Folate or folic acid (FA) is a general name for a group of compounds with a pteroylglutamic acid backbone that have different oxidation states (i.e., folic acid, 5-methyltetrahydrofolate). These components function in one-carbon transfer reactions (Selhub and Rosenberg, 1996; Bagley and Shane, 2005). FA is an essential vitamin that is serving as a cofactor and co-substrate for biological methylation reactions such as those involved in amino acid and nucleic acid synthesis (Bagley and Shane, 2005). Adequate FA status in humans is reported to reduce the risk of neural tube defects in babies (Czeizel and Dudas, 1992; De Wals et al. 2007), stroke (Yang et al. 2006), certain cancers (Kim, 1999), and inflammatory diseases in adults (Wang et al. 2001). Adequate FA nutrition is necessary in human nutrition. Its deficiency results in an increase of plasma homocysteine concentrations, with the latter being linked to an increased risk for cardiovascular (Boushey et al. 1995; Refsum et al. 1998), Alzheimer (Morris et al. 2007), and osteoporosis diseases (Mclean et al. 2004). Therefore, enrichment of poultry products can increase this vitamin consumption in human. The enrichment of eggs with FA has been explored as an additional vehicle to provide FA into the human diet. More than 80% of the total FA composition of eggs is 5-methyltetrahydrofolate (5-MTHF) (Selhub and Rosenberg, 1996). Unlike the synthetically derived folic acid (FA), 5-MTHF is a naturally occurring derivative of FA and therefore unlikely to interfere with normal FA metabolism. Previous studies have shown that dietary FA supplementation can fortify the egg FA. Dietary supplementation by FA can produces the eggs with 45 to 50 μg of dietary FA equivalents or approximately 10% of the recommended dietary allowance for adults (Food and Nutrition Board, 1998). However, further increase of FA level in eggs have not been possible because of saturable processes during intestinal FA absorption which reach a maximum plateau (Said et al. 2000; Said, 2004; Inoue et al. 2008). By dietary FA (6.0-12.0 mg/kg) supplementation, Husseiny et al. (2008) observed no changes of egg production. Keshavarz (2003) reported that certain manipulations of the combination of methionine, choline, folic acid, and vitamin B12 have the potential to reduce egg weight and improve shell quality without affecting egg production during the latter stages of the egg production cycle. Betaine (BET) and other methyl group donors such as choline and methionine play an important role in methylation reactions. Natural BET is obtained from several plants and organisms (Boch et al. 1994) and it is commonly extracted and purified from beetroot. It has three chemically active methyl groups bound to the nitrogen atom of a glycine molecule and classified as a methyl-ammonia (Kidd et al. 1997). BET is considered the readily active methyl-group donor (Kettunen et al. 2001). Previous studies suggested that BET might have a methionine sparing effects in non-ruminant animals (Gudev et al. 2011). BET has been well established as a labile methyl donor and as an osmoprotectant, which is synthesized via the oxidation of BET aldehyde formed by the irreversible oxidation of choline in the mitochondria of the liver (Figure 1) and renal tissues (Ratriyanto et al. 2009). There is a growing interest to using BET and other methyl group donors in the poultry diets (Farrokhyan et al. 2014; Hosseintabar et al. 2015; Jafari-Golrokh et al. 2016). Supplemental dietary BET improved weight gain and feed conversion in some poultry studies (Mathews and Southern, 2000; Hassan et al. 2005) whereas other studies showed minimal or no effect of BET on broilers performance (Zulkifi et al. 2004). Addition of 600-ppm BET to laying hen diets has improved the laying performance (Park et al. 2006). Under heat stress, addition of BET to the poultry feeds (broilers, turkeys and meat ducks) has improved the performance. Furthermore, BT promotes the intestinal microbes against osmotic variations and improves the microbial fermentation activity, which in turn, may enhance the nutrient digestibility in non-ruminant animals (Ratriyanto et al. 2009). The liver is an important site for sulfur amino acid metabolism. Methionine, which is the first-limiting amino acid in commercial poultry diets, is a methyl group donor (transmethylation). Methionine is converted to homocysteine, which lies at the crossroads of sulfur amino acid metabolism. Formation of cysteine may occur if homocysteine proceeds through the irreversible transsulfuration pathway; alternatively, homocysteine may be converted back to methionine after addition of a methyl group by FA-vitamin B12-dependent methionine synthase (MS) or BET - homocysteine methyltransferase (BHMT). The methyl group provided by BHMT is derived from betaine, a product of choline oxidation and the methyl provided by (MS) is derived from 5- methyl tetrahydrofolate (Pillai et al. 2006). Transmethylation is very important in amino acids metabolism, so continued evaluation of sulfur amino acid metabolism is an important part of working toward optimum diet formulation and enrichment poultry feed. This study was carried out to establish dietary supplementation of BET and FA will be able to affect the egg FA content, performance and egg production of Japanese quail.
MATERIALS AND METHODS
A total of 288 forty-two day-old female quails (Coturnix coturnix Japonica) were used in this experiment. The birds were divided into nine treatment groups and 4 replicate (8 birds each) for each treatment. The cage dimensions were 60 × 60 cm, providing 36 cm2 per bird. Cages were equipped with nipple drinkers and trough feeders. Birds were reared under semi controlled environmental conditions in the experimental room with windows and received additional artificial light to provide 16 hour of light and 8 hour of dark. The birds in different experimental treatments were fed by each experimental diets form week 7 to week 12 of age (for 6 weeks).
The basal diets were based on corn-soybean meal to cover the nutrients requirements of laying quails (NRC, 1994). Diets were supplemented with BET at levels of 0, 50 or 100 mg/kg and folic acid at levels of 0, 4 or 8 mg/kg for each BET level ingredients. The chemical compositions of the basal diets are shown in Table 1.
One week before the study, 288 birds (42 day-old healthy female quails) were randomly assigned to 9 treatments, with 4 replicates and 8 birds in each wire cage. At day 49 of age, cages were randomly assigned to receive the dietary treatments. The diets were introduced 7 days before the egg collection period. Experimental diets were fed for a 6-week period.
Figure 1 Metabolism of sulfur amino acids, choline and betaine
Feed intake (FI), egg production (EP) and egg mass (EM) for each cage unit were measured weekly and feed conversion ratio (FCR) calculated. FCR was calculated as grams of feed consumed by quails per gram of egg produced. The number of eggs and egg weight (EW) were recorded daily throughout the experiment by using an electronic scale (0.01 g sensitivity) and calculated as percentage hen-day EP. Random samples of three eggs from each treatment were collected weekly to measure the egg quality. Length and width of the egg were measured with Vernier caliper sensitive. Shape Index (SI) was estimated using the following equation:
Egg shape index (ESI)= [egg width / egg length] × 100
Measurements of the internal components were obtained by carefully making an opening around the sharp end of the egg, large enough to allow passage of both the albumen and the yolk through it without mixing their contents together. The yolk was then carefully separated from the albumen and placed in a petri dish for weighting. Simultaneously, associated albumen was placed on another petri dish and weighed. After each weighing, the petri dishes were washed by clean water and wiped dry before next weighing. The yolk and albumen diameter and albumin height of the egg were measured by Vernier caliper sensitive. The yolk and albumen height were measured by Haugh micrometer. The yolk and albumen pH were measured by sensitive digital pH Metter. The eggshell weight (ESW) with membrane was obtained by carefully placing the opened part in the shell and weighing on the electronic scale. Eggshell thickness (EST, µm) was determined by measuring the thickness mean values taken at three spots on the egg (air cell, equator, and sharp end) using a dial pipe gauge (Mitutoyo, 0.01-20 mm, Japan). Eggshell ratio (ESR) was estimated from the expression:
ESR (%)= (shell weight/egg weight) × 100.
Shell weight unit surface area (SWUSA) (g/cm2) was determined by dividing the shell weight to shell surface area. Egg yolk index (%) was calculated by the expression:
EYI= [yolk height (cm)/yolk diameter (cm)] × 100
Egg yolk percent (EYP) (%)= 100 × (yolk weight/egg weight)
Egg weight ratio (EWR) (%)= (egg weight weight/egg weight) × 100
Albumen index (%)= albumen height (mm) / average of albumen length and width) mm × 100
The egg yolk color (EYC) was recorded according to the yolk color fans (scores 1-15). Egg specific gravity (ESG) was determined by using the saline flotation method as described by Hempe et al. (1998).
Table 1 Composition and calculated analysis of the basal diet
1 Provided (per kg of diet): vitamin A: 8255 IU; vitamin D3: 3500 IU; vitamin E: 30.0 IU; vitamin K3 (menadione with sodium bisulfite): 0.55 mg; vitamin B12: 0.011 mg; Riboflavin: 4.50 mg; Pantothenic acid: 8.24 mg; Niacin: 22.55 mg; Choline: 1201.80 mg; Biotin: 0.100 mg; Thiamine (B1; mononitrate): 2 mg; Pyridoxine (B6; HCl): 3 mg; DL-methionine: 900 mg and Ethoxyquin (antioxidant): 125 mg.
2 Provided (per kg of diet): MnO (manganese oxide): 70 mg; ZnO (zinc oxide): 66 mg; FeSO4 (ferrous sulfate): 50 mg; CuSO4 (copper sulfate): 8.8 mg; NaSeO3 (sodium selenite): 0.3 mg; Ca(IO3)2 premix (calcium iodate premix contained 2.0 g of calcium iodate and 43 g of iodized salt): 0.33 mg and Iodized salt: 0.67 mg.
Determination of dietary FA content
The extraction of the FA-supplemented diets was performed as described by (Wilson and Horne, 1984; Tamura et al. 1997). In brief, feed samples were homogenized with a 50 mM N-(2-cyclohexylamino) ethanesulfonic acid -HEPES buffer with 2% ascorbic acid and 0.2 M 2-mercaptoethanol (pH 7.8) and stored at −80 ˚C until analysis. The extracts were analyzed for FA via reverse-phase HPLC 1100 series made by Agilent Company USA with fluorescence detection, using the method of (Vahteristo et al. 1997). An external standard curve with purified FA was used to quantify feed FA concentration.
Extraction and analysis of egg yolk FA content (EYFC)
The extraction and analysis of the EYFC FA was performed as described previously (House et al. 2002). In brief, eggs were weighed, placed in boiling water for 10 min, cooled, and the yolks were separated, weighed, and retained for analysis by storing at −80 ˚C. Egg FA in the form of 5-MTHF, the major form of FA in eggs, was extracted into an acrobat buffer (pH 7.8). The extracts were analyzed for 5-MTHF via reverse-phase HPLC 1100 series made by Agilent company USA with fluorescence detection, using the method of Vahteristo et al. (1997). An external standard curve with purified 5-MTHF was used to quantify EYFC.
A completely randomized design with 3 level of FA supplementation (0, 0.4 and 0.8 mg/kg of crystalline FA) and 3 level of BET (0, 0.5 and 1 g/kg of betaine) supplementation in a 3 × 3 factorial arrangement was used. Data were subjected to ANOVA, using the PROC GLM procedure of SAS software (SAS, 2008). When evidence of heterogeneity of variance was present, data were log-transformed before analysis.
RESULTS AND DISCUSSION
The results of folic acid and BET effect on laying performance and egg indices of quails are shown in Table 2. None of the performance traits (FCR, EP, EW, EM, ESI and ESG) was affected by dietary treatments.
Table 2 The effects of folic acid and betaine on laying performance and egg indices of quails
FI: feed intake; FCR: feed conversation rate; EP: egg production (hen/day); EW: egg weight and EM: egg mass; ESI: egg shape index and ESG: egg special gravity.
SEM: standard error of the means.
The FI had a trend and was numerically increased by dietary BET (P=0.059). Although there were no significant difference between the treatments for FI but BET fed birds had the higher FI at any levels of folic acid. There were no significant differences between the treatments for feed conversation ratio, egg production percent, egg weight and egg mass throughout the study period (P>0.05). No changes of eggshell indices was observed by dietary supplementation of FA and BET (Table 3). EST decreased numerically (P=0.07) by increasing FA supplementation level. There were no difference between the treatments for ESW, ESR and SWUSA. No significant differences were observed for EW pH and EWR, but HU was affected significantly by folic acid supplementation (P<0.01). High level of FA supplementation (8 mg/kg) decreased the HU. Moreover there was a significant interaction between the FA and BET for HU (P<0.05) and increasing the BET level did not changed the HU at the low and medium levels of betaine, while decreased the HU at the highest level of FA (8 mg/kg). No effects of FA or BET were observed for quail EY pH, EYI and EYP, but EYFC and egg yolk color (EYC) were affected significantly by folic acid supplementation (Table 4) (P<0.01). EYFC increased from a low level of 843.87 µg/kg for birds consuming the basal diet with no added folic acid, up to a high level of 1456.25 µg/kg for birds consuming diets with 4 mg/kg of folic acid.
Performance and egg indices
The earlier studies (Keshavarz, 2003; Turker et al. 2004; Zhan et al. 2006) have reported that BET and any methyl group donors containing-diets significantly improves the EW and FCR in broilers and laying hens because of their role in sulfur amino acids metabolism as a methyl group donors (Kyeong Seon et al. 1955). The results of our study showed that addition of supplemental FA and BET to laying quail diets did not affect the performance, as reflected by insignificant differences in FI (g), FCR, EP (hen/day), EW (g) and EM (g). In the present study, the vitamins contained in the basal diet possibly have met the quail requirements (NRC, 1994), and thus this diet was sufficient for normal EP. Accordingly, Husseiny et al. (2008) indicated no effects of feeding supplemented diets with FA (6.0-12.0 mg/kg) on egg production. In a different way, some previous studies (House et al. 2002; Hebert et al. 2005) have been reported that supplemental FA to laying hen diets significantly increased the FI. Keshavarz (2003) found that reducing dietary FA resulted in decreased EW.
Table 3 The effects of folic acid and betaine supplementation on egg shell and white indices
EST: egg shell thickness; ESW: egg shell weight; ESR: egg shell ratio; SWUSA: shell weight unit surface area; EWpH: egg white pH; EWW: egg white weight; HU: Haugh unit and EWR: egg white ratio.
SEM: standard error of the means.
The lack of changes in performance indices of recent study by BET was in agreement with the results of Waldroup and Fritts (2005) who reported that 100 to 500 mg/kg BET supplementation had not affected the male broilers performance. Enting et al. (2007) showed that dietary addition of 1.0 and 2.0 g/kg BET improved the FCR during 0 to 14 days of age. Moreover, Honarbakhsh et al. (2007) evaluated the effects of different exogenous BET levels (0.0, 0.075, 0.150 and 0.225%) on broiler chickens (Ross) and observed the increased body weight, FI and improved FCR during the grower and finisher periods. In addition, Sayed and Downing (2011) found that addition of BET at an inclusion rate of 500 mg/L in drinking water improved the body weight (BW) of broiler chickens under heat stress. Tollba et al. (2007) showed that dietary supplemental BET for 12 or 16 weeks to Fayoumi laying hens improved FCR and body weight (BW) without significant effect on FI. Baghaei et al. (2009) and Maghoul et al. (2009) have found that 10, 20 and 30% BET replacement for choline had no effect on FI and weight gain (WG), FCR of broilers. Jahanian and Rahmani (2008) found that dietary BET inclusion (0.0, 50 and 100% in substitution for choline) had no effect on FI. In this respect, BET supplementation (800 mg/kg diet) enhanced growth at 21 days and FCR at 42 days of age in broilers (Rao et al. 2011). BET significantly improved BW and FCR of Matrouh poultry strain at a level of 1.0 g/kg diet (Ezzat et al. 2011). Some other researches showed the improved FCR by dietary BET supplementation (Tollba et al. 2007; Enting et al. 2007; Honarbakhsh et al. 2007). The result of current experiment regarding the EW is in agreement with that of Hebert et al. (2005) who researched the supplementation of 2.0, 4.0, 8.0, 16, 32, 64 and 128 mg/kg of FA in 2 Leghorn strains of laying hens and observed no difference in EW during a 3-wk experimental period. Tactacan et al. (2011) noted no effect on EW when Shaver White laying hens were fed diets supplemented with 10 or 100 mg of FA/kg diet. Khalifah and Shahein (2006) determined that the inclusion of 0.0 to 32 mg FA/kg in the Baheij chicken strain did not affected the EW. Bunchasak and Kachana (2009) suggested that FA (0.31 to 10.31 mg/kg diet) added to a corn-soy based diet had no effect on EP of older laying hens (64-72 weeks of age). Our results are in disagreement with those obtained by those of Tollba and El-negar (2008) who observed that increasing BET supplementation (2000 ppm/kg) to the diet caused the higher EP and EM. In addition, Gudev et al. (2011) reported that EP and EM were significantly improved when BET supplemented to laying diets up to 1.5 g/kg for hens during heat stress.
Table 4 The effect of folic acid and betaine supplementation on egg yolk indices
EYpH: egg yolk pH; EYW: egg yolk weight; EYP: egg yolk percent; EYI: egg yolk index egg yolk color and EYFC: egg yolk folate content.
SEM: standard error of the means.
Supplementation of FA showed no effect on ESI in recent experiment. The results agree with Ezzat et al. (2011) who reported that Matrouh poultry strains egg shape index was not affected by FA (1.0 mg/kg diet) supplementation under Egyptian summer condition. Eseceli et al. (2010) investigated the effect of FA inclusion (10 mg/ton diet) to the ration of laying hens and found that supplemental folic acid did not have any significant effect on ESI and ESG. Benkova et al. (2009) increased the dietary content of FA from 0.5 mg/kg to 3.5 mg/kg of feed in laying hens and did not observed any significant effect on ESI.
Egg shell and white indices
No effects of FA or BET were observed on ESW, ESR, EST and SWUSA in recent experiment. The EST decreased from 1.58 µm for control to 1.40 µm and 1.34 µm by increasing the dietary FA levels to 4.0 and 8.0 mg/kg, respectively. The results of this study are in agreement with those of Husseiny et al. (2008) that reported the egg quality parameters did not changed by FA supplementation. Similar results were observed for EST by Hebert et al. (2005) and Khalifah and Shahein (2006). The results of current experiment showed that EW pH, egg white weight (EWW) and egg white ratio (EWR) did not affected by FA and BET supplementation. There was no interaction between FA and BET for HU. However, HU decreased from 95.26 mm for control to 94.94 mm and 95.54 mm for 4.0 and 8.0 mg/kg FA supplemented treatments, respectively. The result of this study are in disagreement with Ezzat et al. (2011) that showed that supplementation of Matrouh poultry strain diets by BET (1.0 g/kg diet) and FA (1.0 mg/kg diet) together from 24-36 weeks of age under Egyptian hot summer significantly increased the HU. Gudev et al. (2011) determined the effect of 0.7 and 1.5 g/kg supplemental BET on egg performance of 2-year-old laying hens. The results of their study indicated no changes in EYI, EYK and HU. Gudev et al. (2011) supplemented basal diets of 2-year-old laying hens with 0.7 and 1.5 g/kg BET and detected no changes for egg quality (albumen weight, albumen index, egg yolk weight (EYW), EYI, EYC, HU, ESG).
Egg yolk indices
EY pH, EYW, EYP and EYI were not affected by BET or FA supplementation in current experiment. Supplementing FA at 4.0 and 8.0 mg/kg to the diet decreased the EYC. Results of this study are in agreement with those of Benkova et al. (2009) who fed laying hens with diets supplemented with 1.5 to 3.5 mg of FA/kg and indicated higher yolk color by dietary supplementation. In an opposite way, Hassanien et al. (2010) showed no effects of dietary FA on BW, EP, FI, FCR, EM, EW, EYC, ESI and EYI of laying hens. Moreover, EYFC increased by supplementing FA at 4.0 and 8.0 mg/kg in our experiment and increased form 886.48 for control to 1002.98 and 1284.77 for 4.0 and 8.0 mg/kg FA, respectively. The results of the present study for EYFC are in agreement with the findings of some other researchers (Sherwood et al. 1993; House et al. 2002; Hebert et al. 2005; Hoey et al. 2009; Tactacan et al. 2010) that showed the possibility of higher FA content in animal products by synthetic folic acid. Our current study regarding the production of enriched quail eggs by FA supports the previous findings in laying hens by feeding the supplemented dietary FA (Sherwood et al. 1993; House et al. 2002; Hebert et al. 2005; Tactacan et al. 2010). The present studies provide strong evidence of the sensitivity of quail egg FA content to dietary FA levels. The results of this study are in disagreement by House et al. (2002) and Hebert et al. (2005) that reported previously, the addition of FA above 4.0 mg/kg in the diet of laying hens yielded no further significant increase in egg FA content. On the other hand, supplementation of 4.0 mg of FA/kg of diet yielded 90% maximal egg FA enrichment. In a disagreement way, House et al. (2002) and Sherwood et al. (1993) reported that egg FA concentrations responded to increasing levels of dietary FA supplementation and maximal egg FA levels were achieved when dietary FA concentrations was 8.0 mg/kg of diet. House et al. (2002) observed that egg FA concentrations increased above a plateau value when the level of FA in the diet was 32 mg/kg of diet. Plasma serves as the precursor pool for egg yolk deposition. Miller and White (1986) have identified the existence of a binding protein-mediated mechanism for the transfer of riboflavin into the egg yolk and there may be a similar mechanism for FA. Plasma FA concentrations may serve as the limiting factor for egg FA concentrations (Sherwood et al. 1993), as both compartments appear to saturate at dietary FA concentrations between 2.0 and 4.0 mg/kg of diet. Plasma FA concentrations are higher in birds consuming 128 mg of folic acid/kg diet than those consuming 4 mg/kg; however, this increase is not sufficient to translate into higher egg FA concentrations. Therefore, a greater understanding of the factors regulating the absorption of dietary FA and its appearance in the systemic circulation in the form of 5-methyltetrahydrofolate is needed. In studies with rats and in vitro intestinal cell model systems, FA is absorbed from the gut via a membrane-bound FA transport system that accepts both oxidized and reduced forms of the monoglutamated forms of FA has been shown to be saturable in a number of model systems and as such represents a potential control point for plasma FA concentrations (Said, 2004). However, the 5- methyltetrahydrofolate form is believed to be the primary circulating form of FA in the plasma. In the enterocyte, FA is reduced via a 2-step process involving the enzyme dihydrofolate reductase to yield tetrahydrofolate (Henderson, 1990). Tetrahydrofolate is then transported to the liver, where it is converted to 5-methyltetrahydrofolate form. Attachment to binding proteins within hepatocytes and transport via the systemic circulation is the putative mechanism whereby FA is transported to the developing yolk (Henderson, 1990). Therefore, multiple control points exist to regulate plasma FA levels, and further studies are required to elucidate the key control point (s), especially in quails. 5-methyltetrahydrofolate participates in a reaction involving the FA-dependent remethylation of the sulfur AA homocysteine to yield methionine. Increasing the crystalline FA level from the 0.0 to 8.0 mg/kg in the basal diet resulted in a significant reduction in plasma homocysteine concentrations in the laying quails of current experiment. This reduction may indicate that the Japanese quail have a higher requirement for FA as compared to the other hens and, indeed, a higher requirement than that given by NRC (1994).
In conclusion, dietary supplementation of betaine and FA have no effects on egg yolk pH, index and percent, but EYFC and color. Supplementation of 8 mg/kg FA decreases the Haugh unit, but enriches the EYFC from 886.48 to 1284.77 µg/100 g (1.45 times).
The authors appreciate all the staffs of the Animal Science Department in Urmia University for the helps during the research.