. Normally distributed data were analyzed using one way ANOVA with Fisher’s LSD multiple comparison tests, while non-normally distributed data were analyzed using the Kruskal-Wallis test with Dunn’s multiple comparison tests. Statistical comparisons among the treatment groups were performed within each sampling time. For gene expression analysis, normally distributed data were analyzed using paired t-test, while non-normally distributed data were analyzed using Wilcoxon rank sum test. P ≤ 0.05 was considered as significantly different.

Results and discussion

One of the immediate consequences of inadequate nutrient reserves and poor embryonic growth is reduced hatchability (Wilson, 1997). In effect, reduced hatchability can result in significant economic loss to the poultry industry. Beyond hatchability, the quality of the day-old birds is critical to subsequent broiler growth and profitability for poultry producers (van de Ven et al., 2012). Hence, the essential objective in hatcheries is to maximize hatchability with good number of high-quality and saleable chicks desired for their high viability and slaughter yield (Decuypere and Bruggeman, 2007). In fact, data show that 2 % to 5 % of neonatal chicks are lost due to limited energy reserves (Uni and Ferket, 2004). Beyond sustenance, inadequate energy reserves impact muscle and intestine development thereby subsequently affecting post-hatch muscle growth, meat yield and quality, nutrient digestion, and overall growth and health (Wang et al., 2022; Nazem et al., 2019; Jha et al., 2019). Hence, establishment of a stable and sufficient energy status is critical to the development of the late term embryo, hatching process and immediate post-hatch development in poultry until feed is consumed (Christensen et al., 2003; Uni et al., 2005). Related to this, in ovo supplementation of probiotics (LR, LP) not only increased hatchability but also improved hatchling quality and morphometry (Gao et al., 2024). Since hatchability and hatchling quality are dependent on optimum embryo development and access to sufficient energy reserves, as a next step we determined the effect of in ovo probiotic supplementation on embryo development and energy status (glycogen reserves) in the developing embryo and hatchling.

Embryo and hatchling weight

Embryo weight (YFBM) is an important indicator for embryonic growth, the heavier YFBM indicates better embryonic development (Meijerhof, 2009; Molenaar, 2011). In this regard, we observed a significant improvement in relative embryo weights in the probiotic groups starting on D14 through D20 when compared to the control (P < 0.05; Fig. 1A). Specifically, LP application improved the relative YFBM by 5.0 %, 8.3 %, 3.4 % and 8.0 % on day 14, 18, 19, and 20, of incubation, respectively. Similar improvements in relative embryo weights were also observed in the LR group with an increase of 8.3 %, 5.9 %, 3.4 %, 6.6 % on day 14, 18, 19, and 20, respectively (Fig. 1A; P < 0.05). In general, in ovo probiotic supplementation led to a significant increase in relative YFBM by 5 – 8 % during mid and late incubation when compared to the control (P < 0.05).

Fig. 1.

Effects of in ovo probiotic supplementation on embryo and hatchling weight. (A) Relative embryo weight starting embryonic day 10 to 20 [percentage of yolk-free-body-mass (YFBM) to starting egg weight]; (B) YFBM (hatchling weight following removal of the residual yolk sac) andHatchling weight at day 21. Data are presented as mean ± SE. ^a,b,cDifferent letters indicate significant difference between groups at each time point (P < 0.05).

On day of hatch (D21), hatchling weight was found to be significantly increased in probiotic groups compared with control (P < 0.05; Fig. 1B). Specifically, hatchling weight in the control, LP and LR groups was 42.50 ± 0.34 g, 47.50 ± 0.58 g and 45.80 ± 0.46 g, respectively. Since chick weight is dependent on egg weight and includes the residual YS, YFBM (chick weight minus residual YS weight) is considered a better indicator of hatchling development (Molenaar, 2011; Bergoug et al., 2015). Hence, we determined YFBM for the hatchlings. We observed that the significant increase in hatchling weight in the probiotic groups was associated with a corresponding increase in YFBM (P < 0.05; Fig. 1B). Overall, in ovo probiotic application led to a 6 – 12 % increase in hatchling weight and associated YFBM when compared to the control at day 21.

Along the same lines, few studies have investigated the effect of in ovo probiotic inoculations on late-term embryo development and subsequent performance. These studies delivered probiotics to the developing embryo mostly via intra-amniotic injections around D18. For instance, in ovo inoculation of different probiotic cocktails on D17.5 did not exert any effect on YFBM at D19 and 21 (de Oliveira et al., 2014). Similarly, in ovo injection of Lactobacillus animalis or Enterococcus faecium to 18-day old broiler embryos was not shown to exert any significant effect on hatchling weight (Beck et al., 2019; Castañeda et al., 2020). To the contrary, in ovo injection of Bacillus subtilis ATCC 6051, Lactobacillus acidophilus ATCC 314 and Bifidobacterium animalis ATCC 27536 at D18 was seen to reduce the chick weight at day of hatch (Triplett et al., 2018; Castañeda et al., 2021). As opposed to these studies, we observed the sustained effect of probiotics in improving embryo development throughout incubation into hatch. Further, these results are in line with our previous research on embryo development in layers (Muyyarikkandy et al., 2023a). Specifically, in ovo probiotic spray application to layer and broiler embryos led to significant increase in hatchability (∼5 – 11 %) which was associated with a corresponding improvement in embryo development (∼6 – 10 % increase in relative embryo weight; Muyyarikkandy et al., 2023a, 2023b; Gao et al., 2024). We hypothesize that the beneficial effects observed in our study may be attributed to the early administration of probiotics, as indicated by the improvement in embryo weight beginning on day 14 of incubation. This contrasts with the effects typically seen following late-term in ovo inoculations, which are often administered during the final stages of incubation. More importantly, our data highlights the potential to support the entire pre-natal period, the benefits of which might be extended into the post-hatch period. This is further corroborated by our previous findings demonstrating improved embryo weight as early as day 10 of incubation (Muyyarikkandy et al., 2023a, 2023b).

In addition, a recent study from our lab demonstrated that in ovo probiotic spray application modulates microbiota development in embryos and its acquisition in hatchlings (Gao et al., 2025). However, due to the complex and interconnected nature of embryonic compartments, variations in probiotic delivery methods including the route of administration (e.g., air cell, amniotic fluid, yolk sac), dosage, and dosing regimen can result in distinct physiological outcomes. Moreover, the specific probiotic strain used may also influence the observed effects. These factors highlight the need for future studies comparing in ovo spray application with other delivery methods and assessing the efficacy of alternative probiotic strains.

Hatching muscle

Hatching muscle is a paired muscle extending from the vertebral column to the dorsal surface of the head, which is called the “M. complexus.” It actively aids in forcing the egg-tooth against the shell (“pipping;” Fisher, 1958). It develops starting from D7 of incubation and continues until D20. This muscle plays a critical role in supporting the pipping and hatching process following which it gradually decreases in size during hatching and disappears after hatch (Fisher, 1958). Since our previous results demonstrate an increase in hatchability (Gao et al., 2024), we determined hatching muscle weight as an indicator of its development and subsequent support of hatching.

Our results demonstrate that the relative hatching muscle weight (relative to YFBM in the embryo/chick weight in the hatchling) was significantly higher in the probiotic groups when compared to the control (P < 0.05; Fig. 2A and 2B). Specifically, LP supplementation increased hatching muscle weight by 30.5 %, 20.0 %, 20.7 %, and 13.9 % on D18, D19, D20 and D21 (day of hatch), respectively. LR application enhanced hatching muscle weight by 28.5 %, 55.0 %, 24.2 %, and 25.5 % on D18, D19, D20 and D21, respectively (Fig. 2A). This increase in hatching muscle weight is significant since it is reported that chicks with smaller hatching muscle had lower hatchability. Further the smaller hatching muscle is hypothesized to be an indicator of suboptimal muscle development and subsequent hatch failure (Fisher, 1958). On the other hand, the significant increase in hatching muscle weight in the LP group could help support our previous findings demonstrating a significant increase in hatchability following in-ovo application of LP (Gao et al., 2024). However, we did not observe a similar outcome in the LR group despite the significant improvement in hatching muscle weight. This highlights the complexity of the hatching process and multitude factors that can impact hatchability, including the perinatal embryo's energy status (Uni et al., 2005; Yadgary et al., 2014).

Fig. 2.

Effects of in ovo probiotic supplementation on (A) relative hatching muscle weight from embryonic day 18 to 21 [percentage of hatching muscle weight to embryo/chick weight] and (B) representative images of the pipping muscle during the perinatal period. Data are presented as mean ± SE. ^a,b,cDifferent letters indicate significant difference between groups at each time point (P < 0.05).

Yolk sac morphometry and glycogen reserves

The YS enclosing the yolk serves as the primary and only source of nutrients to the developing embryo and hatchling (Bauer et al., 2013). In addition, recent studies have demonstrated that YS absorbs, digests and transports nutrients to the developing embryo and the hatchling (Speier et al., 2012; Bauer et al., 2013; Yadgary et al., 2014). This ability to absorb and utilize nutrients by the YS is attributed to the yolk sac membrane (YSM). Also, the YSM serves as the primary site for glycogenesis and gluconeogenesis that is critical to fuel the perinatal embryo (Hu et al., 2017; Wong and Uni, 2021). Once synthesized, besides direct transport into the blood, glycogen can also be secreted into the yolk sac content (YSC; Yadgary and Uni, 2012). Therefore, we measured YS weight and determined glycogen content in all three compartments (YSM, YSC and YS) starting on D10 of incubation.

There was no effect of treatment on the YS weight throughout incubation (P > 0.05; Table 1). To account for variability in SEW, YS weight is represented relative to SEW (D10 – D20). In general, relative YS weight was 39.5 – 41.5 % on D10, 24.7 – 25.2 % on D14, 17.6 – 19.3 % on D18 and 12.9 – 14.1 % on D20. The lack of difference in relative yolk sac weights across treatments suggests that the observed increase in embryo weight in the probiotic groups (in the absence of a proportionate decrease in YS mass) could imply better nutrient utilization in the probiotic group as opposed to the control. Likewise, although the residual yolk sac in the probiotic groups was heavier (LP: 6.99 ± 0.31 g; LR: 7.05 ± 0.31 g) than the control (5.96 ± 0.27 g), we did not observe a significant difference in residual YS weight percentage in the chicks (relative to chick weight) across the three groups (Table 1). Overall, the residual yolk sac represented 13 – 15 % of the hatchling weight. These data are in line with previous reports highlighting that a higher metabolic rate during embryonic development period would imply a lower residual yolk sac weight (van der Wagt et al., 2020).

Table 1.

Morphometry of the yolk sac in the developing embryo and hatchling.

Embryonic day	Relative yolk sac weight (%)			Yolk sac content (g)			Yolk sac membrane (g)
	Control	LP	LR	Control	LP	LR	Control	LP	LR
10	41.51 ± 0.99	39.53 ± 0.63	39.73 ± 0.76	23.51 ± 0.69	22.89 ± 0.50	22.82 ± 0.66	2.54 ± 0.15	2.22 ± 0.09	2.26 ± 0.09
12	27.98 ± 0.58	29.13 ± 0.47	29.31 ± 0.48	13.06 ± 0.58	13.70 ± 0.52	13.74 ± 0.44	4.58 ± 0.21	4.40 ± 0.21	4.42 ± 0.14
14	24.68 ± 0.50	25.27 ± 0.47	25.25 ± 0.53	9.10 ± 0.49	10.45 ± 0.61	10.37 ± 0.51	6.17 ± 0.21	5.55 ± 0.26	5.28 ± 0.21
16	21.50 ± 0.58	21.56 ± 0.45	22.49 ± 0.72	6.73 ± 0.45	7.27 ± 0.42	7.74 ± 0.67	6.40 ± 0.15	6.28 ± 0.21	6.10 ± 0.27
18	17.57 ± 0.54	19.57 ± 0.63	19.27 ± 0.73	4.89 ± 0.43	6.41 ± 0.48*	6.44 ± 0.52*	5.90 ± 0.19	5.86 ± 0.17	5.74 ± 0.16
20	13.22 ± 0.52	12.94 ± 0.39	14.14 ± 0.61	2.73 ± 0.22	3.49 ± 0.21§	4.05 ± 0.37§	5.42 ± 0.18	4.78 ± 0.18	4.78 ± 0.24
21	13.96 ± 0.50	14.49 ± 0.49	15.32 ± 0.61	3.30 ± 0.23	4.43 ± 0.23¥	4.07 ± 0.24¥	2.65 ± 0.18	2.57 ± 0.16	2.91 ± 0.22

Data are represented as means ± SEM.

Relative yolk sac weight is calculated as percentage of embryo weight/hatchling weight (D21).

*, §, ¥ Indicates significant difference from the control within each time point at P ≤ 0.05.

While the YS supports overall embryonic development, it is even more critical to fuel the perinatal embryo development and hatching. Particularly, the YSM is actively involved in glycogen synthesis and the YS serves as a glycogen reserve for the hatching process (Uni et al., 2005; Yadgary and Uni, 2012). Hence, we determined the glycogen concentration (mg/g of wet tissue) and glycogen content (mg of total tissue) in YSM and YSC, and total glycogen in the YS (YSM + YSC). As shown in Fig. 3A, glycogen concentration in the YSM increased from D10 to 18, then decreased after D18 in all groups (Fig. 3A and B). A similar pattern was also observed in total glycogen content of YSM (Fig. 3C). This increase in glycogen content in the YSM likely implies active glycogen synthesis, while the subsequent decrease could reflect its breakdown and utilization by the perinatal embryo (Yadgary and Uni, 2012; Dayan et al., 2023a).

Fig. 3.

Effects of in ovo probiotic supplementation on glycogen reserves in the yolk sac from embryonic day 10 to 21. (A) glycogen concentration in yolk sac membrane (YSM); (B) total glycogen content in YSM; (C) total glycogen content in YSC; (D) total glycogen content in YS. Data are presented as mean ± SE. ^a,b,cDifferent letters indicate significant difference between groups at each time point (P < 0.05).

In-ovo supplementation of LP was seen to significantly improve glycogen concentration (mg/g of wet tissue) in the YSM by ∼1.08 – 1.65 mg/g, specifically, through the mid incubation period (D10 – D16) when compared to the control (P < 0.05; Fig. 3A). Glycogen concentration in the YSM of the LP and control group was determined to be 3.18 ± 0.57 and 1.53 ± 0.18 mg/g on D10, 2.01 ± 0.29 and 0.93 ± 0.12 mg/g on D14, and 5.28 ± 0.51 and 3.61 ± 0.4 mg/g on D16, respectively (Fig. 3A). Following D16, glycogen concentration in all groups ranged between 5 – 10 mg/g. In line with the increased glycogen concentration, total glycogen content in the YSM of the probiotic groups was also found to be significantly higher than the control by 2.1 – 11.2 mg during D10 – D16 of incubation (Fig. 3B).

As previously reported, YSM plays a critical role in glycogen synthesis, storage, breakdown and gluconeogenesis (Wong and Uni, 2021). The increased glycogen content in YSM at the early stage in the LP and LR groups could be due to improved glycogen synthesis via glycogenesis or gluconeogenesis with glycerol and lactate as the substrate and fatty acid oxidation (Dayan et al., 2023a, 2023b). The energy demand from the embryo is very high during the last days of incubation, and the main fuel comes from glycogen breakdown. Between D19 to hatch, glycogen might be degraded in the YSM to supply glucose to other embryonic tissues (Yadgary and Uni, 2012). Thus, the significant decrease in glycogen concentration in YSM during the late incubation period may be due to glycogen breakdown and glucose mobilization to meet the energy demand of the perinatal embryo.

In terms of YSC, we did not observe any significant difference in the glycogen concentration among the different groups at all timepoints. Overall, glycogen concentration (mg/g of YSC) in the YSC ranged from 0.51 – 0.58 mg/g on D14 to 4.2 – 4.7 mg/g on D18, and 5.9 – 6.2 mg/g in the residual YSC on day of hatch (D21; Fig. 3B). However, we observed a significantly higher total glycogen content in the YSC of the LP and LR groups on D18 – D21 (Fig. 3C). Specifically, between D18-D21, the total glycogen content in the YSC was determined to be 38 – 54 % higher in the probiotic groups when compared to the control (Fig. 3C, P < 0.05). This increase in YSC glycogen content can be attributed to the concomitant increase in YSC weight (27 – 35 %) in LP and LR groups (Table 1). Moreover, this increase in glycogen content in the YSC starting on D18 is associated with a concomitant reduction in glycogen content in the YSM (Fig. 3B). This could be due to the mobilization of glycogen from the YSM into the YSC for subsequent use by the perinatal embryo (Dayan et al., 2023a, 2023b; Yadgary and Uni, 2012).

As seen with the glycogen content in the YSM and YSC, we observed a consistent increase in total glycogen levels in the YS in the LP and LR groups (Fig. 3D, P < 0.05). The glycogen content in the YS increased from D10 (Control: 3.33 ± 0.49 mg; LP: 6.24 ± 1.11 mg; LR: 5.46 ± 0.81 mg) to D18 (Control: 73.00 ± 5.61 mg; LP: 90.89 ± 5.50 mg; LR: 84.74 ± 3.3.55 mg), and then decreased from D18 to D21 (Control: 31.22 ± 2.62 mg; LP: 40.15 ± 2.80 mg; LR: 39.67 ± 2.03 mg). Further, as seen in Fig. 3D, probiotic supplementation significantly increased glycogen content in the yolk sac by 22 – 87 % throughout incubation. Overall, in ovo probiotic supplementation led to improved glycogen reserves in the YS thereby providing critical energy and fuel for the developing embryo and hatchling. It is important to note here that although in ovo feeding has been shown to improve glycogen levels in the muscle and liver, most studies did not focus on the YS glycogen reserve (Dang et al., 2022; Kornasio et al., 2011; Yadgary and Uni, 2012; Shafey et al., 2010; Uni et al., 2005; Yu et al., 2018). Moreover, limited research on the effect of in ovo supplementation of creatine on YS glycogen content did not observe any significant effect of the treatment (Dayan et al., 2023a). Contrary to these studies, our data demonstrate a potential role for probiotics in modulating glycogen metabolism in the developing embryo through incubation and hatch, thereby supporting overall embryo developing and hatching.

Breast muscle and glycogen reserves

To determine the effect of in ovo probiotic application on embryo development, besides embryo weight, we also measured breast weight. Results from our study demonstrate a sustained increase in relative breast weight (relative to YFBM in the embryo/chick weight in the hatchling) in the developing embryo corresponding to the observed increases in embryo weight (Figs. 1; Fig. 4A and 4B). In the control group, relative breast weight (as percentage of YFBM/hatchling weight) ranged from 4.32 ± 0.27 % on D10, 5.67 ± 0.24 % on D14, 3.98 ± 0.09 % on D18 to 3.18 ± 0.06 % in the hatchling. In comparison with the control, relative breast weight in the LP and LR groups were significantly higher throughout incubation and at hatch (P < 0.05; Fig. 4A). For instance, the relative breast weight in the LP group on D10, D18, and D21 (hatchling) was 7.23 ± 0.49 %, 5.11 ± 0.05 %, and 4.23 ± 0.07 %, respectively. Overall, in ovo supplementation of probiotics (LP and LR) improved relative breast weight by 8 % on D16, 28 % on D18, and 30 – 34 % in the hatchling. These findings correspond to our previous report on muscle growth and development in the broiler embryo (Muyyarikkandy et al., 2023b). The observed improvement in relative breast weight was associated with an increase in myofiber density by 77 – 80 % on D18 and modulation of key myogenic genes (Muyyarikkandy et al., 2023b). Similarly, in ovo administration of different nutrients including carbohydrates and environmental manipulation was shown to improve perinatal muscle growth and hatchability (Harding et al., 2016; Halevy et al., 2006; Kornasio et al., 2011).

Fig. 4.

Effects of in ovo probiotic supplementation on breast muscle weight and glycogen levels. (A) relative breast muscle weight [percentage of breast muscle weight to embryo/chick weight], (B) representative images of the breast from the perinatal period, (C) glycogen concentration in the breast muscle and (D) total glycogen content in breast muscle from embryonic day 10 to 21. Data are presented as mean ± SE. ^a,b,cDifferent letters indicate significant difference between groups at each time point (P < 0.05).

The pectoral muscle in the perinatal embryo accounts for the greatest quantity of total glycogen stored in the body (Christensen et al., 2001). While glycogen serves as the immediate substrate for glucose, gluconeogenesis via the breakdown of protein helps meet the energy demand once glycogen is depleted (Uni and Ferket, 2004). Towards this, the pectoral muscle is the predominant source of protein for gluconeogenesis in the perinatal embryo (Vieira and Moran, 1999). Therefore, depletion of glycogen reserves may consequently force the embryo to mobilize more muscle protein for gluconeogenesis (Uni et al., 2005; Oliviera et al., 2008; Zhao et al., 2018). Depletion of glycogen reserves during the perinatal period is associated with a reduction in hatching weight, breast muscle yield and market weight (Uni and Ferket, 2004; Uni et al., 2005; Tangara et al., 2010; Kornasio et al., 2011). Given that optimum breast muscle development is critical not only to future performance but also the energy status of the embryo, we determined the effect of in ovo probiotic supplementation on promoting breast muscle growth and improving pectoral muscle glycogen reserves.

As seen in Fig. 4C, glycogen concentration (mg/g of wet tissue) in the embryonic breast muscle varied from ∼ 2.5 mg/g on D10 to ∼ 8 – 9 mg/g in the hatchling (Fig. 4C). This trend in pectoral muscle glycogen concentration corresponds to previous findings as reported by Uni et al. (2005) and Dayan et al. (2023a). Further, similar to the YS, LP and LR supplementation significantly improved glycogen concentration and total glycogen content in the breast muscle when compared to the control group during the perinatal period (Fig. 4C and D, P < 0.05). For instance, glycogen concentration (mg/g of wet tissue) in the LR group ranged from 8 – 9.7 mg/g during the perinatal period as opposed to 6.1 – 6.7 mg/g in the control group (Fig. 4C). This represents a 24 – 47 % increase in glycogen concentration in the breast muscle from probiotic treated embryos and hatchlings.

In association with the glycogen concentration, we observed significantly improved total glycogen content in the breast muscle in the embryos and hatchlings from the probiotic groups in comparison to the control (Fig. 4D, P < 0.05). In the LP group, total breast muscle glycogen content was determined to be 9.3 ± 0.6 mg on D18, 10.83 ± 1.06 mg on D20 and 14.67 ± 1.91 mg in the hatchlings, respectively. Whereas total pectoral glycogen reserves in the control group ranged from 6 – 7 mg between D18 and D21. Overall, LP and LR supplementation improved pectoral muscle glycogen reserves by 10 – 52 % on D10, and 82 – 89 % in the hatchling. This significant increase in muscle glycogen reserves corresponds to the higher breast weight and glycogen concentration observed in the probiotic treated groups. This in turn supports our hypothesis that improved breast muscle growth and muscle mass provides for increased glycogen reserves. On the other hand, the increased glycogen levels in the probiotic group provides more energy reserves, which could limit the need for muscle protein breakdown to generate glucose via gluconeogenesis, resulting in heavier breast muscles (Uni et al., 2005; Hu et al., 2017).

Glycogen metabolism in the liver

As in the adult birds, the liver is responsible for maintaining energy homeostasis via the regulation of carbohydrate, protein, and lipid metabolic pathways in the developing embryo (de Oliveira et al., 2008, Zhai et al., 2011). Particularly in the perinatal embryo, glycogen reserves in the liver play a crucial role in providing much needed energy for perinatal embryo development. Further, towards the end of the incubation period, liver glycogen is rapidly metabolized to glucose, which fuels the hatching process (Moran, 2007; Zhai et al., 2011). Given our previous observations on probiotic mediated improvement in hatchability and hatchling quality (Gao et al., 2024) and the liver’s critical role in glycogen metabolism, we measured relative liver weights, liver glycogen concentration and total glycogen content. In terms of liver morphometry, as embryonic development progressed, relative liver weights increased from 1.2 % on D10 to 2.4 % in the hatchling. In addition, we did not observe any significant difference in relative liver weights between the probiotic and control groups (P > 0.05; data not shown). We then determined the glycogen concentration (mg/g of wet tissue) and total glycogen content (mg of total tissue) in the liver (Fig. 5A and 5B).

Fig. 5.

Effects of in ovo probiotic supplementation on liver glycogen levels. (A) glycogen concentration in the liver and (B) total glycogen content in the liver from embryonic day 10 to 21. Data are presented as mean ± SE. ^a,b,cDifferent letters indicate significant difference between groups at each time point (P < 0.05).

With the liver glycogen concentration (mg/g of wet tissue), we observed an initial increase in glycogen concentration from D10 until D18 [Control: 2.06 ± 0.21 mg/g (D10) to 7.32 ± 0.925 mg/g (D18); LP: 3.74 ± 0.47 mg/g (D10) to 9.82 ± 1.21 mg/g (D18); LR: 2.80 ± 0.33 mg/g (D10) to 7.08 ± 1.42 mg/g (D20)] followed by drastic decrease until D21 (Control: 1.77 ± 0.13 mg/g; LP: 1.96 ± 0.16 mg/g; LR: 2.30 ± 0.10 mg/g; Fig. 5A). As observed in the yolk sac and breast muscle, the probiotic groups exhibited significantly higher liver glycogen concentration and total glycogen content compared to the control throughout incubation. (Fig. 5A). For instance, with the LP group, liver glycogen concentration was higher than the control by 83 %, 93 %, 34 %, and 11 % at D10, 14, 18, and 21. In line with the glycogen concentration, total glycogen content in the liver followed a similar pattern with amounts ranging from 0.08 – 0.2 mg on D10 to 4.5 – 5.7 mg on D18 and 1.7 – 2.4 mg on D21. Further, as seen with the higher glycogen content in the residual YS of the hatchling, probiotic supplementation also led to significantly higher liver glycogen content in the hatchling (P < 0.05, Fig. 5B). LP and LR supplementation led to a 0.5 – 2.2 mg increase in liver glycogen amounts in the hatchlings when compared to the control.

The trend in changing glycogen reserves during incubation is consistent with published literature (Kornasio et al., 2011; Yadgary and Uni, 2012). For example, in ovo injection of carbohydrates improved glycogen content in liver and pectoral muscle (Shafey et al., 2012; Naeem Asa et al., 2022). Similarly, Uni et al. (2005; 2011) used in ovo injection of carbohydrate and β‑hydroxy-β-methylbutyrate, and results indicated an improved glycogen levels in liver and muscle. Additionally, Neves et al. (2020) found that in ovo injection of glycerol and insulin-like growth factor significantly increased the hepatic glycogen levels, with no effect on muscle glycogen. However, the supplements used in the in ovo injection were mostly the substrates for gluconeogenesis, which is the major pathway for glucose synthesis in the late term embryo when glycogen reserves are limited. Moreover, to our knowledge, probiotics have not been previously evaluated for their ability to modulate glycogen reserves in the broiler embryo.

Total glycogen reserves in the developing embryo and hatchling

To elucidate the overall effect of probiotics on the energy status of the embryo, we determined total glycogen reserves available to the embryo. As seen from Fig. 6, total glycogen content increased from D10 (Control: 4.10 ± 0.55 mg; LP: 7.67 ± 1.19 mg; LR: 6.72 ± 0.87 mg) to 18 (Control: 89.30 ± 5.77 mg; LP: 106.52 ± 4.04 mg; LR: 100.68 ± 2.57 mg), and then decreased until D21 (Control: 42.67 ± 3.26 mg; LP: 52.00 ± 2.67 mg; LR: 54.26 ± 3.08 mg). In total, supplementation with LP and LR significantly increased total glycogen reserves in the embryo and hatchling through incubation (P < 0.05). This implies that probiotic-treated embryos had significantly higher glycogen reserves during incubation, the perinatal period and immediately following hatch. This indicates that the improved total glycogen content provides higher energy reserves that support embryo development and hatching. This is critical since hatching is a high energy demanding process, and late-term embryo may suffer from the stress of insufficient energy (van der Wagt et al., 2020).

Fig. 6.

Effects of in ovo probiotic supplementation on total glycogen content in the embryo from embryonic day 10 to 21. Total glycogen content was determined as the summative total of glycogen reserves in the YS, breast muscle and liver. Data are presented as mean ± SE. ^a,b,cDifferent letters indicate significant difference between groups at each time point (P < 0.05).

Beyond hatch, commercial chicks are often subjected to delayed access to feed and therefore must rely on nutrient reserves in the residual YS (Souza Da Silva et al., 2021). Further, nutrient deprivation at this critical stage can have long-term effects on post-hatch muscle and intestine development, thereby impacting overall growth and performance (Nazem et al., 2019; Jha et al., 2019; Wang et al., 2022). Hence, hatchlings that start off with better nutrients reserves will be of higher quality with better survival rates and post-hatch performance. Towards this, in ovo inoculation of different nutrients was seen to improve glycogen reserves in the embryo and hatchling, support hatching and promote breast muscle growth and improve post hatch performance (Kornasio et al., 2011; Zhai et al., 2011; Dayan et al., 2023a, 2023b). In line with these findings, our data demonstrate a strong potential for in ovo early probiotic supplementation to improve energy reserves in the embryo support embryo growth, breast muscle development, hatchability and hatchling (Gao et al., 2024; Muyyarikkandy et al., 2023b). Moreover, the improved glycogen reserves in the probiotic-treated hatchlings can provided sustenance to chicks during the immediate post-hatch phase before feed is available.

Probiotic mediated modulation of glycogenesis, glycogenolysis and gluconeogenic pathways in YSM, breast muscle and liver

Given the observed improvement in glycogen reserves, we examined the effect of probiotics on key genes involved in glycogen metabolism and gluconeogenesis. Specifically, we determined the expression of glycogen synthesis enzymes [glycogen synthase (GYS1 for breast muscle; GYS2 for YSM and liver)], and gluconeogenic enzymes [fructose 1,6-bisphosphatase (FBP1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6PC2), and glycerol kinase (GK)]. Since Lactobacillus paracasei (LP) was more effective than Lactobacillus rhamnosus (LR), gene expression assays were conducted only for the LP group.

In the YSM, GYS2 was significantly upregulated (1.43 – 1.5-fold) on Day 10 and Day 14 in the LP group when compared to the control (Table 2, P < 0.05). This observation aligns with the improved glycogen concentration (∼+1.08 – 1.65mg/g) and total glycogen content (∼+2.9 – 5.5 mg) observed in the YSM of the LP group (Fig. 3B and 3C). During the late-incubation period, the YSM’s absorptive surface decreases leading to a reduction in its functional capacity and eventual structural degeneration and resorption (Yadgary et al., 2014et al). At this stage, metabolic reliance shifts to the liver and muscle to fuel embryo development and the hatching process (Tong et al., 2013; Yadgary et al., 2014). Correspondingly, LP supplementation enhanced glycogen reserves by ∼0.6 – 2.2 mg (liver, Fig. 5B) and 1.08–7 mg (muscle, Fig. 4D) from Days 14 to 21. This was supported by a significant 1.23 – 1.6-fold upregulation of GYS2 (liver) and GYS1 (muscle; Table 3, 4, P < 0.05). However, no consistent differences in GYS expression were observed at other times, despite elevated glycogen levels in late incubation (Figs. 4C, 4D and 5A, 5B). This aligns with previous reports of inconsistent GYS expression and glycogen levels, suggesting a need for comprehensive broader transcriptomic analysis (Yadgary and Uni, 2012).

Table 2.

Effects of in ovo probiotic supplementation on glycogenic, glycogenolytic and gluconeogenic genes in the YSM of the developing embryo and hatchling.

	Embryonic day
	10	14	18	21
Genes	Control	LP	Control	LP	Control	LP	Control	LP
GYS2	1 ± 0.05	1.43 ± 0.40*	1 ± 0.17	1.5 ± 0.30*	1 ± 0.10	−0.7 ± 0.50	1 ± 0.87	−1.9 ± 0.66
PYGL	1 ± 0.07	−1.31 ± 0.21*	1 ± 0.58	−0.93 ± 0.22*	1 ± 0.08	−1.5 ± 0.10*	1 ± 0.55	−1.8 ± 0.20*
FBP1	1 ± 0.12	0.61 ± 0.10	1 ± 0.50	−0.48 ± 0.20	1 ± 0.11	0.05±0.68	1 ± 0.48	−1.2 ± 0.55
G6PC2	1 ± 0.30	−1.66 ± 0.45*	1 ± 0.50	−0.05 ± 0.16	1 ± 0.09	−0.15 ± 0.40	1 ± 0.40	−1.04 ± 0.41
PEPCK-C	1 ± 0.50	−2.36 ± 0.58*	1 ± 0.65	−0.74 ± 0.21*	1 ± 0.30	0.3 ± 0.60	1 ± 0.61	−2.4 ± 0.85*
PEPCK-M	1 ± 0.20	−2.59 ± 0.18*	1 ± 1.20	−1.63 ± 0.80*	1 ± 0.10	−1.86 ± 0.22*	1 ± 0.50	−0.72 ± 0.50
GK	1 ± 0.10	−1.54 ± 0.27*	1 ± 0.50	0.96 ± 0.10	1 ± 0.10	−1 ± 0.45*	1 ± 0.50	−1.05 ± 0.41

Data are represented as mean±SE.

* indicate significant difference from the control for the respective gene within each sampling time at P ≤ 0.05.

▲ upregulation, ▼ downregulation, ○ Neutral.

Table 3.

Effects of in ovo probiotic supplementation on glycogenic, glycogenolytic and gluconeogenic genes in the breast muscle of the developing embryo and hatchling.

	Embryonic day
	14	18	21
Genes	Control	LP	Control	LP	Control	LP
GYS1	1 ± 0.31	1.60 ± 0.56*	1 ± 0.40	0.2 ± 0.54	1 ± 0.54	0.49 ± 0.61
PYGL	1 ± 0.38	1.25 ± 0.10*	1 ± 0.49	0.1 ± 0.50	1 ± 0.40	−0.66 ± 0.45
FBP1	1 ± 0.50	1.71 ± 0.22*	1 ± 0.50	0.82 ± 0.62	1 ± 0.80	0.23 ± 0.71
G6PC2	1 ± 0.50	1.17 ± 0.53	1 ± 0.42	0.6 ± 0.42	1 ± 0.52	−1.26 ± 0.32*
PEPCK-C	1 ± 0.68	2.96 ± 0.41*	1 ± 1.23	2.06 ± 1.12	1 ± 0.93	−2.36 ± 0.71*
PEPCK-M	1 ± 0.41	1.81 ± 0.23*	1 ± 0.53	−0.6 ± 0.52	1 ± 0.43	−0.49 ± 0.52
GK	1 ± 0.40	1.24 ± 0.20*	1 ± 0.44	−1.34 ± 0.11*	1 ± 0.50	0.14 ± 0.60

Data are represented as mean±SE.

* indicate significant difference from the control for the respective gene within each sampling time at P ≤ 0.05.

▲ upregulation, ▼ downregulation, ○ Neutral.

In addition, we also assessed glycogen breakdown by determining PYGL (glycogen phosphorylase) expression. PYGL was significantly downregulated in YSM at D10, 14 and 18 (Table 2; P < 0.05). During the mid-stage of incubation, fatty acid oxidation is the main energy source Since fatty acid oxidation is the primary energy source during mid-incubation (de Oliveira et al., 2008), LP supplementation may have enhanced this pathway, reduced glycogen breakdown and increasing glycogen reserves. Also, embryos primarily accumulate glycogen content before D18 to prepare for hatching instead of consuming glycogen. Similarly, PYGL was significantly downregulated at D18 and D21 in liver (P < 0.05; Table 4), potentially explaining the higher total liver glycogen content in the LP group when compared to the control (Fig. 5A and 5B).

Table 4.

Effects of in ovo probiotic supplementation on glycogenic, glycogenolytic and gluconeogenic genes in the liver of the developing embryo and hatchling.

	Embryonic day
	14	18	21
Genes	Control	LP	Control	LP	Control	LP
GYS2	1 ± 0.58	1.23 ± 0.08*	1 ± 0.13	0.98 ± 0.11	1 ± 0.11	0.93 ± 0.11
PYGL	1 ± 0.63	−0.34 ± 0.68	1 ± 0.10	0.87 ± 0.12*	1 ± 0.20	0.53 ± 0.24*
FBP1	1 ± 0.60	−0.9 ± 0.57*	1 ± 0.14	1.13 ± 0.10	1 ± 0.14	0.79 ± 0.14
G6PC2	1 ± 0.71	−1.69 ± 0.70*	1 ± 0.12	0.92 ± 0.10	1 ± 0.10	1.05 ± 0.10
PEPCK-C	1 ± 0.70	−5.02 ± 1.11*	1 ± 0.94	0.74 ± 0.23	1 ± 0.81	1.16 ± 0.43
PEPCK-M	1 ± 0.51	−0.93 ± 0.52	1 ± 0.22	−1.51 ± 0.22*	1 ± 0.33	0.54 ± 0.11*
GK	1 ± 0.54	−0.16 ± 0.81	1 ± 0.13	0.83 ± 0.13*	1 ± 0.22	0.5 ± 0.14*

Data are represented as mean±SE.

* indicate significant difference from the control for the respective gene within each sampling time at P ≤ 0.05.

▲ upregulation, ▼ downregulation, ○ Neutral.

Lastly, we examined the genes associated with the gluconeogenic pathway, including FBP1, PEPCK, G6PC2, and GK. FBP1 is a key rate-limiting enzyme which converts fructose 1,6-bisphosphate into fructose 6-phosphate (Pilkis and Granner, 1992). PEPCK-C uses amino acids and pyruvate for gluconeogenesis, while PEPCK-M primarily utilizes lactate for gluconeogenesis (Watford et al., 1981; Watford, 1985). G6PC2 converts glucose 6-phosphate to free glucose (Allred and Roehrig, 1970). GK is responsible for gluconeogenesis via glycerol (Yadgary and Uni, 2012). Overall, we observed significant downregulation in gluconeogenic genes in all tissues at different sampling times (Table 2 – 4).

In the YSM, G6PC2, PEPCK, and GK were downregulated by 1.3 – 2.6-fold on Day 10, with further reductions in PEPCK and GK at Days 14, 18, and 21 (P < 0.05, Table 2). In breast muscle, while some gluconeogenic genes (FBP1, PEPCK-C, PEPCK-M, GK) were upregulated at Day 14, the key rate limiting enzyme G6PC2 and PEPCK were significantly downregulated at D21 in the LP group when compared to the control (P < 0.05, Table 3). During D18 and 21, glycogen is used as the main energy source to support the hatching process (Uni and Ferket, 2004). Towards this, our data demonstrate the probiotic application improved glycogen reserves in the perinatal embryo when compared to the control. The improved glycogen reserves in the probiotic treated embryos could in turn spare the need for breakdown of protein, particularly muscle protein to generate glucose for energy. Further, this could support improved muscle development as observed by the improved breast weight in the perinatal embryo and hatchling in the LP group (Fig. 4A and 4B). Although Lactobacillus spp. have been shown to modulate skeletal muscle glucose metabolism and promote hypertrophic growth in broilers characterized by increased muscle fiber diameter and density (Cai et al., 2024; Muyyarikkandy et al., 2023b), additional studies are warranted to elucidate the effects of probiotic spray application. Further investigation is warranted to delineate the influence of probiotics on myogenesis and its interplay with glycogen deposition and glucose homeostasis during embryonic development.

In the liver, FBP1, G6PC2, and PEPCK-C were downregulated on Day 14, while PEPCK-M and GK were repressed at Days 18 and 21 (P < 0.05, Table 4), indicating reduced gluconeogenic activity due to elevated liver glycogen in the LP group. This is in line with the findings of Zhao et al. (2018) demonstrating an increase in PEPCK expression in the liver on D19 and 21 following in ovo supplementation of gluconeogenic substrates like creatine pyruvate. Overall, probiotic supplementation and in particular LP enhanced glycogen reserves via increased glycogenesis rather than gluconeogenesis. This suggests improved energy availability in the perinatal period, reducing reliance on muscle protein breakdown for glucose. Given that perinatal energy imbalances impair hatchability, chick quality, and muscle growth, our findings support early in ovo probiotic supplementation as a strategy to optimize embryonic development and post-hatch performance.

Conclusions

In summary, early in ovo probiotic spray application promoted embryonic growth and enhanced glycogen deposition in both the developing embryo and hatchling. Probiotic administration significantly increased relative embryo and pectoral muscle weights throughout incubation and at hatch. These growth enhancements were associated with elevated glycogen concentrations in the yolk sac, liver, and pectoral muscle, which may underlie the improved hatchability previously observed in the LP group. Post-hatch, probiotic-treated chicks maintained significantly higher tissue glycogen levels, providing a critical energy reserve prior to initial feed intake and potentially contributing to improved hatchling quality. Moreover, increased glycogen availability corresponded with downregulation of key gluconeogenic genes, thereby reducing proteolytic catabolism and supporting muscle accretion and early post-hatch performance. Nonetheless, further transcriptomic analyses are warranted to delineate the molecular mechanisms mediating these probiotic-induced effects.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Mary Anne Amalaradjou has patent #US11497197B2 issued to University of Connecticut. Corresponding author serves as an associate editor for Poultry Science.