Growth performance, nutrients digestibility, carcass characteristics, hematology and serum biochemistry of Japanese quail (Coturnix coturnix japonica) fed diets containing rice gluten meal and betaine supplementation during summer season

Abstract

A total of 375 un-sexed Japanese quail (Coturnix coturnix japonica) chicks, 5 days-old were randomly allotted to 5 experimental groups to examine the effect of the inclusion of rice gluten meal (RGM) at levels of 2.5 and 5% and supplementation of betaine at levels of 0.5 and 1.0 g/kg diet on the growth performance, carcass characteristics, hematology, serum biochemistry and gastrointestinal tract microbiota of growing Japanese quail under summer season. Quails were fed a basal diet and the other four diets contained 2.5 and 5% RGM with 0.5 or 1.0 g betaine supplementation. The results revealed quails fed 5%RGM + 0.5 or 1.0 g betaine achieved the highest (P < 0.05) body weight at 5 weeks of age, quails fed 5%RGM + 0.5 or 1.0 g betaine recorded the highest (P < 0.05) body weight gain at 1–5 weeks of age. Quails fed 5%RGM + 1.0 g betaine improved (P < 0.05) DFI at all periods. 5%RGM + 0.5 or 1.0 g betaine groups recorded the best FCR at 1–5 weeks of age. RGM and betaine improved CP and EE digestibilities. RGM and betaine was lower (P < 0.05) in abdominal fat. Quails fed 5%RGM + 0.5 or 1.0 g betaine increased (P < 0.05) WBC count. HB level of the quails group fed diet contained 5%RGM + 0.5 or 1.0 g betaine was higher (P < 0.05). Betaine supplementation increased (P < 0.05) the lactic acid bacteria count and decreased (P < 0.05) E-coli and coliform counts. In conclusion, the combination of RGM and betaine could maintain the growth performance, intestinal health and improve nutrients digestibility of Japanese quails during summer season.

Introduction

The Japanese quail (Coturnix coturnix japonica) breeding industry has exploded in recent years. Quail production is a new branch of the poultry industry that adds variety to poultry meat (Sabow, 2020). The meat of quail has high nutritional value and adds variety to the human diet, which is highly valued by consumers (Quaresma et al. 2022). Moreover, quail meat is low in lipids, making it a more cost-effective source of animal protein (Fakolade 2015). The main obstacle to the long-term viability of intensive quail production is the high cost of feed, which accounts for about 70% of overall production expenses (Mnisi et al. 2021). Indeed, Mnisi and Mlambo (2018) stated that the effectiveness of protein feedstuffs for poultry is determined by its ability to provide the bird with an adequate amount of essential amino acids. Dietary amino acids are used to build protein for muscle growth, membrane glycoproteins, and enzymes are involved in a variety of biochemical processes, as well as act as DNA or RNA precursors. Amino acids are essential precursors for hormone synthesis as well as other nitrogenous elements with significant biological significance (Qaid and Al-Garadi, 2021). Soybean meal is a high-quality source of protein for poultry. Alternative sources of dietary protein will help to reduce the reliance of the industry on soybean meal, also they can help to reduce the feed costs in quail production (Mnisi and Mlambo, 2018). Rice gluten meal (RGM) is a by-product of rice wet milling which is produced during starch extraction and syrup preparation. It is a new feedstuff with a brownish colored and a coarse powdery texture (Dinani et al. 2020). Because it contains 50% CP, it has the potential to be utilized as an elective to soybean in poultry diets for economic production (Wani et al. 2018) and 3330 kcal/kg metabolizable energy (ME) (Metwally and Farhat, 2015). Some previous studies of using RGM as alternative protein source were done in broiler (Metwally and Farahat, 2015; Wani, 2018 and Dinani et al. 2020) and Cattle (Malik et al., 2017). Betaine (N, N, N-trimethylglycine) is a non-toxic amino acid derivative that is present in sugar beets, wheat, spinach, and aquatic invertebrates (Fernández et al., 2000) and also has a vital role in biological processes like osmoprotective, methionine and choline sparing, immunity and fat distribution (Graham, 2002), and has a role in maintaining the proteins' stability. A common topic of betaine research is the idea of betaine saving some methionine (Alagawany et al. 2022). Many studies have shown that betaine supplementation in Japanese quail diets improves health status and productivity (Ratriyanto et al. 2017; Ratriyanto and Prastowo, 2019; Arif et al. 2021). Betaine is a methyl group donor and organic osmolyte that improves quail performance in tropical environments (Masykur et al. 2021). EKlund et al. (2006) reported that the methionine sparing effect of betaine might improve the efficient use of dietary protein. Betaine supplementation to low protein and low methionine diets could enhance the efficiency of protein utilization and the performance of broiler chickens (Park and Kim, 2019). A seasonal impact with high mortality rates of broiler occurring in the summer months (Lara and Rostagno, 2013). High ambient temperature could negatively impact on reproductive performance, antioxidant status, immunity in breeder quails (Kalvand et al. 2022). The overall objective of the study was to examine the impact of graded levels of 2.5 and 5% RGM with 0.5 or 1% of dietary betaine supplementation on growth performance, nutrient digestibility, carcass characteristics, hematology, serum biochemistry, gastrointestinal tract microbiota of Japanese quail (Coturnix coturnix japonica).

Materials And Methods

The experiment was carried out at El-Fayoum Poultry Research Station, El-Fayoum Governorate, Animal Production Research Institute (APRI), Egypt.

Birds, management and experimental design

A total of 375 five day old, unsexed Japanese quails (Coturnix coturnix japonica) chicks (5 day old) with an average initial body weight at hatch was 16.18 ±0.04 g and randomly divided into five groups. Each group was divided into 3 replicates with 25 unsexed chicks each. The five diets were formulated to meet Japanese quail's requirements according to NRC (1994). Quails were fed a basal diet (without RGM and betaine addition) (control) and the other four diets contained 2.5 and 5% RGM with 0.5 and 1.0% betaine supplementation. Rice gluten meal (RGM) was obtained from TIBA Starch & Glucose Manufacturing Company, located in El-Sharkia Governorate, Egypt. Betaine was provided as Betafin S4 (betaine anhydrous minimum 93% feed grade, manufactured by Finnfeeds Finland Oy), manufactured for Danisco Animal Nutrition. Composition and calculated analysis of the basal diet are presented in Table 1. The birds were housed in a conventional type cage (50 × 30 × 50 cm³) with feed and fresh water provided ad libitum throughout the experimental period. Birds were maintained in 24h light period throughout the trial. All chicks were kept under the same managerial, hygienic, and environmental conditions. Body weight (BW) and feed intake (FI) of each group were recorded weekly, and then body weight gain (BWG) and feed conversion ratio (FCR) were computed. Dead quails were recorded daily and were expressed as mortality (%). The trial lasted for 5 weeks. During the experimental period (June−August 2021), in the quail farm the minimum and maximum temperatures, the relative humidity and the temperature humidity index ranged 25.5– 33.5°C, 62–75% and 87.5–93.5, respectively.

Digestibility trail

Six 5-wk-old quails from each treatment were used in digestibility trails to evaluate the nutrients digestibility of the experimental diets. Birds were housed individually in metabolic cages and fed the tested diets. The chicks were allowed to acclimate for 3 d, then the daily feed intake was recorded and feces were collected every 24 h for 5 d and excreta output was collected daily, oven dried at 70°C for 36 hrs, weighed and ground). The representative samples were used for analysis. The Diet and feces analyses were carried out directly according to AOAC (2007).

Slaughter procedure and carcass parameters

At the end of the experimental period (35 days of age), three fasted birds from each group were randomly taken for slaughter, fasted for 12 hours, individually weighed and slaughtered to complete bleeding then liver, heart, gizzard, intestine, stomach, breast, thigh, abdominal fat were separated then weighed and their relative weights were calculated as a percentage of live body weight. Intestine and cecum length were determined. Carcasses were manually eviscerated and weighed.

Biochemical Analysis

Blood samples were collected during slaughtering using three quail chicks from each group and divided into two parts. The first part was collected in heparin tubes while the second part was collected in non-heparin tubes to obtain serum. Fresh blood aliquots were used to determine hematological parameters like white blood cells (WBCs), red blood cells (RBCs), hemoglobin (HB), lymphocytes, neutrophils (NEO), eosinophils (EO), monocytes (Mono), HCT %, MCV %, MCH % and MCHC %. Serum was separated by centrifugation at 5,000 rpm (3,354 g force) for 15 min and stored at −20°C until biochemical analysis. Plasma total protein, albumin, urea, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glucose were colorimetrically determined using commercial kits (purchased from Bio-Diagnostic, Cairo, Egypt), according to the manufacturers’ guidelines. Plasma globulin concentration was calculated by the difference between plasma total protein and albumin, and then the albumin/globulin ratio was calculated.

Microbiological determination

The contents of the gastrointestinal tract of each bird were collected and stored in the freezer at −4°C. The cultures were prepared. The ileal contents from the gut were serially diluted. From the prepared intestinal content, 0.1 ml was inoculated in suitable medium and then incubated in aerobic and anaerobic conditions. Each sample was serially diluted from 10^-1 to 10^-6. Dilutions were subsequently plated on duplicate selective agar media for enumeration of selective bacteria (Lactobacillus bacteria, E-coli and coliform). The plate media used were MRS agar for Lactobacillus spp., Mackonkey for E-coli and coliform. The plates were then incubated at 37°C for 48 to 72h aerobically and colonies were counted. Results were expressed as log₁₀ colony-forming units per gram of ileum digesta (log₁₀ CFU/g) (Hartemink and Rombouts, 1999). Total coliform counts of intestinal content were carried out according to the American Public Health Association (A.P.H.A, 1985).

Statistical Analysis

The experiment was conducted in a completely randomized design. All data were analyzed with one-way ANOVA using General Linear Model procedure of Statistical Analysis System (SAS, 2009). Significant differences among the means were determined by Duncan’s multiple range test (Duncan, 1955) at the significance level (p < 0.05). According to the statistical model:

Yij = m + Ti + eij where Yij = the observation, m = the overall mean, Ti = the treatment effect and eij = random error.

Results

Growth performance

As shown in Table 2, Japanese quails chicks fed diets contained 2.5% RGM+ 1.0 g betaine and 5% RGM+0.5 g betaine recorded higher (P<0.05) LBW compared to the other experimental groups and the control group at 3 weeks of age. While, group of quails chicks fed diets included 5%RGM +0.5 or 1.0 g betaine achieved the highest (P<0.05) LBW among all the experimental groups and the control group at 5 weeks of age. During the period of 1-3 weeks of age, DBWG was the greatest (P<0.05) with quails groups fed diet contained 2.5% RGM+1.0 g betaine and 5% RGM +0.5 g betaine compared to all the experimental groups and the control group. Moreover, at the whole period of 1-5 weeks of age, the quails group fed diet contained 5%RGM+0.5 or 1.0 g betaine recorded the highest (P<0.05) DBWG compared to all the experimental groups and the control group. Concerning DFI, the quails group fed diet contained 5%RGM+1.0 g betaine improved (P<0.05) DFI at 1-3, 3-5 and 1-5 weeks of age compared to the other experimental groups. However, there was no significant difference in DFI between the experimental groups and the control group at 3-5 weeks of age. In the same time, the quails group fed diet contained 5% RGM+ 0.5 g betaine recorded the best (P<0.05) FCR among all the experimental groups at 1-3. While the quails group fed diet included 5%RGM+1.0 betaine was the best (P<0.05) in FCR at 3-5 weeks of age. also, 5%RGM+0.5 or 1.0 g betaine groups recorded the best FCR at 1-5 weeks of age compared to the control group and the other experimental groups.

Nutrients digestibility

The effect of the inclusion of two different levels of RGM and betaine on nutrients digestibility was listed in Table 3. Improved (P<0.05) digestibility of CP and EE were observed in all treatment groups compared to the control group. Quails fed diets contained 2.5%RGM+1.0 g betaine, 5%RGM+0.5g betaine and 5% RGM+1.0 g betaine recorded higher (P<0.05) CF digestibility compared to the control group. Digestibility of DM and OM were negatively correlated with 5% RGM+1.0 g betaine. Therefore, the quail group fed diet included 5%RGM+1.0 g betaine was lower (P<0.05) in digestibility of DM and OM than the control group. At the same time, the quails group fed the control diet was the highest (P<0.05) digestibility of NFE compared to the other treatment groups.

Carcass characteristics

The results presented in Table 4. showed insignificant changes in most measured carcass traits except cecum length (cm), abdominal fat and test weight (g). Meanwhile, the quails group fed diet contained 2.5%RGM+0.5g betaine was higher (P<0.05) in carcass weight (g) than the quails group fed diet contained 2.5%RGM+1.0g betaine. Furthermore, the quails group fed diet included 2.5% RGM+ 0.5g betaine was higher (P<0.05) in cecum length (cm) than quails group fed diet contained 2.5%RGM+1.0 g betaine, 5%RGM+1.0 betaine and the control diet. Otherwise, the inclusion of RGM and betaine tended to lower (P<0.05) abdominal fat compared to the control group.

Serum Biochemical Parameters

The results of serum biochemistry are in Table 5. revealed that the inclusion of RGM and betaine had no significant effect on total protein, albumin, creatinine and AST concentrations. While, quails group fed diets contained 5%RGM+0.5 g betaine and 5% RGM+1.0 g betaine increased (P<0.05) globulin level compared to the control group. Moreover, quails group fed diet included 5% RGM+1.0 g betaine was significantly lower (P<0.05) in A/G ratio than the control group. However, the quails group fed diet included 2.5% RGM+1.0 g betaine and 5% RGM+0.5 g betaine tended to increase (P<0.05) urea levels compared to the control group. Regarding the liver functions, the activity of ALT was higher (P<0.05) with the group of quails fed diet contained 5% RGM +0.5 g betaine compared to the other treatment group and the control group. furthermore, the inclusion of RGM and betaine supplementation gradually increased (P<0.05) the glucose levels compared to the control group.

Hematological profile

From data in Table 6, the quails group fed diet contained 5%RGM+ 0.5 g betaine and 5%RGM+ 1.0 g betaine significantly increased (P<0.05) WBC count compared to the group of quails fed diet included 2.5% RGM+1.0 betaine. As well, HB level of the quails group fed diet contained 5%RGM+ 0.5 g betaine and 5%RGM+ 1.0 g betaine tended to be higher (P<0.05) than the control group. Additionally, no significant differences were observed between the control group and the other treatments in RBC, lymphocyte, NEO and HCT counts. Meanwhile, the quails group fed diet contained 5%RGM+1.0 g betaine was the highest (P<0.05) in EO and MCV counts compared to all treatment groups. With the Mono counts of quails group fed diets of 5%RGM+ 0.5 g betaine and 5% RGM+1.0 g betaine recorded higher (P<0.05) than the quails group fed 2.5% RGM+1.0 g betaine diet. Counts of MCH and MCHC of the quails group fed diet of 5%RGM+1.0 g betaine were higher (P<0.05) than that recorded by the other treatment groups and the control group.

Microbiological determination

Regarding the gut microflora activity in Table 7, the combination of RGM and betaine affected positively the microbial counts of the quails gut. It is clear to observe that the inclusion of RGM and dietary supplementation of betaine gradually increased (P<0.05) the lactic acid bacteria count and also decreased (P<0.05) the E-coli and coliform counts compared to the control group.

Discussion

Growth performance

Betaine is one of the promising feed additives to improve performance of Japanese quail (Arif et al. 2021; Abuoghaba et al. 2021). Because betaine is a donor of methyl group and organic osmolyte that improves quail performance in tropical environments (Masykur et al. 2021). In the poultry feed industry, RGM can replace corn and corn byproducts as an alternate protein source (Sherazi et al. 1995; Sittiya et al., 2011, Metwally and Farahat, 2015; Wani, 2018). Our observations revealed that the combination of two graded levels of RGM and the dietary supplementation of betaine improved LBW especially the mix of 5%RGM+1.0 g betaine achieved the highest (P<0.05) LBW at 5 weeks of age. Similar findings have also been described by Metwally and Farahat (2015) who found that the inclusion of RGM up to 12.5% did not significantly affect BW, BWG, FI and FCR for 42 days. Furthermore, Agena et al. (2019) stated that broilers fed diets included RGM at levels of 3, 6, 9 and 12% for 6 weeks and observed that body weight, daily weight gain and feed intake did not significantly change during the whole experimental period. The high nutritional value of RGM, such as its high amount of crude protein (57.6%) should be taken into account and good amino acid composition has a lower level of crude fiber (1.45%) and a moderate level of ether extract (3.16%) the percentage of total amino acid relative to crude protein in rice gluten meal was found to be higher for most of the essential amino acids (lysine, methionine, arginine, valine, histidine), and nearly similar for isoleucine and threonine (Metwally and Farahat, 2015). So it could be an effective alternative unconventional feed ingredient in the broiler's diet (Agena et al. 2019). Regarding, Betaine supplementation, current results showed that 1.0 g betaine/kg diet improved the DWG, DFI and FCR of quails diets. This effect has been attributed to betaine has benefits for the broiler industry, involving enhanced intestinal morphology as a feed additive (Lui et al. 2019; Dos Santos et al. 2019), otherwise, it protects cellular water homeostasis by acting as an osmoprotectant and as a methyl group donor in the methioninehomocysteine cycle (Pillai et al. 2006). Such results agreed with the findings obtained by Arif et al. (2021) who reported that dietary supplementation at levels of 0.75, 1.5 and 2.25 g/kg in quails diets showed a higher significant impact on weight gain and FCR when compared to the control diet. As well, Attia et al. (2009) reported that betaine supplementation at levels of 0.5 or 1.0 g/kg improved the BWG and FI whereas it decreased the FCR of slow-growing white-feathered broilers under heat stress. In addition, supplementation of graded levels of betaine improved the BWG and FI (p<0.05) of broilers under heat stress (Liu et al. 2019). In contrast, supplementation of betaine at levels of 0.05% and 0.075% had no significant effect on feed intake, body weight gain and FCR of broiler chickens (Sakomura et al., 2013). In the current study, the results indicated that growth performance improved in parallel with the mixture between the inclusion of RGM and betaine supplementation.

Nutrients digestibility

Determining the nutrient digestibility of quail diets is essential to assess the effectiveness of diets in optimizing quail performance without compromising quail health. However, the inclusion of RGM and betaine improved the digestibility of CP and EE in all experimental diets. 5%RGM plus 0.5 or 1.0 g betaine has higher CF digestibility compared to the control group. Additionally, the digestibility of DM did not differ significantly with 2.5 or 5% RGM. These findings are in consensus with Wani et al. (2018) who observed insignificant differences between DM digestibility of broiler due to the dietary levels of RGM (50, 75, 100, 125, and 150 g/kg). This confirms that including RGM at levels of 2.5 and 5.0 % in quail diets may be necessary to ensure adequate digestible nutrients. These are confirmed by Amagliani et al. (2019) who demonstrated that rice protein meal has excellent physicochemical properties which make it as functional and value-added ingredient. As well, it contains favorably good amino acids profile with relatively higher methionine (Malik et al. 2017). The improvement of CP digestibility is related to the methionine sparing effect of betaine may improve the effective use of dietary protein Eklund et al. (2005). With betaine addition, these results are in line with reports that betaine supplementation at levels of 0.5 or 1.0 g/kg recouped the CP digestibility coefficients from the negative effects of heat stress on slow-growing chicks (Attia et al. 2009). In this respect, Ratriyanto et al. (2017) found that the supplementation of 0.06 and 0.12% betaine in Japanese quails enhanced DM, CP, CF and EE digestibilities. This enhancement in nutrients digestibility may be due to the increase of nutrients availability in the gastrointestinal tract leading to better performance. Dietary betaine supplementation increased the activities of amylase and trypsin of
jejunal digesta and improved absorption of nutrients, which might contribute to the improvement of nutrient digestibility in broiler so improved the growth performance (Song et al. 2021). According to Wang et al. (2018), betaine administration raised the small intestine's enzymes activities like amylase, lipase, trypsin, and chymotrypsin activity in stressed rats. Also, Ratriyanto et al., (2014) confirmed that supplementation of betaine improved CF digestibility of broilers diets possibly due to stimulation of microbial fermentation in the digestive tract. Contrariwise, Eklund et al. (2005) revealed that supplementation of betaine increased protein, methionine, and crude fat digestibilities in broilers’ diet. On the contrary, the inclusion of rice gluten meal at graded levels of 25, 50, 75, 100, and 125 g/kg did not affect nutrients digestibility of DM, CP, EE, starch, and NDF % in the broiler. It can be deduced from the results that betaine supplementation enhanced the efficiency of protein utilization and the performance of experimental quails.

Carcass characteristics

The present study showed that 2.5%RGM+1.0g betaine diet increased (P<0.05) carcass weight (g) of quails, Also, the abdominal fat was reduced (P<0.05) in response to the inclusion of RGM and betaine compared to the control group. These findings are in consensus with Liu et al. (2019) who pointed out that betaine supplementation supplemented 500, 1000, 2000 mg/kg betaine improved (linear, p < 0.05) carcass and breast weight of heat stressed broilers. In this direction, Arif et al. (2021) observed that the carcass weight and breast yield were highest (p<0.05) in the quails group fed diet supplemented with betaine at the rates of 2.25 g/kg diet. While, intestinal length and weight were significantly higher. This effect might be given by that betaine could enhance intestinal structural integrity of broilers which, in turn, improved the broilers growth performance (Eklund et al. 2005). Also, fat weight was lower in the same group than the other untreated group. Concerning to RGM effect, Agena et al. (2019) stated that feeding broiler on the RGM did not influence on carcass dressing and relative organ weights were detected among dietary treatments. Conversely, Filgueira et al. (2014) demonstrated that the inclusion of broken rice in the quail's diets at levels of 20, 40, 60, 80 and 100% in replacement of corn did not influence dressing percentage or breast, liver, and gizzard yields. In the current study, better carcass weight in 2.5% RGM diets may be associated with type and composition of amino acids particularly methionine present in RGM. Accordingly, the actual metabolizable energy and digestible amino acid content of RGM should be considered in order to prevent excessive fat accumulation. Furthermore, betaine is lipotropic that has been shown to reduce fat deposition in the liver (Craig, 2004).

Serum Biochemical Parameters

Our results showed that the inclusion of RGM and dietary betaine supplementation did not impact the investigated blood biochemicals,
except globulin , urea, ALT levels and A/G ratio. These previous observations were supported by Agena et al. (2019) who reported that no significant change in the serum glucose, total protein, albumin, globulin, ALT, AST, creatinine and urea in dietary groups fed on diets contained RGM. Also, Metwally and Farahat (2015) observed that the serum biochemical parameters of broiler chickens at 6 weeks of age didn't differ significantly after feeding graded levels of RGM. It is clear to note that the inclusion of 5%RGM with 0.5 and1.0 g betaine supplementation increased (P<0.05) globulin level compared to the control group. These were confirmed by Brigotti et al. (2003) who demonstrated that betaine addition to rabbit improved mRNA translation of globin. As well, the increase in globulin due to betaine supplementation may be related to its ability as a methyl group donor, which is frequently used in the metabolism of proteins (Nutautaite et al., 2020). So the betaine supplementation improved the immunity. In the current study, the incorporation of 2.5 or 5% RGM with 0.5 or1.0 g betaine tended to increase (P<0.05) urea level compared to the control group. Interestingly, the elevated serum urea levels may be partially attributed to the efficient use of dietary protein related to the protein content of RGM (Wani, 2017). Moreover, betaine is a methyl group donor (Masykur et al. 2021). Betaine's ability to save methionine has been suggested by EKlund et al. (2006) to improve the use of dietary protein. The increased levels of urea are associated with AA degradation occurred in the feed (Al-Sagan et al. 2021). Meanwhile, the increased levels of glucose are directly correlated with the betaine supplementation. These results are consistent with Al-Sagan et al. (2021) who found that betaine supplementation at a level of 0.15% in broilers diets increased the serum glucose level compared to the other treatments. It is well known that betaine may have transferred a methyl group to homocysteine molecule to synthesize methionine in the methionine cycle (Choline, 1999). Methionine is a glucogenic amino acid that may be used in the process of gluconeogenesis to produce glucose.

Hematological profile

Hematological parameters were affected by the inclusion of RGM and betaine supplementation. 5%RGM+ 0.5 or 1.0 g betaine significantly increased (P<0.05) WBC count and HB level. While, no significant differences were observed between the control group and the other treatments in RBC, lymphocyte, NEO and HCT counts. Similarity, Al-Sagan et al. (2021b) demonstrated that broilers fed diet supplemented with betaine at levels of 0.075%, 0.10%,
and 0.15% had no changes in heterophils, lymphocytes, and their ratio (H:L ratio). Likewise, Park and Kim (2017) stated that betaine addition in meat-type ducks diets at levels of 700, 1000 and 1300 ppm betaine improved RBCs and platelet counts. All values were considered
in the normal range for growing Japanese quails (Mihailov et al. 1999). Additionally, Egbuniwe et al. (2021) observed that 2 g betaine promoted the RBC counts but decreased MCV, and MCH in female quails. These findings are matched with the results of Hamidi et al. (2010) who suggested that betaine promoted an immune system of broiler chickens because sparing dietary methionine with betaine may affect the immune responses (Rao et al. 2011).

Microbiological determination

Diet composition can have a strong effect on the gastrointestinal tract microbiome (Borda-Molina et al. 2021). In the current findings, the incorporation of RGM and betaine had a positive effect on reducing the pathogenic bacteria such as E. coli and coliform and promoted lactic acid bacteria count as beneficial bacteria. Such results are confirmed by Uyanga et al. (2022) who postulated that betaine plays a crucial role in maintaining the gut functions during heat stress conditions. Furthermore, the beneficial impact of betaine on Lactobacillus plantarum survival (Kets and de Bont, 1994) may explain the enhancement in lactic acid bacteria count. Being widely known that Lactobacillus index is an indicator of healthy gut (Hassan et al. 2021). These results are agreed with the findings obtained by Ahmed et al. (2018) who reported that duck diet supplemented with the combination of prebiotic and betaine decreased (P<0.001) the total E.coli bacteria count and increased (P<0.05) the total lactobacillus bacteria count. From current results, the dietary supplementation of betaine may control and reduce the growth of pathogenic bacteria like E-coli and promoted the beneficial bacteria like lactobacillus in the gut of experimental Japanese quails. This helps maintain the gut health which plays an important role in maintaing of overall health and the production of broilers (Oviedo-Rondon, 2019). Additionally, broilers and quail commonly have Lactobacillus colonise their ileum. According to Crisol-Martnez et al. (2016), they are known to maintain bacterial stability, limit pathogen adherence, and enhance bird health. While, Escherichia coli is a common colonizer of the avian digestive tract (Dipineto et al. 2014), it is an enteropathogenic bacteria that can caused the disease.

Conclusion

The outcomes obtained in this experiment showed that the inclusion of RGM at levels of 2.5 and 5% with a combination dietary supplementation of betaine at levels of 0.5 and 1.0 g/kg diet could maintain the growth performance, carcass characteristics, intestinal health and hematological and biochemical blood parameters of Japanese quail chicks. An improvement in nutrients digestibility and gastrointestinal tract microbiota are the major issues in experimental quails.

Declarations

Acknowledgements; The authors thank the stuff of El-Fayoum research station for providing the research facility.

Author Contributions: Conceptualization and writing Fawzia, A.; performed the experiments, Abeer R.; investigation, Marawa.; resources, Abdlatif H.; lab analysis, Samia A. and Ibrahim H.

Data availability: The data are available upon request.

Ethics approval: The experiment was conducted following animal welfare regulations at Animal Production Research Institute, Egypt with all procedures approved by the Institutional Animal Care and Use Committee.

Consent to participate: Consent was obtained from all authors.

Consent for publication: All participants have consented to the submission of the article to the journal.

Conflict of interest: The authors declare no competing interests.

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Tables

Table (1). Composition and calculated analysis of the experimental diets (as fed basis).

¹each kg contain Vit. A, 12000.000 IU; Vit. D3, 2000.000 IU; Vit E,10g; Vit. K2, 1g; Vit. B1, 1g, Vit. B2, 4g, Vit. B6, 1. 5g; Vit. B12, 10g; Pantathenic acid, 10g; Nicotinic acid, 20g; Folic acid, 1000 mg; Biotin, 50g; Choline chloride, 500g; Copper, 10g; Iodine, 1g; Iron, 30g; Manganese, 55g; Zinc, 55g; Selenium, 0.1g.

²Calculated according the basis of the ingredients composition.

Table 2. The impact of inclusion of RGM and betaine on growth performance of Japanese quails chicks

^{a, b,c,d} Means having different superscripts within each effect in the same row are significantly different at p<0.05.

SEM = standard error mean.

2.5RGM, 5RGM stand for diets containing 2.5 and 5% Rice Gluten Meal; 0.5B, 1.0B stand for diets having 0.5 and 1.0 g/Kg betaine

Table 3. Effect of inclusion of RGM and betaine on nutrients digestibility of Japanese quails. 

^{a, b,c,d} Means having different superscripts within each effect in the same row are significantly different at p<0.05.

SEM = standard error mean.

2.5RGM, 5RGM stand for diets containing 2.5 and 5% Rice Gluten Meal; 0.5B, 1.0B stand for diets having 0.5 and 1.0 g/Kg betaine

Table 4. Effect of inclusion of RGM and betaine on carcass characteristics of Japanese quails. 

^{a, b} Means having different superscripts within each effect in the same row are significantly different at p<0.05.

SEM = standard error mean.

2.5RGM, 5RGM stand for diets containing 2.5 and 5% Rice Gluten Meal; 0.5B, 1.0B stand for diets having 0.5 and 1.0 g/Kg betaine

Table 5. Effect of inclusion of RGM and betaine on serum biochemistry of Japanese quails. 

^{a, b,c} Means having different superscripts within each effect in the same row are significantly different at p<0.05.

SEM = standard error mean.

2.5RGM, 5RGM stand for diets containing 2.5 and 5% Rice Gluten Meal; 0.5B, 1.0B stand for diets having 0.5 and 1.0 g/Kg betaine

Table 6. Effect of inclusion of RGM and betaine on hematology of Japanese quails. 

^{a, b,c,d} Means having different superscripts within each effect in the same row are significantly different at p<0.05.

SEM = standard error mean.

2.5RGM, 5RGM stand for diets containing 2.5 and 5% Rice Gluten Meal; 0.5B, 1.0B stand for diets having 0.5 and 1.0 g/Kg betaine

Table 7. Effect of inclusion of RGM and betaine on Microbiological count of the gut (Log CFU/g) of Japanese quail.