The effect of methionine and folic acid administered in ovo on the blood biochemical parameters of chickens (Gallus gallus domesticus)

Abstract

Methionine is one of the most frequently supplemented amino acids in raising of poultry. However, an overdose of methionine can cause hyperhomocysteinemia. Folic acid, taking part in the process of homocysteine remethylation, is a factor affecting the reduction of the concentration of this amino acid. The study was carried out in 2 stages. The experiment of step I was to investigate the effect of methionine and/or folic acid administration in ovo in the early stage of embryogenesis (E4), and the experiment of the second stage – in the late stage of embryogenesis (E17) on the following biochemical parameters of chicken blood: glucose concentration in whole blood and concentration of homocysteine and uric acid in plasma of domestic chickens (Gallus gallus domesticus). Our results confirm that methionine supplementation may increase the concentration of uric acid and homocysteine. Moreover, we demonstrated that folic acid administered during embryogenesis decreased homocysteine concentration, also in groups simultaneously supplemented with methionine, especially in the initial stage of postnatal life of the bird.

INTRODUCTION

The chick embryo is a commonly used model in physiological and toxicological studies. A bird embryo develops outside the mother's body, which makes it possible to track the direct impact of the studied factor on the developing body. The proper development of the bird embryo depends primarily on the incubation parameters and nutrients contained in the egg. Deposition of nutrients into the egg is possible only during its formation. Amino acids, which are the basic component of proteins, which in turn are the main building material of the body, are deposited in the egg in a constant amount, regardless of the direction of use, breed or line (Fisher, 1998; Ohta et al., 2001, 2004; Uni et al., 2005; Molenaar et al., 2010; Ekmay at al., 2013). Comparing the amino acid pattern of a chicken egg published by FAO in the 1970s with the present determinations, it seems that the egg amino acid profile remains virtually unchanged (FAO, 1970; Lunven et al., 1973; The official Danish Food Composition Database 1984 (Health Canada); Fisher, 1998; Ohta et al., 1999, 2001, 2004). Contemporary lines of meat chickens are the result of many years of sharp selection towards rapid growth, which is connected with their faster metabolism and a significant increase in nutritional requirements. According to numerous authors, the content of nutrients accumulated in the egg of meat chickens may be insufficient for proper growth as well as pre- and postnatal development, as indicated by the reduced hatching rate of broilers (Naber, 1993; Fisher, 1998; Ohta et al., 2001, 2004; Uni and Ferket, 2004; Weber, 2009). Proteins contained in food are broken down by catabolic enzymes to free amino acids, and the activity of these enzymes is regulated by feedback and depends on the concentration of free amino acids in blood. In the case of amino acid deficiency, catabolic processes intensify, and the body begins to break down its own proteins. This process may be prevented by administering preparations containing free amino acids that inhibit the activity of catabolic enzymes (Boujendar et al., 2003; Witek et al., 2008).

Methionine is one of the most frequently supplemented amino acids in the poultry industry (Baker, 2006; Kalbande et al., 2009; Urllich et al., 2019). As an amino acid that starts translation, it is considered a factor that limits the use of all other amino acids contained in food. Moreover, it has an enormous impact on the proper course of organogenesis in vertebrates. Its deficiency interferes with the division of mesenchymal cells, which differentiate into connective tissues and participate in the formation of the cardiovascular and lymphatic system (Coelho and Klein, 1990; Christ and Ordahl 1995; Brosnan and Brosnan, 2006). It also plays an important role in the body's production of tubulin embryo, neurofilament L and actin (Coelho and Klein, 1990; Moephuli et al., 1997; Afman et al., 2003), and is an important element of the antioxidant system (Atmaca and Fry, 2005). The increase in methionine concentration stimulates the transport and absorption of lysine and arginine (Badzian, 2007). It can be assumed that supplementation with methionine in ovo improves the embryo's use of other amino acids found in the egg (Fisher, 1998; Molenaar et al., 2010) and has a positive effect on embryonic development, and particularly on the formation of the nervous system and muscle tissue. As a transcription and translation regulator, it can reduce the number of mutations and developmental anomalies, and by taking part in the synthesis of GSH, prevent the negative effects of oxidative stress. However, it needs to be emphasized that methionine is demethylated to homocysteine (Hcy), and an overdose of this amino acid can cause hyperhomocysteinemia. Hyperhomocysteinemia is considered to be a factor of increased risk of cardiovascular diseases, it occurs in states of impaired renal function (Orzechowska-Pawiłojć et al., 2005; Tinelli et al., 2019; Muzurović et al., 2021), and causes oxidative stress in liver cells, leading to their steatosis and inflammatory processes (Yan et al., 2020). Muzurović et al. (2021) report that animals fed with high methionine feed exhibited elevated Hcy concentrations in the plasma, pathological changes in arterial walls, and the occurrence of thromboembolic events. Hyperhomocysteinemia in humans is often associated with a deficiency of folic acid and vitamin B₆ (Orzechowska-Pawiłojć et al., 2005). Folic acid, taking part in the process of homocysteine remethylation, is a factor affecting the reduction of the concentration of this amino acid in disorders caused by its excessive concentration (Holmes et al., 2005; Orzechowska-Pawiłojć et al., 2005; Czeczot, 2008).

The purpose of this study was to investigate whether the in ovo administration of folic acid reduces the concentration of homocysteine in the blood of domestic chicks (Gallus gallus domesticus) under conditions of increased methionine availability and prevents possible hyperhomocysteinemia.

MATERIAL AND METHODS

This study was carried out in accordance with the NIH Animal Care Guide, and were approved by the 1st Local Ethical Commission affiliated to the Jagiellonian University in Krakow (No. 25/OP/2004).

A total of 1,200 eggs (62 ± 5.2 g) from the ROSS 308 line (Aviagen, Zębowo, Poland) of 42-wk-old parental broiler stock were used in the experiment; 275 eggs were subjected to experimental procedures in stage I and 560 eggs – in stage II. Before the first stage, 5 randomly selected eggs were subjected to the anlysis of the amino acid composition. Analysis of hatching eggs in terms of amino acid composition as well as quantitative determination of individual amino acids in egg yolk and albumen was carried out by ion exchange chromatography using an INGOS AAA-400 amino acid analyzer (the Czech Republic), working on the basis of liquid chromatography with ion exchange column and pre-column derivatization with ninhydrin. Buffers and column-based separation programmes were used in accordance with the manufacturer's recommendations (INGOS, Czech Republic). Calculations were conducted using the CHROMuLAN program. Previously prepared samples were subjected to oxidative hydrolysis with formic acid and perhydrol for the determination of sulfur amino acids (Cys, Met), and hydrolysis in 6M HCl for the determination of other amino acids (Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, Ile, Leu, Tyr, Phe, His, Lys, Arg). Hydrolysis was carried out for 24 h, at a temperature of 110°C. The hydrolyzate, after filtration and then evaporation on an evaporator (temp. 58°C), was solubilized in a buffer of pH = 2.2.

Hatching Eggs

The eggs were stored for about 72 h at a temperature (t) of 17 ± 0.5°C and relative humidity (RH) of 70%; they were then gradually heated to 25°C for 12 h before the planned start of incubation, and fumigated immediately before setting (according to Fasenko, 2007).

Incubation Technology

The eggs were set in Masalles 65 Digite laboratory incubators and incubated at 37.8 ± 0.1°C and 50% ± 1% RH for d 1 to 18 d of incubation (E1–E18) and at 37.2 ± 0.1°C and 55 to 70% RH between E19–E21. Until E18, the eggs were set on trays tilted at an angle of 45° and turned by 90° every hour; they were then transferred to hatching baskets on E19 (according to Tombarkiewicz et al., 2020). The candling of eggs to remove the ones that were damaged, unfertilized or contained dead embryos, was carried out at E4, E6 and E18 (stage I) or E6 and E17 (stage II).

Experimental Procedures

The study on the effect of in ovo injections of folic acid and methionine on the course of embryogenesis and selected biochemical and hematological parameters of the blood of broiler chicks was performed in 2 stages.

In the first stage of the study, the effect of early administration of the tested substances on embryogenesis of the chicken embryo was assessed. The experimental material consisted of 275 eggs (selected randomly after additional candling conducted on the 4th d of incubation from among the embryos which had started to grow) divided into 11 equal groups (n = 25), according to the scheme presented in Table 1, where: O – uninjected control group; C – control group injected with physiological saline solution; F – groups injected with folic acid, M – groups injected with methionine, MF – groups injected with a mixture of folic acid and methionine. The in ovo injection of methionine and/or folic acid solutions in 0.7% saline solution was performed on the 4th d of incubation.

Table 1.

Scheme of in ovo administration of methionine and/or folic acid to chicken embryos on 4th d of emryogenesis.

Group	Eggs [n]	Dose of methionine/egg	Dose of folic acid/egg	Diluent 0.7% NaCl /egg
O	25	–	–	–
C	25	–	–	250 µL
F3	25	–	3 mg	250 µL
F15	25	–	15 mg	250 µL
F30	25	–	30 mg	250 µL
MF5/3	25	5 mg	3 mg	250 µL
MF25/15	25	25 mg	15 mg	250 µL
MF50/30	25	50 mg	30 mg	250 µL
M5	25	5 mg	–	250 µL
M25	25	25 mg	–	250 µL
M50	25	50 mg	–	250 µL
total	275

Injections were performed into albumen, under the inner shell membrane near the blastodisk, according to the method described by Ohta and Kidd (2001) with subsequent modifications of Lis et al. (2009). Before injection, the eggshells were disinfected with a 70% ethanol solution, and then a hole was made at the blunt end of the egg using a G18 needle. A G25 needle was inserted through the hole made, at an angle of about 45°, and using an insulin syringe (1 mL), 250 µL of the selected solution was introduced. After injection, the holes were sealed with hot paraffin.

On the 6th and 18th d of incubation, routine egg candlings were performed, during which dead embryos were eliminated, which, like embryos from unhatched eggs, after hatching were subjected to embryo-pathological analysis. The number and percentage of eggs hatched in relation to the eggs laid was recorded, and all the hatchlings were weighed in D1 on a laboratory scale to the nearest 0.1 g.

From 1-day-old chicks (D1) from each group (after prior decapitation), blood was drawn from the jugular vein for biochemical analyses (n = 10). The exceptions were groups M25 and M50, where, due to high mortality, blood was collected from 6 and 2 specimens, respectively. Blood was collected into EDTA-coated tubes to analyze homocysteine concentrations in plasma, and into sodium heparin-coated tubes – to determine uric acid concentrations in plasma. The samples were centrifuged for 10 min (2,000 × g), and plasma samples were kept at −20°C until determination (Pawlak et al. 2018). In the whole blood, glucose concentration was also measured.

The experiment of stage II was to investigate the effect of methionine and/or folic acid administration in late embryogenesis (E17) on the selected biochemical parameters of chick blood. In stage II, 560 hatching eggs of Ross 308 meat line chickens were used; they were selected after candling on the 17th d of incubation and divided into 8 equal groups (n = 70) according to the scheme presented in Table 2, where: O – control group, not injected, C – control group injected with a physiological saline solution 0.7%, F – groups injected with folic acid, M – groups injected with methionine, MF – groups injected with a mixture of folic acid and methionine. Methionine and/or folic acid solutions in 0.7% NaCl were injected into eggs on the 17th d of incubation. Due to the high mortality of embryos recorded in the group injected with the highest dose of methionine in stage I, the following groups were abandoned in stage II: M50, MF50/30 and F30.

Table 2.

Scheme of in ovo administration of methionine and/or folic acid to chicken embryos on 17th d of emryogenesis.

Group	Eggs [n]	Dose of methionine /egg	Dose of folic acid /egg	Diluent 0.7% NaCl /egg
O	70	–	–	–
C	70	–	–	250 µL
F3	70	–	3 mg	250 µL
F15	70	–	15 mg	250 µL
MF5/3	70	5 mg	3 mg	250 µL
MF25/15	70	25 mg	15 mg	250 µL
M5	70	5 mg	–	250 µL
M25	70	25 mg	–	250 µL
Total	560

Injections of the tested substances were performed in ovo to the amniotic cavity (Uni and Ferket, 2004; Foye et al., 2006; Lis et al., 2009; Bakyaraj et al., 2012) according to the procedure described above.

After hatching, 20 chickens (10 male and 10 female) were randomly selected from each group, from which blood was taken from the jugular vein for biochemical analyses after decapitation (n = 20). Of the remaining chicks, 40 individuals (20♀ and 20♂) were selected from each group. They were marked with individual wing stamps and intended for breeding under production conditions, in compliance with the rules of welfare and the instructions for rearing chickens of the Ross 308 line (Aviagen). On the 7th d of rearing, blood was taken from the jugular vein from 20 chicks from each group (10♀ and 10♂) to perform analyzes identical to those on the first day. From a wing vein, blood was also taken from 20 birds (10♀ and 10♂) on the 35th d of life. All biochemical determinations made in stage II were carried out for n=20.

The broilers were fed commercial pellet (De Heus-Polska, Łęczyca, Poland) and water according to their age. The composition of the pellet was provided in our previous paper (Tombarkiewicz et al., 2020). The broiler chickens were reared on deep litter for 35 d under production conditions in accordance with welfare regulations and instructions for rearing chickens of the Ross 308 line (Aviagen).

Biochemical Measurements

Blood glucose level was more fully determined using the Blood Glucose Monitoring System manufactured by Eumed Biotech Co. Ltd, Taiwan. The operation of the system is based on the electrochemical biosensor technology and the action of capillary forces. The system includes the EUSURE Plus glucometer and appropriate test strips.

Determination of homocysteine concentration in blood plasma was carried out by the immunoenzymatic method (ELISA) using commercial kits FHCY100 produced by Axis-Shield Diagnostics, Ltd., Scotland. The reading was carried out using the LisaScan EM microplate reader manufactured by Erba Mannheim, Germany.

Uric acid concentration in blood plasma was determined by colorimetric method using Uric Acid kits manufactured by BioSystems (Barcelona, Spain). The absorbance reading was carried out using a spectrophotometer with a Cecil CE 2021 2000 series thermostatic chamber (Great Britain).

Statistical Analysis

The compliance of the obtained results with the normal distribution was tested using the Shapiro–Wilk test, while the assumption of the homogeneity of variances was verified using the Levene test. Then, the studied biochemical parameters of blood (homocysteine and uric acid concentration in plasma and whole blood glucose concentration) were subjected to analysis of variance (ANOVA), and differences between the groups were examined by Duncan's test. Pearson's linear correlation coefficient (r) between individual variables was also calculated. The significance level was set at 0.05 each time. Statistical calculations were carried out using the SAS statistical package (ANOVA, post hoc) and the Statistica 13.3 package (Pearson's correlation).

RESULTS

Analysis of the Amino Acid Composition of Chicken Egg

The determined content of amino acids in the contents of eggs of the Ross 308 meat chickens was presented per fresh weight, in the form of total nitrogen and the percentage of amino acids in the albumen, yolk and egg contents in relation to lysine (Table 3). The determined values did not deviate from those presented in the available literature (FAO, 1970; Lunven et al., 1973; The official Danish Food Composition Database 1984 (Health Canada); Fisher, 1998; Ohta et al., 1999, 2001, 2004).

Table 3.

Amino acid content (AA) in the albumen, yolk and egg content in Ross 308 line chickens, per mass of fresh material [mg/g fresh material], total nitrogen [mg/g N], and as a percentage in relation to lysine (Lys) [%].

AA	Per mass of fresh material [mg/g of fresh material]			Per total nitrogen [mg/g N]			In relation to lysine [%]
	Albumen ± SD	Yolk ± SD	Content ± SD	Albumen ± SD	Yolk ± SD	Content ± SD	Albumen	Yolk	Content
Asp	10.3 ± 0.31	5.33 ± 0.6	12.6 ± 0.43	685 ± 69	587 ± 34	622 ± 39	158	130	140
Thr	4.1 ± 0.14	2.12 ± 0.34	5.8 ± 0.24	272 ± 29	294 ± 17	286 ± 19	63	65	64
Ser	5.9 ± 0.21	3.04 ± 0.33	8.4 ± 0.11	388 ± 33	432 ± 27	415 ± 18	89	96	93
Glu	12.9 ± 0.18	6.51 ± 0.81	15.4 ± 0.32	849 ± 52	702 ± 48	756 ± 39	196	155	170
Pro	3.1 ± 0.02	1.57 ± 0.25	4.3 ± 0.14	206 ± 15	219 ± 18	214 ± 13	47	48	48
Gly	3.3 ± 0.06	1.68 ± 0.16	3.9 ± 0.12	218 ± 20	175 ± 11	191 ± 11	50	39	43
Ala	5.2 ± 0.09	2.64 ± 0.37	6 ± 0.15	343 ± 18	268 ± 20	295 ± 17	79	59	66
Val	6.7 ± 0.19	3.43 ± 0.37	7.8 ± 0.15	440 ± 19	351 ± 24	384 ± 19	102	78	86
Ile	4.6 ± 0.15	2.38 ± 0.27	5.8 ± 0.06	305 ± 12	277 ± 17	287 ± 12	70	61	65
Leu	8 ± 0.09	4.02 ± 0.36	10.3 ± 0.13	526 ± 32	498 ± 25	508 ± 23	121	110	114
Tyr	2.8 ± 0.08	1.45 ± 0.1	3.6 ± 0.06	187 ± 17	168 ± 6	175 ± 3	43	37	39
Phe	5.7 ± 0.16	2.94 ± 0.28	6.1 ± 0.06	378 ± 16	253 ± 15	300 ± 12	87	56	67
His	1.3 ± 0.2	0.76 ± 0.38	1.8 ± 0.24	87 ± 13	91 ± 16	90 ± 14	20	20	20
Lys	6.6 ± 0.14	3.35 ± 0.51	9 ± 0.32	434 ± 36	452 ± 30	445 ± 30	–	–	–
Arg	5.8 ± 0.15	2.95 ± 0.6	8.5 ± 0.24	380 ± 17	440 ± 32	417 ± 25	88	97	94
Cys	2.1 ± 0.18	1.16 ± 0.06	2.6 ± 0.06	142 ± 22	122 ± 3	129 ± 7	33	27	29
Met	3.2 ± 0.14	1.64 ± 0.07	3.2 ± 0.04	208 ± 23	130 ± 6	159 ± 5	48	29	36

Biochemical Parameters of Blood

Glucose Concentration in Whole Blood of Chicks After in ovo Injection in E4

In ovo injection of methionine and/or folic acid on the 4th d of embryogenesis did not affect the concentration of glucose in whole blood of chicks on the 1st d after hatching; in contrast, statistical analyses showed a negative correlation between blood glucose and methionine dose (r = -0.210; P ≤ 0.05) (Table 4).

Table 4.

Glucose concentration in whole blood, homocysteine and uric acid concentration in plasma on the 1st d after hatching (D1) after in ovo injection of methionine and/or folic acid in E4.

Group	Glucose [mg/dL]	uric acid [mg/dL]	homocysteine [µmol/L]
	mean ± SD	mean ± SD	mean ± SD
O	267.9 ± 53.39A	4.4 ± 1.38AB	35.2 ± 11.90ABC
C	240.7 ± 51.34A	3.5 ± 1.62ABC	36.9 ± 8.43AB
M5	261.5 ± 30.31A	2.8 ± 1.17ABC	36.3 ± 6.16ABC
M25	264.3 ± 47.05A	3.8 ± 1.03ABC	38.1 ± 15.87AB
M50	228.5 ± 6.361	3.0 ± 2.621	41.2 ± 6.451
MF5/3	255.4 ± 52.98A	3.9 ± 1.67AB	32.4 ± 4.41ABC
MF25/15	233.8 ± 74.82A	2.3 ± 1.66BC	32.2 ± 8.59ABC
M50/30	248.4 ± 30.82A	2.4 ± 2.30BC	38.5 ± 11.25A
F3	210.7 ± 58.22A	1.3 ± 1.20C	31.8 ± 6.30ABC
F15	265.0 ± 36.81A	5.1 ± 2.27A	26.4 ± 9.73BC
F30	251.4 ± 32.31A	3.7 ± 1.72ABC	25.0 ± 3.03C

AB – values marked with different letters within a given column differ statistically significantly (P ≤ 0.05).

¹The M50 group was not included in the statistical calculations due to the high mortality in this group.

Glucose Concentration in Whole Blood of Chicks After in ovo Injection in E17

On the first day of life, glucose concentration was the lowest in the blood of chicks of group F15 and MF5/3, and differed statistically significantly (P ≤ 0.05) from the M25 group in which it was the highest. Groups F15 and MF5/3 also differed at a statistically significant level from the non-injected control group O (P ≤ 0.05) (Table 5). On the 7th d of life, glucose concentration in the blood of the chicks increased from 3 to 27% compared to the 1st d (P ≤ 0.05), while it did not differ between groups at the level of statistical significance (Table 5). At the end of the rearing, glucose concentration in the blood of the chicks decreased, assuming values close to those of 1-day-old chicks. The mean glucose concentrations in the blood of chicks from different groups did not differ at the level of statistical significance except for groups O and F3, between which the significance of differences at the (P ≤ 0.5) level was demonstrated (Table 5).

Table 5.

Glucose concentration in whole blood, on the 1st d after hatching (D1) after in ovo injection of methionine and/or folic acid in E17.

Group	Glucose [mg/dL]
	Day of life
	D1	D7	D35
	Mean ± SD	Mean ± SD	Mean ± SD
O	271.0 ± 18.20AB	283.0 ± 21.33A	264.3 ± 25.83A
C	262.6 ± 32.57BCD	275.3 ± 30.30A	222.3 ± 25.29ABC
M5	244.6 ± 36.83BCD	287.3 ± 30.82A	232.3 ± 21.54ABC
M25	295.8 ± 41.64AB	303.5 ± 47.26A	246.2 ± 22.31ABC
MF5/3	232.8 ± 43.04CD	287.1 ± 21.75A	246.4 ± 32.60ABC
MF25/15	254.7 ± 19.42BCD	289.3 ± 22.83A	253.0 ± 27.33ABC
F3	244.2 ± 19.45BCD	282.4 ± 34.37A	220.0 ± 49.97C
F15	226.7 ± 21.47D	280.6 ± 39.19A	242.8 ± 33.34ABC

ABCD – values marked with different letters within a given column differ statistically significantly (P≤0.05).

Statistical analyzes also showed a positive correlation between blood glucose and methionine dose (r = 0.193; P ≤ 0.01) as well as glucose concentration and homocysteine concentration in blood plasma (r = 0.372 P ≤ 0.001).

Uric Acid Concentration in the Blood Plasma of the Chicks After in ovo Injection in E4

In experiment I, with in ovo injection on the 4th d of embryogenesis, uric acid concentration in the blood of the chicks was affected the most by folic acid, which, when given at the lowest dose (F3), significantly reduced the uric acid level compared to other chick groups (Table 4).

Uric Acid Concentration in the Blood Plasma of the Chicks After in ovo Injection in E17

After in ovo administration of methionine and/or folic acid on the 17th d of embryogenesis, the uric acid level was highest on the first day after hatching and decreased with the age of the birds. On the first day after hatching, the lowest level of uric acid was recorded in group O, where it was comparable to its level in the non-injected control group of the first experiment (injection in E4). In other groups, uric acid concentration in the blood was much higher, not only in relation to the control group (often at the level of statistical significance), but also to the values obtained in respective groups at the in ovo injection in E4 (Table 4). On the 7th d of life, there was a decrease in the concentration of uric acid in the blood of the chicks from all experimental groups was reduced to the level of the control group, and noticeably the highest concentration of uric acid in the blood of the chicks was recorded in the group supplemented with a higher dose of methionine (M25). A similar trend persisted until the last days of chick rearing, with a significant difference between the concentration of uric acid in the blood of the M25 chicks and the M5 group chicks (Table 6).

Table 6.

Uric acid concentration in the blood plasma on the 1st, 7th and 35th d after hatching (D1, D7 and D35), following in ovo injection of methionine and/or folic acid in E17 [mg/dL].

Group	Uric acid [mg/dL]
	Day of life
	D1	D7	D35
	Mean ± SD	Mean ± SD	Mean ± SD
O	5.2 ± 0.56A	5.0 ± 1.75A	4.9 ± 0.73AB
C	6.8 ± 1.41AB	5.3 ± 2.73A	4.6 ± 2.06AB
M5	8.8 ± 2.16B	5.0 ± 2.82A	4.1 ± 1.23A
M25	8.0 ± 2.77B	6.5 ± 2.76A	5.9 ± 1.53B
MF5/3	7.4 ± 2.14AB	5.0 ± 1.53A	5.2 ± 1.47AB
MF25/15	8.4 ± 2.80B	6.4 ± 1.80A	4.8 ± 1.01AB
F3	7.8 ± 1.77B	4.6 ± 2.88A	4.2 ± 1.60A
F15	8.2 ± 2.89B	4.6 ± 1.81A	5.0 ± 2.10AB

ABCD – values marked with different letters within a given column differ statistically significantly (P ≤ 0.05).

Statistical calculations also showed positive correlations between the uric acid level and methionine dose (r = 0.138; P ≤ 0.05) and folic acid dose (r = 0.147; P ≤ 0.05) when these compounds were administered separately, and no correlation was found in groups supplemented with a mixture of these substances. The calculations also showed a negative correlation between uric acid concentration and the age of birds (r = -0.373; P ≤ 0.01).

Concentration of Homocysteine in the Blood Plasma of the Chickens

Homocysteine Concentration in the Blood Plasma of the Chicks After in ovo Injection in E4

When injected early in embryogenesis (E4), lower levels of homocysteine in the blood of the chicks were observed in the groups receiving folic acid compared to other groups, however, in the F30 group it was lower at the level statistically significant in relation to the injected control and groups receiving higher doses of methionine (M25 and MF 50/30) (P ≤ 0.05). The lowering effect of folic acid on homocysteine concentration in the blood plasma of chicks is also confirmed by a negative correlation between the dose of folic acid and homocysteine concentration (r = -0.414; P ≤ 0.05). The blood of the chicks supplemented with methionine showed an increase in homocysteine concentration, but it was not statistically significant in relation to any of the control groups (Table 4).

Homocysteine Concentration in the Blood Plasma of the Chickens After in ovo Injection in E17

Statistical analyzes showed positive correlations of the concentration of this metabolite with glucose concentration (r = 0.372; P ≤ 0.01) and uric acid concentration (r = 0.193; P ≤ 0.01) in the blood of the chicks. The highest dose of methionine increased (at a statistically significant level) the concentration of homocysteine in the blood plasma of 1- and 7-day-old chicks (P ≤ 0.05), and considerably, but beyond the level of statistical significance, in the blood of 35-day-old chickens (Table 7).

Table 7.

Homocysteine concentration in the blood plasma on the 1st, 7th and 35th d after hatching (D1, D7 and D35), following in ovo injection of methionine and/or folic acid in E17 [µmol/L].

Group	Homocysteine [µmol/L]
	Day of life
	D1	D7	D35
	Mean ± SD	Mean ± SD	Mean ± SD
O	27.7 ± 11.49AD	21.3 ± 12.21A	0.6 ± 0.28A
C	22.6 ± 9.64AF	25.3 ± 11.66A	0.9 ± 0.46A
M5	20.6 ± 9.81AB	27.3 ± 14.15A	1.4 ± 0.48A
M25	66.9 ± 27.39C	39.2 ± 14.77B	1.8 ± 1.02A
MF5/3	18.4 ± 8.96BDF	32.0 ± 13.18A	0.9 ± 0.66A
MF25/15	34.2 ± 21.16DE	38.5 ± 18.22B	1.0 ± 0.62A
F3	31.9 ± 14.23AE	43.3 ± 16.79B	1.0 ± 0.63A
F15	14.0 ± 4.78BF	26.9 ± 10.09A	1.9 ± 1.37A

ABCD – values marked with different letters within a given column differ statistically significantly (P ≤ 0.05).

DISCUSSION

A bird embryo, unlike mammalian, develops outside the mother's body, therefore its development depends primarily on the substances deposited in the egg. Its composition can be partially modified by maternal nutrition, but in terms of amino acid composition it is very stable and does not change under the influence of nutritional factors (Fisher, 1998; Ohta et al., 2001, 2004). The amino acid composition of the contents of eggs of the Ross 308 meat line determined in our own study (Table 3) did not deviate from the values presented in the literature, regardless of the type of chickens (FAO, 1970; Lunven et al., 1973; Fisher, 1998; The official Danish Food Composition Database 1984 (Health Canada); Ohta et al., 1999, 2001, 2004).

Critical in embryonic development of birds is the perinatal period during which the embryo begins pulmonary respiration and gradually prepares for the transition to nutrition with exogenous components. During this time, to rebuild the exhausting energy reserves needed to survive in the first hours and days after hatching, the embryo often uses its own proteins. Administration of preparations with free amino acids during this period may inhibit the activity of catabolic enzymes and prevent the decomposition of own proteins (Uni and Ferket, 2004; Ferket, 2006; Bakyaraj et al., 2012). Around the 14th d of incubation, the egg white enters the amniotic cavity, and then during the following days it is taken per os by the embryo and digested. Supplementation of chicken eggs with selected compounds about the 17th d of embryogenesis is called “in ovo nutrition”. In ovo nutrition may prevent the use of own proteins, as well as accelerate the development of the digestive system, and thus postnatal development, enabling the achievement of a greater body weight (Uni and Ferket, 2004; Ferket, 2006; Bakyaraj et al., 2012).

Excessive consumption of methionine is extremely dangerous for the body. Even doubling the recommended daily intake of this amino acid may lead to various complications, including kidney damage, pathological changes in the pancreas and liver (Kumagai et al., 2002; Troen et al., 2003, 2007). The toxic effect of methionine is usually attributed to its derivative – homocysteine (Hcy), which is a toxic metabolite of methionine, and its harmful effects are confirmed by both in vivo and in vitro studies (Olinescu et al., 1996; Ventura et al., 2004; Toue et al., 2006). This amino acid is produced in all types of cells found in the body, while it is mainly neutralized in the liver and kidneys through remethylation or transsulfuration. Folic acid, the availability of which is a prerequisite for the removal of excess homocysteine, plays a key role in this process (Swain and St. Clair, 1997; Ventura et al., 2004; Kraczkowska et al., 2005). Methionine consumption may increase homocysteine plasma concentrations (Zhang et al., 2004; Toue et al., 2006). In our study, Hcy concentration in plasma was investigated to determine whether in ovo administration of methionine affected its content in plasma, and if so, whether folic acid has a regulating effect on this amino acid. An increase in homocysteine concentration in the blood of the chicks supplemented in ovo with methionine alone (at the highest doses) regardless of the date of injection was found. However, in the case of animals injected in E4, unlike E17, it was not statistically significant in relation to any of the control groups (Table 4). The belief that folic acid has a lowering effect on homocysteine concetration in the blood is confirmed by a negative correlation between the supplementation of embryos with folic acid in E4 and the concentration of homocysteine in the blood plasma of 1-day-old chicks (r = -0.414; P ≤ 0.05). The result obtained in the own study is consistent with the results obtained by Swain and St. Clair (1997), who stated that homocysteine concentration in blood can be reduced by supplementing the body with folic acid. There was a negative relationship between the age of broiler chickens and the concentration of homocysteine in blood plasma (r = -0.618; P ≤ 0.01). These results find confirmation in the research of Kraczkowska et al. (2005) and Ventura et al. (2004), according to which homocysteine concentration in blood plasma depends on gender, age, folate supply in the diet and the efficiency of organs responsible for detoxification, such as the liver and kidneys. The kidneys are an extremely important organ affecting homocysteine concentration in blood. Decreased renal function leads to an increased concentration of this metabolite in plasma (Refsum et al., 2004; Baszczuk et al., 2014), and at the same time, an elevated (as a result of an increase in the amount of methionine in the diet) homocysteine concentration causes damage to the kidneys and other organs (Kumagai et al., 2002). The mechanism through which this damage occurs is not yet fully understood. This may have to do with damage to blood vessels (Kumagai et al., 2002). In our own study, with the late injection of the tested substances, a positive correlation was found between the concentration of this metabolite in blood plasma and uric acid concentration in the blood plasma of the chicks (r = 0.193; P ≤ 0.01), the increased presence of which may indicate impaired renal function (Krauze and Grela, 2008). Supplementation with folic acid in the early stage of embryogenesis resulted in a decrease in homocysteine concentration in the blood, which may be indicative of its beneficial effect in the case of a diet rich in methionine.

According to numerous authors, glucose concentration is one of the easiest to determine physiological parameters that are an indicator of stress in chickens (Puvadolpirod and Thaxton, 2000; Elston et al., 2000; Campo et al., 2008; Zaręba, 2008). In our own study, in 1-day-old chicks, hatched from eggs injected on the 4th d of embryogenesis (Stage I), a negative correlation was found between glucose concentration and the dose of methionine administered (r = -0.210; P ≤ 0.05). Such dependence did not occur in 1-day-old chicks hatched from eggs injected in late embryogenesis, and in the context of the entire study period (up to the 35th d of life), correlation between these factors is positive (r = 0.193; P ≤ 0.01). Adeyemo et al. (2010) point out that there may be large differences in glucose concentration in birds, and in their nutritional studies, birds receiving methionine had higher blood glucose levels than those not receiving the addition of the amino acid tested.

In our study, chicks supplemented in ovo on the 17th d of embryogenesis showed positive correlations between the dose of methionine and glucose concentration (for D1 r = 0.322; P ≤ 0.01; throughout the course of the experience r = 0.193; P ≤ 0.01). It is also worth noting that in the first day of bird life, glucose concentration was the lowest in the group injected with 15 mg of folic acid (Table 5).

In the study by Tani et al. (1990) it was shown that high doses of essential amino acids, including methionine, may cause damage to the rat pancreas, whereas Włochal et al. (2012) report that people with pancreatitis have folic acid deficiencies leading to hyperhomocysteinemia. Inadequate supply of methionine and folic acid in the prenatal period causes methylation disorders and affects the differentiation of pancreatic cells, increasing the risk of its damage and inflammation (Longnecker, 2002).

In papers discussing issues related to methionine supplementation, attention is paid not only to the process of methionine translation leading to the formation of new proteins of the body, but also to their breakdown associated with the need to remove nitrogen from the body. Nitrogen is removed from birds mainly in the form of uric acid, which is their main end metabolite of protein, and its amount may increase with an increase in the total protein content in the serum as a result of dehydration (Krauze and Grela, 2008). The level of this compound in blood plasma is an indicator of the overall metabolism of amino acids, and indirectly also allows for the assessment of the functional status of kidneys (Krauze and Grela, 2008). In chickens fed a diet with methionine deficiency, increased feed intake with reduced efficiency of its use, reduced nitrogen retention, and increased synthesis of uric acid manifested by its increased concentration in blood plasma can be observed (Solberg et al., 1971).

In humans, uric acid is usually considered a metabolically neutral end product of purine metabolism. This compound is also a selective antioxidant, particularly in reactions with hydroxyl radicals and hypochlorous acid, and is considered a marker of cardiovascular diseases (Kalay et al., 2011). Administration of small amounts of folic acid (3 mg) in an early stage of embryogenesis resulted in a decrease in uric acid concentration – significant compared to group O (Table 4). In contrast, with supplementation in the final stage of embryonic development, there was a positive correlation between these substances in the blood plasma of 1-day-old chicks (r = 0.147; P ≤ 0.05). The study also showed a positive correlation between the level of uric acid and the dose of supplemented methionine (r = 0.138; P ≤ 0.05).

CONCLUSIONS

The effect of methionine addition on blood glucose depended on the date of administration of this amino acid: early administration lowered, and later supplementation led to an increase in glucose concentration. Our results suggest that methionine supplementation may increase the concentration of uric acid and homocysteine. Folic acid administered in ovo during embryogenesis decreased homocysteine concentration, also in groups simultaneously supplemented with methionine, especially in the initial period of postnatal life of the bird. Folic acid given at a low dose in the initial stage of embryogenesis caused a decrease in uric acid concentration in the blood of the chicks. However, when given in the final phase of embryonic development resulted in an increase in the concentration of this metabolite in the initial period of the animal's postnatal life. The rsults of the current study indicate that folic acid can have ameliorative effect in methionine-suplemented chicks.

DISCLOSURES

The authors declare no conflicts of interest.