An investigation of the effect of folic acid and its delivery routes on broiler chickens’ hatch and growth performance, blood biochemistry, anti-oxidant status, and intestinal morphology

Abstract

This study investigated the effect of folic acid (FA) and its delivery routes (in-feed or in ovo) on broiler chicken’s hatch and growth performance, blood biochemistry, anti-oxidant status, and intestinal morphology. A total of 1,860 Cobb 500 hatching eggs were incubated for 21 d. On day 12 of incubation, viable eggs were randomly allotted to four groups: the noninjected group, in ovo saline (injected with 0.1 mL/egg of saline solution), in ovo FA 1 (injected with 0.1 ml FA containing 0.1 mg/egg; FA1), and in ovo FA 2 (injected with 0.1 ml FA containing 0.15 mg/egg). All in ovo treatments were delivered via the amnion. At hatch, chicks were re-allotted to five new treatment groups: FA1, FA2, in-feed FA (FA 3; 5mg/kg in feed), in-feed bacitracin methylene disalicylate (BMD; 55 mg/kg in feed), and negative control (NC; corn-wheat-soybean diet) in 6 replicate pens (22 birds/pen) and raised in starter (days 0 to14), grower (days 15 to 24), and finisher (days 25 to 35) phases. Hatch parameters were assessed on day 0, and body weight and feed intake (FI) were determined weekly. On day 25, 1 bird/cage was euthanized, immune organs weighed, and intestinal tissues harvested. Blood samples were collected for biochemistry and anti-oxidant (Superoxide dismutase-SOD and Malondialdehyde-MDA) analysis. Data were analyzed in a randomized complete block design. While FA1 and FA2 decreased (P < 0.001) hatchability in a dose-dependent manner, FA2 caused a 2% increase (P < 0.05) in average chick weight compared to the noninjected group. Compared to the BMD treatment, FA3 decreased (P < 0.05) average FI across all feeding phases. At the end of the trial on day 35, FA2 had similar feed conversion ratio as the BMD treatment while recording less (P < 0.001) FI. FA1 and FA2 recorded a tendency (P < 0.1) to increase MDA levels and SOD activity by 50% and 19%, respectively, compared to the NC treatment. Compared to NC treatment, FA2 increased (P < 0.01) villus height, width, and villus height to crypt depth ratio in the duodenum, and villus width in the jejunum. Besides its negative effect on hatchability, FA2 may help improve embryonic development and anti-oxidant status in broiler chickens.

This study reveals the potential of in ovo delivery of folic acid to improve broiler chicken’s hatch weight, intestinal morphology, anti-oxidant status, and growth performance.

Introduction

Antibiotic growth promoters (AGP) are renowned in animal production, especially poultry production, for their roles in disease prevention and growth promotion. However, issues related to the emergence of antibiotic-resistant bacteria and increased consumer demand for antibiotic-free poultry product have stirred public outcry against AGP use in poultry production. This public outcry has necessitated the search for potent alternatives that ensure disease prevention and growth promotion in the poultry industry. For the poultry industry to successfully depart from AGP use, it is pertinent that all strategies that ensure bird growth promotion and disease prevention are employed to meet the rising demand for poultry products.

Moreover, the avian embryonic development is unique compared to their mammalian counterparts, as a continuous maternal supply of nutrients is lacking. This limits the embryo’s nutritional requirements to what can be supplied by the egg alone. Several indicators suggest the inadequacies of avian embryo nutrition via the egg. For instance, Ohta et al. (2001) have reported that only 25% to 30% of nutrients, including vitamins supplied to breeder diets, are incorporated into their eggs, suggesting that the embryo might require an external supply of nutrients for optimum growth. Additionally, an imbalance in available nutrients is triggered by excessive metabolic heat produced by the growing embryo during the late incubation phase (Janke et al., 2004). Interestingly, these nutrient deficiencies occur at a time (especially the late stage of incubation) when embryo energy requirements are usually high. Hence, it is unsurprising that a reduction in embryo growth rate and increased mortality due to nutrient and energy deficiencies at this time point have been recorded in broiler chickens (Zhai et al., 2011a; Ebrahimi et al., 2017). Furthermore, under current commercial poultry settings, chicks often encounter other perinatal nutritional deficiencies that include delayed access to feed that could last about 24 to 36 h due to long hatch window and other time-consuming hatchery activities that could include chick sexing, sorting, and transportation. Hatchlings are, thus, unable to meet the energy and thermoregulation nutritional requirements, making them predisposed to immunosuppression, dysbiosis and reduced growth rate (Gholami et al., 2015; Momeneh and Torki, 2018; Nouri et al., 2018). As the late incubation period is critical for enteric embryo development and posthatch growth development (Uni and Ferket, 2003; Luqman et al., 2019), an optimal supply of nutrients at this time-point must be ensured.

In ovo delivery technology, defined as “the direct inoculation of bioactive substances to the developing embryo to elicit superior lifelong effects, while considering the dynamic physiology of the chicken embryo”, offers an opportunity to mitigate the perinatal nutritional deficiencies that chicks encounter (Oladokun and Adewole, 2020). The in ovo delivery of nutrients has been established as one possible way to enhance hatchability and posthatch performance in poultry (Najih Jabir Al-Shamery and Mohammed Baqur S. Al-Shuhaib, 2015; Joanna et al., 2017; Bhattacharyya et al., 2018; Zhang et al., 2018). Oladokun and Adewole (2020) have also recently revealed the potential application of in ovo technology to deliver several bioactive substances with immunomodulatory properties as alternatives to AGP. To substantiate the efficacy of in ovo-delivered bioactive substances replacing AGP, the in ovo delivery of several nutrients, including trace elements, amino acids, and vitamins, continues to gain interest across several poultry studies (Bakyaraj et al., 2012; Hou and Tako, 2018; Oladokun and Adewole, 2020).

Although scarcely researched, folic acid (FA) is one of several vitamins whose in ovo delivery is currently being researched. FA belongs to the water-soluble vitamin B-complex group. It is physiologically important for its role in deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein methylation, as well as acting as a coenzyme involved in nucleic and amino acids synthesis and metabolism (Bailey and Gregory, 1999; Choi and Mason, 2000; Leung et al., 2013; El-Azeem et al., 2014). It also plays a crucial role in embryo development and is essential for embryo brain and nerve cell development (Viera, 2007; Hussian et al., 2019). Moreover, breeder hens are reported to have higher FA requirement compared to laying hens (Viera, 2007). The role of FA in red blood cell synthesis and immunocompetence in domestic animals have also been reported (Feng et al., 2011; Asif, 2016). Folate deficiency has been associated with cardiovascular disease, intra-uterine growth retardation in humans (Boushey et al., 1995), short bones, curved tibia, and beak defects in poultry (Ezzat and Shoeib, 2011). Long-term storage of hatching eggs has been implicated as a possible cause of FA deficiency (Whitehead et al., 1985; Liu et al., 2016).

A few studies have highlighted the potential of the in ovo delivery of FA to improve embryo growth and organ development, growth performance indices, immune status, and blood biochemical properties, including plasma cholesterol, glucose, and phosphorus levels in broiler chickens (Bekhet, 2013; El-Azeem et al., 2014; Liu et al., 2016; Nouri et al., 2018; Ismail et al., 2019; Gouda et al., 2022; Tufarelli et al., 2022). Aside from the paucity of studies involving the in ovo delivery of FA, conflicting results on the effect of FA on broiler chicken performance exist in the literature. For instance, varying doses of in ovo delivered FA have been reported to improve hatchability in poultry (Robel, 2002; Li et al., 2016; Hussian et al., 2019; Ismail et al., 2019). Also, some studies have reported that hatchability and growth performance were not affected by FA supplementation (Robel, 1993a; Nouri et al., 2018; Tufarelli et al., 2022). Therefore, this study sought to investigate the effect of the supplementation of two doses of FA (0.1 and 0.15 mg per egg) and its delivery routes (in ovo vs. in-feed) on hatch and growth performance, intestinal morphology, blood biochemistry, immune, and anti-oxidant status of broiler chickens, compared to in-feed antibiotics. To our knowledge, this is the first direct comparison of in ovo and in-feed delivered FA in poultry studies.

Materials and Methods

Ethics statement

The experiment was carried out at the hatchery facility of the Agricultural Campus of Dalhousie University and the broiler rearing facility of the Atlantic Poultry Research Center, Dalhousie Faculty of Agriculture. The experiment was conducted following guidelines recommended by the Canadian Council on Animal Care (Rowsell, 1990) and approved by the Animal Care and Use Committee of Dalhousie University (Protocol number: 2021-032).

Egg incubation and in ovo injection procedure

Hatching broiler eggs (Cobb 500, 52 wk old breeders, average weight = 63 ± 1.27 g, N = 1860) were obtained from a commercial hatchery (Cox Atlantic Chick hatchery, Nova scotia) and incubated in a ChickMaster single-stage incubator (ChickMaster G09, Cresskill, NJ, USA), under standard conditions (37.5 °C, 55% relative humidity) from embryonic days (ED) 1 to 19, and then to an average of 32 °C and 68% from ED 19 to 21. Eggs were candled on ED12, and unviable eggs were discarded. Viable eggs were subsequently assigned to one of four experimental groups: 1) noninjected eggs (control; 166 eggs); 2) in ovo saline eggs (38 eggs; injected with 0.2 mL of physiological saline, i.e., 0.9% NaCl, Baxter Corporation, ON, Canada); 3) in ovo FA group 1 (53 eggs; injected with 0.1 mL FA (FA; ≥97%; Sigma, USA) at 0.1 mg per egg) and 4) in ovo FA group 2 (53 eggs; injected with 0.1 mL FA at 0.15 mg per egg). The injection of FA on ED 12 was via the amnion. Treatments were replicated in six similar incubators operated under similar conditions. The injection procedure utilized in this study has been previously described by Oladokun et al. (2021). Briefly, all eggs were disinfected by cleaning with 70% alcohol swabs (BD alcohol swabs-catalogue 326910, ON, Canada), followed by careful punching of the air cell (the blunt end of the egg) using an 18-gauge needle. The injected FA treatments were then delivered to the amnion using a selfrefilling injector (Socorex ultra-1810.2.05005, Ecublens, Switzerland) equipped with a 22-gauge needle (injection needle length - 3 cm) at a 45° angle. After injection, the injection sites were sealed with sterile medical tapes (Nexcare Flexible Clear Tape-7100187758, 3M, MN, USA). The noninjected eggs were taken out and returned to the incubator simultaneously with other injected treatment groups.

Birds, housing, and diets

Hatchlings were weighed and randomly assigned to five new treatment groups (Figure 1). Chicks from the initial noninjection group were randomly allocated into three new treatment groups consisting of 1) chicks fed with a basal corn-soybean meal-wheat-based diet (Negative Control treatment; NC); 2) chicks fed with NC + 0.05% bacitracin methylene disalicylate (in-feed antibiotics); and 3) chicks fed NC + 5 mg/kg FA (in-feed FA). The in ovo FA treatments were placed on the control diet to form treatments 4) in ovo FA group 1, and 5) in ovo FA group 2. Chicks (mixed sex, N = 22) were weighed and assigned to 6 replicate floor pens (0.93 m × 2.14 m)/treatment at a stocking density of 0.076 m²/bird. There were two broiler production rooms. The temperature in the broiler room was monitored daily and was gradually reduced from 32 to 22.5 °C from days 0 through 35. The lighting program was set to 18 h of light and 6 h of darkness throughout the experimental period, and illumination was gradually reduced from 20 lx on day 0 to 5 lx on day 35. Dietary treatments, ingredients, and nutritional composition are presented in Table 1. Birds were provided with feed and water ad libitum; diets were fed as mash in the starter (0 to 14 d) phase and pellets in the grower (15 to 25 d) and finisher (26 to 35 d) phases. Diets met or exceeded the NRC nutritional requirements for broiler chickens (NRC, 1994).

Figure 1.

Schematic presentation of experimental structure in the hatchery and barn. In ovo folic acid group 1 - eggs injected with 0.1 mg folic acid per egg; In ovo folic acid group 2 - eggs injected with 0.15 mg folic acid per egg; In ovo saline group - injected with 0.2 mL of physiological saline (0.9% NaCl); In-feed antibiotics - chicks fed NC + 0.05% bacitracin methylene disalicylate; In-feed folic acid - chicks fed NC + 5 mg/kg folic acid; NC - Negative control treatment - chicks fed a basal corn-soybean meal-wheat–based diet.

Table 1.

Composition and nutritional contents of experimental diets¹ (as-fed basis, percentage (%), unless otherwise stated)

Ingredients	Phases
Starter (0 to 14 d)			Grower (15 to 25 d)			Finisher (26 to s35 d)
Negative control	In-feed antibiotics	In-feed folic acid	Negative control	In-feed antibiotics	In-feed folic acid	Negative control	In-feed antibiotics	In-feed folic acid	Ingredient composition
Corn (ground)	46.63	46.53	46.63	51.16	51.06	51.16	53.63	53.53	53.62
Soybean meal -46.5	37.12	37.14	37.13	31.87	31.89	31.87	29.2	29.22	29.21
Wheat	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Soybean oil (young or mature)	1.80	1.83	1.80	2.18	2.21	2.18	2.75	2.78	2.75
Limestone	1.37	1.37	1.37	1.30	1.30	1.30	1.19	1.19	1.19
Dicalcium phosphate 21 P	1.45	1.45	1.45	1.35	1.35	1.35	1.18	1.18	1.18
DL Methionine premix2	0.58	0.58	0.58	0.57	0.57	0.57	0.52	0.52	0.52
Vitamin/Mineral premix 3,4	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Salt	0.38	0.38	0.38	0.36	0.36	0.36	0.36	0.36	0.36
Lysine HCl	0.17	0.17	0.17	0.21	0.21	0.21	0.17	0.17	0.17
Pellet binding agent	–	–	–	–	0.50	0.50	0.50	0.50	0.50
BMD 110G5	–	0.05	–	–	0.05	–	–	0.05	–
Folic acid sigma	–	–	0.0005	–		0.0005	–	–	0.0005
Total	100	100	100	100	100	100	100	100	100
Nutrient Calculated Composition
Metabolizable energy, kcal/kg	2,975	2,975	2,975	3,025	3,025	3,025	3,100	3,100	3,100
Crude protein	22	22	22	20	20	20	19	19	19
Calcium	0.90	0.90	0.90	0.84	0.84	0.84	0.76	0.76	0.76
Available phosphorus	0.45	0.45	0.45	0.42	0.42	0.42	0.38	0.38	0.38
Sodium	0.18	0.18	0.18	0.17	0.17	0.17	0.17	0.17	0.17
Digestible lysine	1.22	1.22	1.22	1.12	1.12	1.12	1.02	1.02	1.02
Digestible methionine + cysteine	0.91	0.91	0.91	0.85	0.85	0.85	0.80	0.80	0.80
Digestible Tryptophan	0.24	0.24	0.24	0.22	0.22	0.22	0.20	0.20	0.20
Digestible Threonine	0.84	0.84	0.84	0.76	0.76	0.76	0.72	0.72	0.72
Analyzed composition
Dry matter	92.2	92.2	91.6	91.5	92.1	91.4	91.7	91.8
Crude protein	24.5	24.7	24.3	21.3	21.2	21.6	19.3	20.9	91.6
Crude fat	4.05	4.31	4.09	4.86	4.69	3.67	4.81	4.25	21.3
Calcium	0.81	0.80	1.08	0.89	0.90	0.83	0.83	0.75	4.92
Potassium	1.05	1.00	1.07	0.94	0.91	0.95	0.84	0.92	0.83
Phosphorus	0.62	0.65	0.72	0.66	0.65	0.65	0.57	0.60	0.95
Sodium	0.14	0.15	0.19	0.17	0.17	0.17	0.16	0.15	0.63

¹Basal diet (NC); in-feed antibiotic diet containing NC + 0.05% bacitracin methylene disalicylate (BMD); in-feed folic acid diet containing NC + 5 mg/kg FA. ²Supplied/kg premix: DL-Methionine, 0.5 kg; wheat middling, 0.5 kg. ³Starter vitamin-mineral premix contained the following per kg of diet: 9750 IU vitamin A; 2000 IU vitamin D3; 25 IU vitamin E; 2.97 mg vitamin K; 7.6 mg riboflavin; 13.5 mg Dl Ca-pantothenate; 0.012 mg vitamin B12; 29.7 mg niacin; 1.0 mg folic acid, 801 mg choline; 0.3 mg biotin; 4.9 mg pyridoxine; 2.9 mg thiamin; 70.2 mg manganese; 80.0 mg zinc; 25 mg copper; 0.15 mg selenium; 50 mg ethoxyquin; 1543 mg wheat middling’s; 500 mg ground limestone. ⁴Grower and finisher vitamin-mineral premix contained the following per kg of diet: 9750 IU vitamin A; 2000 IU vitamin D3; 25 IU vitamin E; 2.97 mg vitamin K; 7.6 mg riboflavin; 13.5 mg Dl Ca-pantothenate; 0.012 mg vitamin B12; 29.7 mg niacin; 1.0 mg folic acid, 801 mg choline; 0.3 mg biotin; 4.9 mg pyridoxine; 2.9 mg thiamin; 70.2 mg manganese; 80.0 mg zinc; 25 mg copper; 0.15 mg selenium; 50 mg ethoxyquin; 1543 mg wheat middling’s; 500 mg ground limestone.⁵Bacitracin methylene disalicylate (providing 55 mg/kg mixed feed); Alpharma, Inc., Fort Lee, NJ, USA.

Measurements

Hatch parameters and chick quality

Hatched chicks were counted and weighed individually. Hatchability was calculated as the percentage of hatched chicks to fertile incubated eggs per replicate. Chick navel quality was evaluated by adopting the scoring method by Reijrink et al. (2009). Navel quality was scored 1 - when the navel was completely closed and clean; scored 2 - when the navel was discolored (i.e., when the navel color differs from the chick’s skin color) with a maximum 2 mm opening; and scored 3 - when the navel was discolored and with more than a 2 mm opening. Chick length was also determined by placing the chick on its ventral side and recording its length from the tip of the beak to the middle toe on the right leg.

Growth performance parameters

Feed intake (FI) and average body weight (BW) were measured on a pen basis weekly. The obtained data were then used to calculate the average feed intake (AFI), average body weight gain (ABWG), and feed conversion ratio (FCR). The FCR was calculated as the amount of feed consumed per unit of BW gain. Mortality was recorded daily, and dead birds were subsequently weighed and sent to the Nova Scotia Agriculture, Animal Health Laboratory for necropsy. Mortality was then used to correct the FCR.

Sampling

On day 25, 1 bird per pen (6 replicate birds per treatment group) was randomly selected, weighed, and euthanized by electrical stunning and exsanguination. After euthanasia of the bird, blood samples were collected from each bird into 10 mL blood serum collection tubes (BD Vacutainer Serum Tubes, fisher scientific- BD366430) for further serum assays and 10 mL heparinized tubes (BD Vacutainer Glass Blood Collection Tubes with Sodium Heparin, fisher scientific- BD366480) for further blood plasma assays. Blood serum and plasma were centrifuged at 1200 g × 10 min × 18 °C. The resulting supernatants were stored in aliquots at −80 °C until further analysis.

After slaughter, the weights of the bursa of Fabricius and the spleen were also determined by trained personnel. The small intestinal segments, including the duodenum (region from the gizzard junction to the pancreatic and bile ducts), jejunum (1.5-cm length midway between the point of entry of the bile ducts and Meckel’s diverticulum) and ileum (1.5-cm length midway between Meckel’s diverticulum and the ileocecal junction), were excised and fixed in neutral buffered formalin (10%) for further histomorphological processing.

Relative weight of organs

The weights of bursa of Fabricius and spleen were recorded and then specified as a fraction of the live BW (g/Kg BW) of the slaughtered chicken.

Serum immunoglobulins

The concentrations of immunoglobulins (IgG and IgM) in the serum were quantified using chicken-specific immunoglobulins enzyme-link immunosorbent assay (ELISA) kits (Bethyl Laboratories, Montgomery, TX, USA; catalog numbers E33-104-200218 and E33-102-180410, respectively) following manufacturer instructions. The values were determined on a microplate reader (Bio-Tek Instrument Inc., Wonooski, VT, USA) using a software program (KC4, version #3.3, Bio Tek Instruments). The four-parameter logistic model was used to extrapolate immunoglobulins concentration from absorbance readings.

Blood biochemistry

Samples for blood biochemical analysis were shipped on ice to Atlantic Veterinary College, University of Prince Edward Island Pathology Laboratory, for analysis using cobas 6000 analyzer series (Roche Diagnostics, Indianapolis, IN, USA).

Anti-oxidant Indexes

The activitiy of superoxide dismutase (SOD), and the concentration of Malondialdehyde (MDA) in the serum were measured according to the manufacturer’s instructions using Cayman’s SOD assay kit (catalog number 706002, Cayman Chemical, Ann Arbor, MI, USA) and chicken MDA ELISA kit (catalog number MBS260816, MyBioSource, San Diego, CA, USA), respectively. The total anti-oxidant capacity (TAC) in blood plasma was analyzed using the Oxiselect Total Anti-oxidant Capacity assay kit (catalog number STA360; Cell BioLabs Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Absorbance for all analysis was measured at recommended wavelengths on a microplate reader (Bio-Tek Instrument Inc., Wonooski, VT, USA) using a software program (KC4, version #3.3, Bio Tek Instruments).

Intestinal morphology

The procedure for intestinal morphometric analysis was as described by Oladokun et al. (2021). Briefly, fixed intestinal tissues were embedded in paraffin, sectioned (0.5 μm thick), and stained with hematoxylin and eosin for morphological examinations. In each cross-sectioned tissue, 10 morphometric measurements including the villus height (from the base of the intestinal mucosa to the tip of the villus excluding the intestinal crypt), villus width (halfway between the base and the tip), crypt depth (from the base upward to the region of transition between the crypt and villi) (Ozdogan et al., 2014) per slide were carried out using Leica 1CC50 W microscope at 4 × Magnification (Leica Microsystems, Wetzlay, Germany) and an image processing and analysis system (Leica Application Suite, Version 3.4.0, Leica Microsystems).

Statistical analysis

Hatch data were analyzed as a randomized complete block design, with the incubator as the blocking factor. Datasets from the grow-out trial were also analyzed in a randomized complete block design, with broiler production rooms being the blocking factor. The normality of all data sets was ascertained by testing residuals by the Anderson–Darling test in Minitab statistical package (v.18.1). Data were analyzed using the generalized linear model in the same statistical package. Significant means were separated using Tukey’s honest significant difference test in the same statistical package. Analyzed data were presented as means ± SEM and probability values. Values were considered statistically different at P ≤ 0.05 and considered a statistical trend at P < 0.1.

Results

Hatch performance and chick quality

The results on hatch performance and chick quality are presented in Table 2. The in ovo delivered FA reduced (P < 0.001) hatchability in a dose-dependent manner, with the highest reduction observed in the in ovo FA group 2 treatment, having 43% reduction in hatchability compared to the noninjected eggs. In contrast, chicks in the in ovo FA group 2 treatment had at least 2% heavier weight (P = 0.02) than those in the noninjected eggs group. All other evaluated parameters, including average chick length and average navel score, were observed to be similar across all treatment groups.

Table 2.

Effect of in ovo delivery of folic acid on hatch performance and chick quality

Hatch parameters		Treatments1
	Noninjected	In ovo saline	In ovo folic acid 1	In ovo folic acid 2	SEM2	P-value3
Hatchability, %	96.1a	95.2a	75.2b	54.5c	3.43	<0.001
Average chick weight, g	43.1b	43.8a,b	43.8a,b	44.0a	0.14	0.023
Average chick length, cm	19.0	18.9	19.1	19.0	0.18	0.872
Average navel score	1.45	1.38	1.51	1.45	0.07	0.739

¹Treatments include: 1) noninjected egg (N =166 eggs); 2) in ovo saline group (N = 38 eggs) - injected with 0.2 mL of physiological saline (0.9% NaCl); 3) In ovo folic acid group 1 (N = 53 eggs) injected with 0.1 mg folic acid per egg and 4) In ovo folic acid group 2 (N = 53 eggs) injected with 0.15 mg folic acid per egg. All treatment were replicated in similar incubators operated under similar conditions. ²SEM = Standard error of means.^.3Means within a row with different superscripts. ^a,b,cSignificantly differ.

Growth performance

Table 3 highlights the results observed for growth performance parameters across all feeding phases. In the starter phase (days 0 to 14), only the birds in the in-feed antibiotics treatment consumed more feed (27.5%; P = 0.01) than the in-feed FA treatment; other treatments were statistically similar to the in-feed antibiotics treatment. No differences in ABWG and FCR amongst treatment groups were recorded in the starter phase. In the grower (days 15 to 25) and finisher (days 26 to 35) phases, the in-feed antibiotic treatment recorded higher (P < 0.001) AFI compared to the in-feed FA and in ovo FA group 2 treatments. Other treatments recorded statistically similar AFI values compared to the in-feed antibiotic treatment. The in-feed antibiotic treatment also recorded higher (P = 0.05) FCR compared to the in-feed FA treatment; other treatments recorded statistically similar FCR values. At the end of the total trial period (days 0 to 35), of all the evaluated growth performance parameters evaluated, only the AFI values were found to be significantly different among treatment groups. The in-feed antibiotic treatment recorded higher (P < 0.001) AFI compared to the in-feed FA and in ovo FA group 2 treatments. All treatment groups had similar values for ABWG and FCR.

Table 3.

Effect of folic acid and its delivery routes on the growth performance of broiler chickens raised for 35 d

Growth performance parameters	Treatments1
	Negative control	In-feed antibiotics	In-feed folic acid	In ovo folic acid 1	In ovo folic acid 2	SEM2	P-value3
Starter (0 to 14 d)
Average feed intake, g	353a,b	348a	273b	336a,b	340a,b	6.16	0.013
Average body weight gain, g	291	314	284	309	250	4.57	0.155
FCR4	1.21	1.11	0.97	1.10	1.34	0.02	0.058
Grower (15 to 25 d)
Average feed intake, g	1,479a,b,c	1,643a	1,246b,c	1,415a,b	1,072c	22.8	<0.001
Average body weight gain, g	1,376	1,331	1,220	1,166	1,067	15.6	0.265
FCR4	1.07a,b	1.19a	1.03b	1.21a,b	1.02a,b	0.02	0.045
Finisher (26 to 35 d)
Average feed intake, g	2,310a,b,c	2,348a	1,922b,c	2,117a,b	1,780c	25.1	<0.001
Average body weight gain, g	826	908	797	1068	1000	294	0.922
FCR4	2.69	2.49	2.41	2.03	1.77	0.11	0.981
Total trial period (1 to 35 d)
Average feed intake, g	3,252a,b,c	3,332a	2,610b,c	2,930a,b	2,418c	38	<0.001
Average body weight gain, g	2,566	2,643	2,349	2,548	2,300	292	0.683
FCR4	1.24	1.24	1.13	1.20	1.06	0.03	0.305

¹Treatments include: - 1) Negative control treatment - chicks fed a basal corn-soybean meal-wheat–based diet; 2) In-feed antibiotics - chicks fed NC + 0.05% bacitracin methylene disalicylate; 3) In-feed folic acid- chicks fed NC + 5 mg/kg folic acid; 4)In ovo folic acid group 1 - eggs injected with 0.1 mg folic acid per egg and 4) In ovo folic acid group 2 - eggs injected with 0.15 mg folic acid per egg. Each treatment group had chicks (N = 22) raised in 6 replicate pens. ²SEM, standard error of means. ³Means within a row with different superscripts. ^a,b,cSignificantly differ. ⁴FCR, Feed conversion ratio.

Immune organ weight, serum immunoglobulin concentration, and anti-oxidant indexes

Results on the relative weight of immune organs (Bursa of Fabricius and spleen), serum immunoglobulin concentration and anti-oxidant (SOD, MDA, and TAC) indexes are presented in Table 4. Of the two immune organs evaluated, only the relative weight of the Bursa of Fabricius (g/Kg BW) recorded a tendency to be increased by the in ovo FA group 1 treatment. The relative weight of the Bursa of Fabricius in the in ovo FA group 1 was at least 37.8% higher (P = 0.08) than those of other treatment groups. In terms of anti-oxidant indexes, while the in ovo FA group 2 treatment recorded a tendency to increase (P = 0.08) serum SOD activity by at least 50%, compared to other treatments; a marginal increase (P = 0.07) in serum MDA concentration was observed in the In ovo FA group 1 treatment, compared to other treatments. There was no effect of treatment on the concentrations of IgG and IgM in the serum.

Table 4.

Effect of folic acid and its delivery routes on relative weight of immune organs, serum immunoglobulin concentrations, and anti-oxidant indexes

Parameters	Treatments1
	Negative control	In-feed antibiotics	In-feed folic acid	In ovo folic acid 1	In ovo folic acid 2	SEM2	P-value
Bursa weight, g/Kg BW	1.86	1.85	2.00	2.55	2.22	0.09	0.076
Spleen weight, g/Kg BW	0.72	0.78	0.81	0.83	0.69	0.03	0.474
Immunoglobulin G, Mg/mL	10.9	2.53	1.18	0.80	1.71	0.32	0.120
Immunoglobulin M, Mg/mL	0.86	0.34	0.24	0.30	0.17	0.05	0.589
SOD3activity, U/ml	0.34	0.36	0.36	0.35	0.51	0.02	0.081
MDA4, ng/ml	20.9	11.9	13.1	24.8	20.7	1.90	0.068
TAC5, mM uric acid equivalents (UAE)	0.80	0.68	0.84	0.89	0.76	0.04	0.353

¹Treatments include: 1) Negative control treatment - chicks fed a basal corn-soybean meal-wheat–based diet; 2) In-feed antibiotics - chicks fed NC + 0.05% bacitracin methylene disalicylate; 3) In-feed folic acid- chicks fed NC + 5 mg/kg folic acid; 4) In ovo folic acid group 1 - eggs injected with 0.1 mg folic acid per egg and 5) In ovo folic acid group 2 - eggs injected with 0.15 mg folic acid per egg. N = 6 birds per treatment group. ²SEM, standard errr of means. ³SOD, superoxide dismutase. ⁴MDA, smlondialdehyde. ⁵TAC, total anti-oxidant capacity.

Blood biochemistry

Table 5 shows the results on blood plasma biochemistry indexes. Only the concentrations of plasma electrolyte minerals—sodium and chloride were affected by the evaluated treatment groups. Sodium concentration (mmol. L^-1) in the in-feed antibiotics treatment was higher (P = 0.04) than those of the NC treatment. Other treatment groups recorded statistically intermediate values for sodium concentration in the blood plasma. Conversely, chloride concentration (mmol. L^-1) in the in-feed antibiotics treatment was higher (P = 0.001) than in the NC and in-feed FA treatment groups. Both levels of in ovo delivered FA treatment groups recorded statistically intermediate values for chloride concentration in the blood plasma.

Table 5.

Effect of folic acid and its delivery routes on broiler chicken plasma biochemistry indices

Parameters	Treatments1
	Negative control	In-feed antibiotics	In- feed folic acid	In ovo folic acid 1	In ovo folic acid 2	SEM2	P-value3
Electrolytes, mmol/L
Sodium	149b	152a	150a,b	152a,b	151a,b	0.58	0.036
Potassium	6.96	6.62	6.86	6.62	6.78	0.09	0.666
Sodium: potassium	21.4	23.0	22.1	23.0	22.5	0.30	0.382
Chloride	109b	113a	110b	111a,b	112a,b	0.50	0.001
Calcium	3.06	2.76	3.05	2.99	3.01	0.01	0.373
Phosphorus	2.46	2.16	2.41	2.42	2.37	0.06	0.221
Magnesium	0.87	0.80	0.82	0.80	0.81	0.01	0.206
Metabolites, mmol/L
Urea	0.30	0.29	0.32	0.28	0.29	0.10	0.692
Glucose	15.5	15.3	15.5	16.2	15.1	0.14	0.231
Cholesterol	3.48	3.51	3.61	3.46	3.91	0.06	0.215
Iron	18.5	20.6	20.2	19.7	20.2	0.01	0.784
Bile acids	22.2	24.0	21.6	19.3	20.5	0.96	0.716
Uric acid	361	371	371	424	365	0.01	0.641
Creatinine	1.95	0.95	1.82	1.17	1.56	0.09	0.117
Enzymes, U/L
Amylase	589	686	715	889	758	49.4	0.540
Lipase	21.0	22.5	18.3	21.4	20.4	0.03	0.839
Creatine kinase	6,218	7,937	5,229	8,218	4,791	0.04	0.414
Alkaline phosphatase	9,813	7,383	10,506	17,443	10,841	1104	0.112
Alanine transaminase	2.56	2.21	2.60	2.40	2.88	0.04	0.888
Aspartate Aminotransferase	164	190	166	173	164	0.01	0.388
Gamma-glutamyl transferase	9.07	10.63	10.52	8.67	9.67	0.27	0.128
Proteins, g/L
Total proteins	28.6	27.3	29.2	28.8	29.2	0.001	0.508
Albumin	11.8	11.9	12.1	11.7	12.2	0.14	0.82
Globulin	16.8	15.4	17	17	17	0.002	0.343
Albumin: Globulin	0.70	0.78	0.71	0.69	0.71	0.01	0.310

¹Treatments include: 1) Negative Control treatment- chicks fed a basal corn-soybean meal-wheat–based diet; 2) In-feed antibiotics- chicks fed NC + 0.05% bacitracin methylene disalicylate; 3) In-feed folic acid- chicks fed NC + 5 mg/kg folic acid; 4) In ovo folic acid group 1 - eggs injected with 0.1 mg folic acid per egg and 5) In ovo folic acid group 2 - eggs injected with 0.15 mg folic acid per egg. N = 6 birds per treatment group. ²SEM = standard error of means. ³Means within a row with different superscripts. ^a,bSignificantly differ

Intestinal morphology

Table 6 shows the effect of FA and its delivery routes on the three intestinal segments. All FA treatments (irrespective of delivery routes) seemed to improve duodenal morphology in this study, as evidenced by increased (P < 0.01) villus height, villus width, villus height to crypt depth ratio, and reduced (P = 0.04) crypt depth, compared to the control treatment. Nonetheless, the in ovo FA 2 group recorded the highest increase in villus width, at least 13.6 % wider than other treatments and only statistically comparable to the antibiotic treatment.

Table 6.

Effect of folic acid and its delivery routes on broiler chicken intestinal morphology

Parameters	Treatments1
	Negative control	In-feed antibiotics	In-feed folic acid	In ovo folic acid 1	In ovo folic acid 2	SEM2	P-value3
Duodenum
Villus height, mm	2.04b	2.14a,b	2.15a,b	2.28a	2.21a	0.02	<0.001
Villus width, mm	0.22b	0.24a,b	0.22b	0.22b	0.25a	0.00	0.003
Crypt depth, mm	0.16a	0.14b	0.14b	0.15ab	0.15a,b	0.00	0.004
Villus height: Crypt depth	12.5b	15.0a	14.7a	15.5a	14.4a	0.19	<0.001
Jejunum
Villus height, mm	1.15	1.17	1.12	1.16	1.11	0.01	0.497
Villus width, mm	0.25a	0.20b	0.23a	0.24a	0.26a	0.00	<0.001
Crypt depth, mm	0.11a,b	0.10c	0.10b,c	0.12a	0.11a,b.c	0.00	<0.001
Villus height: Crypt depth	10.2b,c	11.8a	10.7a,b	9.30b,c	9.88c	0.18	<0.001
Ileum
Villus height, mm	0.76a,b	0.77a,b	0.82a	0.70b	0.74a,b	0.01	0.001
Villus width, mm	0.18a,b	0.18a,b	0.17b	0.20a	0.19a,b	0.00	0.007
Crypt depth, mm	0.15a	0.13b,c	0.14a,b	0.13b,c	0.14a,b,c	0.00	0.002
Villus height: Crypt depth	5.01b	5.74a	5.68a,b	5.47a,b	5.21a,b	0.10	0.028

¹ Treatments include: 1) Negative control treatment - chicks fed a basal corn-soybean meal-wheat–based diet; 2) In-feed antibiotics - chicks fed NC + 0.05% bacitracin methylene disalicylate; 3) In-feed folic acid- chicks fed NC + 5 mg/kg folic acid; 4) In ovo folic acid group 1 - eggs injected with 0.1 mg folic acid per egg, and 4) In ovo folic acid group 2 - eggs injected with 0.15 mg folic acid per egg. N = 6 birds per treatment group; 10 morphometric measurements that were taken from each histological slide. ²SEM = Standard error of means. ³Means within a row with different superscripts. ^a,b,cSignificantly differ.

Conversely, in the jejunum, the antibiotic treatment recorded the least (P < 0.001) crypt depth and highest villus height to crypt depth ratio compared to the control treatment. Interestingly, the antibiotic treatment equally recorded reduced (P < 0.001) jejunal villus width compared to the control treatment. All other treatments mostly recorded statistically intermediate values for villus width, crypt depth, and villus height to crypt depth ratio in the jejunum. No difference in the jejunal villus height was recorded for all treatments in this study.

In the ileum, results on villus height and width were variable. Significant differences were recorded between the in-feed FA group and the in ovo FA 1 group for both parameters. While the former increased (P = 0.001) ileal villus height compared to the latter, the latter increased (P = 0.007) the villus width compared to the former. Nonetheless, the antibiotic treatment recorded the lowest (P = 0.002) crypt depth and the highest (P = 0.03) villus height to crypt depth ratio compared to the control treatment. Other treatments had statically intermediate crypt depth and villus height to crypt depth ratios.

Discussion

While a few studies have reported the potential of the in ovo delivery of FA to improve embryo growth, bird growth performance, and immune and blood biochemical indexes (El-Azeem et al., 2014; Li et al., 2016; Nouri et al., 2018; Ismail et al., 2019; Gouda et al., 2022), we present here the first comparison of FA (across both in ovo and in-feed ­delivery routes) with a classic antibiotic (bacitracin methylene disalicylate) in broiler chicken study, to our knowledge.

This study reports contrasting results on chick hatchability and hatchling weight. While in ovo delivered FA reduced hatchability with increasing dosage, the highest dose (0.15 mg/egg) recorded the highest hatchling weight compared to the noninjected treatment. Conflicting results on the effect of in ovo delivered FA on hatchability are reported in the few available literature. While a few studies have reported improved hatchability with in ovo delivered FA (Li et al., 2016; Liu et al., 2016; Gouda et al. 2021, 2022), other studies have recorded no effect of in ovo delivered FA on hatchability (Nouri et al., 2018; Ismail et al., 2019). Abdel-Halim et al. (2020) observed decreased hatchability (91% vs. 76%) with in ovo injected eggs (0.2 mg FA/egg via the albumen), compared to noninjected eggs in their study. Similarly, Abdel-Fattah and Shourrap (2013) recorded decreased hatchability values following in ovo delivery of FA (1 mg FA/egg via the air cell). Several factors, including the dosage of injected substance, form or solubility of injected substance, the volume of injection, site of injection, injection needle length, and time of injection, have been reported to influence hatchability outcomes across in ovo studies (Ohta and Kidd, 2001; Zhai et al., 2011b; Abdel-Halim et al., 2020; Oladokun and Adewole, 2020). Of all these factors, the form of injected substance, injection needle length and time of injection are speculated to be the key factors that could have contributed to the hatchability outcome observed in this study. While we utilized commercially available FA in its powder form in this study, it would be important for further studies to consider evaluating liquid FA forms, if available. This might aid easy embryo absorption and utilization of this bioactive substance. Also, Ohta and Kidd (2001) have earlier documented that a 13-mm needle length, as opposed to a 19-mm, is optimum for in ovo delivery of amino acids. Our laboratory has successfully established the procedure for the in ovo delivery of probiotics (B. subtilis) in broiler chickens using a 3-cm (30-mm) injection needle length, recording 91% hatchability rate (Oladokun et al., 2021). While we utilized a similar needle length (3 cm) in this study, it is probable that a shorter needle length might be more relevant for the in ovo delivery of FA to ensure increased chances of embryo viability. Regarding the time of injection, Sunde et al. (1950) pointed out that embryo mortality would not be affected by FA deficiency until embryonic day 17, suggesting that day 17 onwards might be an ideal time for the in ovo delivery of FA. Furthermore, our result on increased hatchling weights following in ovo delivery of FA is consistent with previous reports on in ovo-delivered FA (Abdel-Fattah and Shourrap, 2013; El-Azeem et al., 2014; El Said, 2017; Gouda et al., 2022). Li et al. (2016) equally recorded increased hatchling weight with a similar in ovo delivered FA dose as this study. This positive effect of in ovo-delivered FA on hatchling weight has been attributed to the critical role folate plays in DNA and RNA synthesis, cell replication, blood protein synthesis, thyroid activity, hepatic expression of insulin-like growth factor 2 (IGF2), anti-oxidant activities, and nutrient utilization (Robel, 1993b; Abdel-Fattah and Shourrap, 2013; El-Azeem et al., 2014; Liu et al., 2016; Ismail et al., 2019).

The growth performance dataset from this study shows the in-feed antibiotic treatment consistently recording higher AFI values across all feeding phases. Even more interesting, birds in the in ovo FA 2 treatment (0.15 mg/egg) were observed to record similar FCR values as the in-feed antibiotic treatment while consistently consuming less quantity of feed at the grower, finisher, and overall feeding phases, suggesting that this treatment possessed some sort of nutrient utilization efficiency. The growth promotion potential of AGP (especially BMD) to improve AFI and FCR values are well-substantiated across the literature (Ao and Choct, 2013; Murugesan et al., 2015; Crisol-Martínez et al., 2017; Gadde et al., 2017; Manafi et al., 2017; Walters et al., 2019). Several theories including inhibition of the synthesis of bacteria cell wall (Smith and Weinberg, 1962; Butaye et al., 2003), improved FI resulting from enhanced nutrient digestibility by virtue of improved gut microflora structure (Dibner and Richards, 2005; Naveenkumar et al., 2017), and an anti-inflammatory effect via reduced production of cytokines and chemokines resulting in reduced incidence of anorexia (Niewold, 2007) have been posited to explain AGP growth-promoting effect. The results of FA on FI in the literature are contradictory. While reports of increased FI in broiler chickens (Meremikwu et al. 2021) and layer chickens (House et al. 2002) offered FA exist, other studies have also reported reduced FI following FA supplementation in pigs (Ying et al. 2013), broilers (Liu et al. 2023), and layer chickens (Terčič and Pestotnik, 2014). It is hypothesized that FA is capable of synthesis or reverse action of certain brain neuropeptides and neurotransmitters (such as gamma-aminobutyric acid) responsible for the control of FI (Matte et al. 1990). Hence FA can act both as an appetite stimulant or hunger deregulator depending on multiple factors that include the nutritional composition of basal diet, age of the bird, physiological status, dose and duration of FA supplementation and environmental factors (Terčič and Pestotnik, 2014). More research is needed to provide more insight on how these factors contribute to FA’s effect on FI. Regarding FCR, similar to the result reported here, Gamboa et al. (2022) observed improved FCR in ovo FA injected birds (0.15 mg/egg) on days 1 to 42 compared to control treatment in their study, with similar AFI values and no change in ABWG values. Other studies have also affirmed the capacity of in ovo delivered FA to improve FCR in poultry (El-Azeem et al., 2014; Li et al., 2016; Nouri et al., 2018). This nutrient conversion efficiency afforded by higher dosage of in ovo delivered FA is traceable to folate’s anti-oxidant and thyroid activity, as well as its effect on muscular metabolism (Joshi et al., 2001; El-Azeem et al., 2014; Nouri et al., 2018).

Furthermore, no treatment effect on serum immunoglobulins G and M levels was recorded in this study. Although immunoglobulins are a critical component of humoral response to infection and oxidative stress, the results recorded were not surprising as birds were raised under experimentally controlled conditions with no strain on their immune system. Nonetheless, one study (Li et al., 2016) reported that in ovo delivered (100 and 150 µg/egg) FA increased plasma IgG and IgM concentration in broiler chickens at days 21 and 42. Based on the limited and conflicting results observed, it would be worthwhile for more studies to research the effect of in ovo delivered FA on immunoglobulin concentrations and other factors, including sampling time and differences in quantification assays that could potentially affect detectable immunoglobulin concentrations. Contrary to the result recorded on serum immunoglobulins concentration, a tendency for the in ovo FA 1 treatment to increase the relative weight of the bursa was also observed. This is consistent with the result of El Said (2017), which showed that in ovo delivery of FA (10%, 0.1 ml/egg) increased bursa weight (%). The positive effect of in ovo-delivered FA on lymphoid organ hypertrophy has been linked to the anti-oxidant properties of FA (Joshi et al., 2001; Akinyemi and Adewole, 2022). Consistent with this result, we also record a tendency for a higher dosage of in ovo delivered FA to increase serum SOD activity, alongside a tendency to reduce lipid peroxidation product (MDA), compared to a lower dosage of the in ovo delivered FA. Oxidative stress at the cellular or tissue levels are a product of an imbalance between free radical production and endogenous anti-oxidant defense system (Mishra and Jha, 2019). This potentially leads to lipid peroxidation, protein nitration, DNA damage, and apoptosis. During physiological oxygen metabolism, cells are constantly exposed to free radicals generated (Estévez, 2015). Excessive production of these free radicals (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) or their inefficient removal leads to oxidative stress. These ROS include superoxide, hydrogen peroxide, and the hydroxyl radicals. The primary mechanism involved in the intracellular scavenging of ROS is via the use of anti-oxidant enzymes, which include SOD, catalase (CAT), and glutathione peroxidase (GPx; Kurutas, 2015). SOD is thought to be the first line of anti-oxidant enzyme that converts ROS, the superoxide anion, into hydrogen peroxide and molecular oxygen; the resulting hydrogen peroxide is converted to water by the enzymes CAT and GPx (Kurutas, 2015). The levels of these anti-oxidant enzymes are thus useful indicators of the anti-oxidant status of a bird. Additionally, in situations where oxidative stress is already occurring, lipids are common targets of oxidative stress, hence malondialdehyde (MDA) referred to as “the biosignature of oxidative damage” is the main product of lipid peroxidation (Oladokun and Adewole, 2022). It is the main product derived from peroxidation of polyunsaturated fatty acid. This could also be a useful biomarker of the oxidative stress encountered by birds. Despite the reported anti-oxidant potential of FA (Joshi et al., 2001; Gliszczyńska-Świgło, 2007), it is interesting to note that this is the first study to actually evaluate the effect of in ovo delivered FA on anti-oxidant indexes, to our knowledge. Several other studies (El-Din et al., 2008; Gouda et al., 2020; Li et al., 2021; Savaram et al., 2022) have affirmed that in-feed supplementation of FA improves various anti-oxidant indexes.

Blood sampling continues to be an important diagnostic approach in both human and avian research. In this study, all observed blood biochemical parameters were within the normal physiological range for broiler chickens (Ilo S U et al., 2019). Nonetheless, blood plasma sodium and chloride level in the in-feed antibiotic treatment was observed to be significantly increased compared to the control treatment. Both electrolyte minerals were observed to be within the upper limit of recommended physiological ranges for broiler chickens (Leeson and Summers, 2001). Conditions involving high concentrations of sodium and chloride in the blood are referred to as hypernatremia and hyperchloremia, respectively. Considering the role of these minerals in maintaining acid-base balance and osmotic pressure in body fluids, excessively high levels of these electrolyte minerals have been implicated in the incidences of dehydration, edema, acidosis, poor bone development (tibial dyschondroplasia), and decreased humoral immunity (Pimentel and Cook, 1987; Ruíz-López et al., 1993; Oviedo-Rondón et al., 2001; Pohl et al., 2013). The effect of antibiotics on the concentrations of these blood minerals is relatively unreported in the literature; this study perhaps provides another justification to encourage the discontinued use of AGP in poultry production.

Additionally, a higher dosage (0.15 mg/egg) of the in ovo delivered FA enhanced duodenal, jejunal, and ileal morphology in this study; in most cases, as comparable to or even better than the in-feed antibiotic treatment. Considering that an increased villus height, villus width, and reduced crypt depth are frequently linked to improved nutrient absorptive functions, these results could explain the similar trend in growth performance observed for both treatments. Despite the paucity of studies that have evaluated the effect of in ovo-delivered FA on the intestinal morphology of poultry, Li et al. (2020) have previously reported enhanced intestinal morphology in lamb’s offspring with increasing maternal FA supplementation. A similar positive effect of improved intestinal morphology with antibiotics (especially BMD) supplementation is also well-documented in the literature (Viveros et al., 2011; Khodambashi Emami et al., 2012; Adewole and Akinyemi, 2021; Akinyemi and Adewole, 2022). This positive effect of antibiotics on intestinal morphology is theorized to occur as a result of their antibacterial and gut microbiota modulating properties (Marković et al., 2009; Khodambashi Emami et al., 2012). Given that new cells possess shorter villus height and higher crypt depth, by virtue of a shift in gut microbiota, destruction and the subsequent renewal of gut cells are thus reduced.

Conclusions

This study showed that both dosages (0.1 and 0.15 mg FA/egg) of in ovo delivered FA reduced hatchability in a dose-dependent manner. However, in ovo-delivered FA at a higher dosage (0.15 mg FA/egg) afforded heavier hatchling weight. The same dosage of in ovo-delivered FA also enhanced broiler chicken intestinal morphology and FCR in a similar capacity as the in-feed antibiotic treatment, with birds consuming less quantity of feed. A marginal tendency to increase serum SOD activity was equally observed in the same in ovo-delivered FA treatment. Taken together, the results presented here suggest that in ovo delivered FA at 0.15 mg/egg could offer a similar growth-promoting effect as antibiotics in broiler production. Notwithstanding, it would be important to optimize all possible injection and incubation conditions through further research in order to yield favorable hatchability outcomes.

Glossary

Abbreviations

ABWG

average body weight gain

AFI

average feed intake

AGP

antibiotic growth promoters

BW

body weight

DNA

deoxyribonucleic acid

ED

embryonic days

ELISA

enzyme-link immunosorbent assay

FA

folic acid

FCR

feed conversion ratio

FI

feed intake

IgG

immunoglobulin G

IgM

immunoglobulin M

MDA

malondialdehyde

RNA

ribonucleic acid

SOD

superoxide dismutase

TAC

total anti-oxidant capacity

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.