Effects of Dietary Protein Level and Betaine Supplementation on Nutrient Digestibility and Performance of Japanese Quails

This study investigated the effects of dietary protein levels and betaine supplementation on nutrient digestibility and performance of Japanese quails. In total, 765 Japanese quails were randomly assigned to a 3×3 factorial arrangement, with five replicates of 17 quails each. Three basal diets were formulated to contain three crude protein levels (16.5, 18.0, and 19.5%). Each protein level was supplemented with 0, 0.06, and 0.12% betaine. The diet with 16.5% dietary crude protein with no betaine supplementation resulted in the lowest crude fiber digestibility, while the 18.0% CP diet supplemented with 0.12% betaine generated the highest crude fiber digestibility (p<0.05). The diets with 18.0 and 19.5% crude protein increased crude fiber digestibility, but reduced ether extract digestibility (p<0.01). Moreover, betaine supplementation increased dry matter, crude protein, crude fiber, and crude ash (p<0.01) digestibility and tended to increase ether extract digestibility (p=0.09). The increase in egg weight for the 18.0 and 19.5% protein diets was correlated with a decrease in feed conversion ratio (p<0.05). However, feed intake and egg production were not affected by protein levels. Betaine supplementation enhanced all performance variables (p<0.01). The diets with 18.0 and 19.5% crude protein resulted in heavier yolks and eggshells than the 16.5% crude protein diet (p<0.05), whereas betaine supplementation increased yolk, albumen, and eggshell weight (p<0.01). The 18.0 and 19.5% protein diets produced similar responses in most evaluated parameters. Laying Japanese quails can be fed diets with 18.0% crude protein. Moreover, betaine supplementation provided several benefits, and particularly improved nutrient digestibility, performance, and egg quality.


INTRODUCTION
Layers are susceptible to heat stress because egg formation results in a high level of metabolic heat production (Blem, 2000).Moreover, the high ambient temperature in the tropics may limit protein synthesis, negatively affecting poultry production (Rashid et al., 2012).Proteinrich diets do not have a positive impact on poultry performance in hot climates and may even depress performance due to the high heat increment of protein digestion (Daghir, 2009).Although many studies have been performed to evaluate the effects of dietary crude protein (CP) in layers, the responses have been inconsistent among studies, possibly due to differences in experimental conditions, bird strains and age, laying period, and parameters evaluated (Li et al., 2013;Ding et al., 2016).Previous studies demonstrated that dietary CP influences laying performance and egg quality (Nahashon et al., 2007;Gunawardana et al., 2008;Li et al., 2013).Under tropical conditions, Musa et al. (2008) recommended 18% CP for laying quails, which is lower than the 20% recommended by the NRC (1994).Garcia et al. (2005) observed that, in quails, increasing dietary CP from 16% to 18% improved egg production and feed conversion ratio, while increasing dietary CP to 20% did not result in the further performance improvements.According to Junqueira et al. (2006), for brown hens, feeding with 16% CP was adequate and resulted in better performance and egg quality than 18% and 20% CP.
Betaine is a trimethyl derivative of the amino acid glycine.It is a methyl group donor, donating its labile methyl group (CH 3 ), and plays an important role in the metabolism of protein and energy (Metzler-Zebeli et al., 2009;Ratriyanto et al., 2009a).Because poultry cannot synthesize the methyl group, it must be supplied in their diet.Unlike other methyl group donors, such as choline and methionine, betaine can act directly as a methyl group donor.The methyl groups of choline become available when choline is oxidized to betaine, while methionine must be converted to S-adenosylmethionine to donate its methyl group (Ratriyanto et al., 2009a).Betaine has the potential to substitute for some part of methionine in the diet by providing a methyl group during the conversion of homocysteine to methionine (Metzler-Zebeli et al., 2009).Consequently, betaine may improve methionine availability for protein synthesis, thus achieving optimal performance (Rao et al., 2011).Moreover, due to its osmotic properties, betaine has the potential to improve nutrient digestibility by supporting the growth and survival of intestinal cells, as well as intestinal microbes (Kettunen et al., 2001;Ratriyanto et al., 2009a).It has been shown that betaine supplementation alters the microbial population of the digestive tract of poultry (Kettunen et al., 1999).Dietary betaine may protect intestinal cells and intestinal microbes from osmotic variation along the digestive tract (Ratriyanto et al., 2009a).
Furthermore, there are only few studies evaluating dietary CP levels and betaine supplementation in relation to nutrient digestibility in poultry, particularly in laying quails, even though dietary CP levels or betaine supplementation affect nutrient digestibility in quails (Li et al., 2011;Ratriyanto et al., 2012).Therefore, the objective of this study was to determine the effects of dietary CP levels and betaine supplementation on nutrient digestibility and performance in quails.

MATERIAL AND METHODS
In total, 765 42-day-old female Japanese quails (Coturnix coturnix japonica), with an average body weight of 126.34 ± 7.29 g, were used in the study.The birds were randomly distributed according to a completely randomized experimental design in a 3 × 3 factorial arrangement: CP levels (16.5, 18.0, and 19.5%) and betaine supplementation (0, 0.06, and 0.12%).Each treatment included five replicates of 17 quails each.
The birds were reared in 45 colony battery cages (75×50 cm, one cage per replicate).A uniform managemental practice was applied to all the experimental groups.
The three basal diets were formulated to contain the same amount of metabolizable energy: 2,800 kcal/ kg (Table 1).Betaine (anhydrous betaine 96%) was supplemented in the basal diets at the expense of corn, according to the procedure of Ratriyanto et al. (2009b).The birds were housed under natural temperature conditions, with an average ambient temperature during experiment at 25.4 ºC in the morning, 32.9 ºC in the afternoon, and 28.7 ºC in the evening.During the pre-experimental period (42 days until 50% hen day average -HDA), the birds were fed the same diet, which contained 18% CP.The dietary treatments

Effects of Dietary Protein Level and Betaine Supplementation on Nutrient Digestibility and
Performance of Japanese Quails started to be applied after egg production reached 50% HDA.Water and feed were provided ad libitum.The treatment diets were fed for two periods of 28 days each (2×28 days).Feed intake, egg production, and egg weight were recorded daily.Feed conversion ratio (FCR) was calculated as the ratio of feed intake per egg mass (Junqueira et al., 2006).Protein efficiency ratio (PER) and energy efficiency ratio (EER) were also calculated for each experimental period.PER was calculated as grams of egg mass per gram of protein intake, and EER was calculated as grams of egg mass×100/total ME intake (Suprijatna et al., 2009).Physical egg quality was assessed during the last three days of each period (days 26, 27, and 28).In total, 405 eggs from each period (nine eggs per replicate) were randomly collected to evaluate yolk weight, albumen weight, and eggshell weight and thickness.The eggs were weighed and cracked, and the egg components were weighed thereafter.Eggshell thickness was measured with a digital micrometer (0-25 × 0.001 mm).
At the end of experiment, 90 birds (two per replicate) were randomly selected to determine nutrient digestibility.The digestibility trial was performed over a 5-day total excreta collection, following the procedure proposed by Ratriyanto et al. (2014a).During the collection period, the excreta were periodically spread with 0.2 N H 2 SO 4 to minimize further bacterial fermentation.The excreta collected during the 5 days were dried under the sun.The samples of excreta for the various diets were milled through a 0.5 mm mesh screen prior to analysis.Excreta CP content was determined by the Kjeldahl method, while dry matter (DM), ether extract (EE), crude fiber (CF), and crude ash (CA) analyses were performed as outlined by the AOAC (1990).The nutrient digestibility coefficients were calculated according to Emamzadeh & Yaghobfar (2009).
The data were checked for outliers and tested to ensure the homogeneity of variances (Bartlett test).Then, the data were submitted to analysis of variance using the R statistic (R Core Team, 2015).Statistically different means were compared via Duncan's multiplerange test at 5% probability.

Nutrient Digestibility
There was no influence of the interaction between protein levels and betaine supplementation on nutrient digestibility (Table 2), except for CF.The16.5% CP diet without betaine supplementation resulted in lower CF digestibility than the other dietary treatments (p<0.05).Also, for the three CP levels, 0.12% betaine supplementation increased CF digestibility, which ranged between 12.87 and 26.89%, as compared with the non-supplemented diets (p<0.05).Regardless of the CP level in the diets, these findings indicate that betaine supplementation improved CF digestibility possibly due to stimulation of microbial fermentation in the digestive tract (Ratriyanto et al., 2014a).
Dietary CP levels did not affect the digestibility of DM, CP, CA, or nitrogen free extracts (NFE).This result is in agreement with Li et al. (2011), who did not find any effect of dietary CP levels (17.75 or 19.95% CP) on organic matter (OM) digestibility in yellow quails.Feeding 17 and 20% CP did not affect CP digestibility in broilers either (Faria Filho et al., 2007).In Japanese quails, during the rearing period, Omidiwura et al. (2016) did not find any effect of CP levels on the digestibility of DM and CP.
In the present experiment, dietary 18.0 and 19.5% CP decreased (p<0.01)EE digestibility by 7.10 and 7.56%, respectively, as compared with 16.5% CP, indicating better EE utilization at lower dietary CP levels.According to Rosebrough et al. (1999), dietary CP levels modulate the metabolic effects of dietary fat, as increasing dietary CP reduces lipogenesis and the levels of precursors of fat synthesis, which may explain the decrease in the digestibility coefficient of EE with increasing dietary CP levels in the present experiment.Moreover, dietary 18.0 and 19.5% CP improved (p<0.01)CF digestibility by 7.12 and 6.45%, respectively, as compared with 16.5% CP, indicating more efficient CF degradation and absorption (Faria Filho et al., 2007).This result was in agreement with the previous observation that CF digestibility increased as dietary CP increased (Ratriyanto et al., 2014a).In addition, according to Li et al. (2011), the effect of CP level on nutrient digestibility can be attributed to an imbalance between protein and energy in the diet.

Effects of Dietary Protein Level and Betaine Supplementation on Nutrient Digestibility and
Performance of Japanese Quails CF (El-Husseiny et al., 2007;Ratriyanto et al., 2014a), EE, and NFE digestibilities (El-Husseiny et al., 2007).These results indicate that betaine supplementation promotes better nutrient utilization, as confirmed by the improvement in production performance in the present experiment (Table 3).
There is growing evidence that betaine supplementation improves nutrient digestibility due to its osmoprotective properties, supporting intestinal cells and the growth of intestinal microbes (Ratriyanto et al., 2009a;Ratriyanto et al., 2010).Previous reports have shown that betaine decreased crypt:villus height ratio in broilers, which may partially explain the increased digestibility of nutrients (Kettunen et al., 2001;Ratriyanto et al., 2014b).Moreover, because poultry do not produce fiber-degrading enzymes, the improvement in CF digestibility indicates that betaine has the potential to stimulate the bacterial fermentation of dietary fiber (Ratriyanto et al., 2010;Ratriyanto 2014a).These results confirm previous observation that intestinal bacteria require compatible osmolytes, such as betaine (Ratriyanto et al., 2010).The higher CF digestibility due to betaine supplementation corresponded to higher CA digestibility.Previous studies have shown the interaction between fiber fermentation and mineral absorption (Ohta et al., 1995;Aulrich & Flachowsky, 1997).Obviously, the fiber fraction holds nutrients that can be released during fiber degradation (Aulrich & Flachowsky, 1997).Higher mineral absorption, as indicated by improved CA digestibility, supports the previous observations that betaine reduces the osmolality of the digesta of chickens (Klasing et al., 2002;Hamidi et al., 2010).

Production Performance
There was no significant interaction between dietary CP levels and betaine supplementation levels for the evaluated performance parameters.Dietary CP levels did not affect feed intake or egg production, but the19.5% CP diet improved feed conversion ratio (p<0.05).These results are in agreement with those of Dos Santos et al. (2016), who did not find any significant effect on feed intake or egg production of quails fed 16 or 20% CP.In addition, previous observations in Japanese quails determined that increasing dietary CP from 17 to 20% (Tuleun et al., 2013) or from 15 to 20% (Muhammad et al., 2016) did not affect egg production, but did improve feed conversion ratio.A recent study showed that increasing dietary CP from 18 to 24% did not affect egg production, egg weight, or feed conversion ratio in Japanese quails (Agboola et al., 2016).On the other hand, Azghadi et al. (2014) found   ,b,c Means in the same column and treatment with no common superscript differ significantly (p<0.05).

Effects of Dietary Protein Level and Betaine Supplementation on Nutrient Digestibility and Performance of Japanese Quails
that increasing dietary CP from 18 to 22% increased egg production, but did not affect feed intake, egg weight, or feed conversion ratio.In addition, Garcia et al. (2005) observed that feeding with 18% CP resulted in the best performance of laying quails.
In the present study, dietary 18.0 and 19.5% CP resulted in higher egg weight compared with 16.5% CP (p<0.05), which may be attributed to the fact that birds fed 18.0 and 19.5% CP consumed more protein than those fed the 16.5% CP diet.It is wellestablished that protein is the main nutrient required for egg formation (Ding et al., 2016).Egg weight is highly correlated with daily CP intake because laying hens are not able to store large amounts of protein (Li et al., 2011).Moreover, feed intake and CP levels are important in controlling protein intake (Soares et al., 2003).The discrepancies in the results obtained by different studies may be due to differences in experimental conditions and diets.
The observed increase (p<0.01) in egg mass in birds fed 18.0 and 19.5% CP compared with those fed a 16.5% CP diet may be due to higher egg weight, and not to higher egg production, which was not significantly influenced by dietary CP level.
Consequently, the diets containing 18.0 and 19.5% CP were more effective in improving egg mass production.In addition, the egg mass obtained with the 18.0% CP diet was similar to that observed with the 19.5% CP diet.According to Rashid et al. (2012), feeding poultry reared in hot climates with high protein levels is not recommended because of the high heat increment resulting from protein digestion.The egg mass results of the present study are in line with those of Azghadi et al. (2014), who did not observe any increase in egg mass of Japanese quails when dietary CP levels were increased from 18 to 22%.In laying chickens, previous observations indicated that increasing dietary CP from 14 to 16% increased egg mass (Park & Ryu, 2011).
The reduction (p<0.05) of PER with increasing dietary CP levels may be attributed to higher CP intake (Dos Santos et al. 2016), as high-protein diets increase protein excretion, resulting in lower PER.This result is in agreement with previous findings (Li et al., 2011;Ratriyanto et al., 2014b;Dos Santos et al., 2016).
The 19.5% CP diet increased EER compared with the 16.5% CP (p<0.05).Because the birds were fed isocaloric diets, this result is explained by the higher egg mass produced by the birds fed 19.5% CP.The dietary supplementation with 0.06 and 0.12% betaine improved the production performance of quails (p<0.05), which is in accordance with previous observations in laying chickens (Lu & Zou, 2006;Park et al., 2006;Ezzat et al., 2011;Gudev et al., 2011).Moreover, except for feed intake and egg production, the dietary supplementation with 0.12% betaine promoted better responses compared with 0.06% betaine (p<0.05).Compared with the diet with no betaine, the higher feed intakes observed when birds were fed 0.06 and 0.12% betaine (p<0.01) were correlated with 10.45 and 15.17% higher egg production, and 4.23 and 6.46% higher egg weights, respectively.
The performance improvement obtained with the dietary supplementation of betaine has been previously described and may be attributed to its osmotic properties and its role as a methyl group donor (Metzler-Zebeli et al., 2009;Ratriyanto et al., 2009a;Ezzat et al., 2011).Kettunen et al. (2001) observed that betaine increased the length of the small intestine and reduced the crypt:villi ratio of broilers, indicating a larger surface area for nutrient absorption, resulting in better live performance.Moreover, betaine is involved in the synthesis of metabolically-active substances and in protein and energy metabolism (Metzler-Zebeli et al., 2009).This was confirmed by the enhanced PER and EER in this study, indicating better protein and energy utilization.In addition, betaine stimulates the anterior pituitary to secrete follicle-stimulating hormone and luteinizing hormone.These hormones promote follicle growth and ovulation, increasing egg production (Zou & Feng, 2002;Xing & Jiang, 2012).Previous observations indicated that betaine supplementation in the diets of laying hens increased egg production (Lu & Zou, 2006;Ezzat et al., 2011;Gudev et al., 2011) and egg weight (Park et al., 2006), and improved feed conversion ratio (Ezzat et al., 2011).In contrast, Harms & Russel (2002) did not find any effect of betaine on the performance of laying hens that were fed a diet containing adequate choline and methionine as additional sources of methyl group donors.In addition, Park & Ryu (2011) showed that 0.06% betaine supplementation did not affect the performance of laying hens.

Egg Quality
The interaction between protein levels and betaine supplementation influenced yolk weight (Table 4), but not the other evaluated egg-quality parameters.For the three CP levels, betaine supplementation increased yolk weight (p<0.01).Regardless of CP level, this finding indicates that betaine improved the synthesis of yolk precursors, such as very-low-density lipoproteins and vitellogenin, as described previously by Lu & Zou (2006).
The diets with 18.0 and 19.5% CP increased yolk and eggshell weight (p<0.01),compared with the 16.5% CP diet, and these results may be attributed to higher egg weight in those groups.Akbar et al. (1983) also observed that higher dietary CP levels increased yolk percentage in laying hens.Previous studies showed that increasing dietary CP from 16 to 20% did not affect albumen, yolk, or eggshell weight (Garcia et al., 2005;Abdel-Azeem, 2011), neither eggshell thickness in quails (Abdel-Azeem, 2011).In addition, Agboola et al. (2016) reported that increasing dietary CP from 18 to 24% did not affect yolk or albumen weight or eggshell thickness in quails.A similar result was obtained by Muhammad et al. (2016), who observed that dietary CP did not affect yolk or albumen weight in quails.
The observed increase (p<0.01) in the weight of egg components, such as the yolk, albumen, and eggshell with betaine supplementation was correlated with higher egg weight.These results may be attributed to the methyl donor function of betaine, which is involved in protein and energy metabolism (Ratriyanto et al., 2009a).The higher albumen weight when betaine was supplemented suggests an increase in protein synthesis, was in agreement with previous studies showing that betaine supplementation improved protein synthesis (Apicella et al., 2013), which increased albumen weight (Joseph et al., 2000).Moreover, the 4.26 and 5.18% increases in eggshell weight determined with the supplementation of 0.06 and 0.12% betaine, respectively, may be due the higher availability of minerals for eggshell formation, which is related to improved CA digestibility.However, eggshell thickness was not affected by betaine supplementation in this study, which was in line with previous observations in laying hens (Park & Ryu, 2011) and ducks (Awad et al., 2014).

CONCLUSIONS
Dietary protein levels had minor effects on nutrient digestibility coefficients.The diets containing 18.0 and 19.5% crude protein promoted similar production performance, which was better than that obtained with the 16.5% crude protein diet, independently of dietary betaine level.The dietary supplementation of 0.06 and 0.12% betaine improved nutrient

Table 1 -
Ingredient composition (%) and calculated nutrient content of the basal diets.

Table 2 -
Nutrient digestibility in Japanese quails (%) fed diets with varying levels of crude protein and betaine.

Table 3 -
Production performance of Japanese quails fed diets with varying levels of crude protein and betaine.

Nuhriawangsa AMP Effects of Dietary Protein Level and Betaine Supplementation on Nutrient Digestibility and Performance of Japanese Quails
Means in the same column and treatment with no common superscript differ significantly (p<0.05). a,b,c

Table 4 -
Egg quality of Japanese quails fed diets with varying levels of crude protein and betaine.