Effects of feeding male and female broiler chickens on low-protein diets fortified with different dietary glycine levels under the hot and humid tropical climate

Abstract

This study was conducted to evaluate the effect of sex and level of glycine (Gly) fortification to low-protein diets on growth performance, selected serum metabolites, carcass yield and litter characteristics in broiler chickens under tropical climate. Day-old male (n = 400) and female (n = 400) broiler chicks were assigned to five isocaloric diets (positive control, PC; negative control, NC; and more three diets obtained by fortifying NC diet with higher Gly concentrations). No significant diet × sex interaction was observed for all the parameters measured. Comapered to NC group, weight gain (WG) significantly increased (p<.0001) by feeding Gly fortified low-CP diets throughout the experiment; however, WG and feed intake (FI) of birds fed NC or NC + Gly diets were significantly lower (p<.0001) compared to PC group. During the overall period, fortification of NC diet with Gly significantly improved (p<.0001) the feed conversion ratio (FCR) compared to NC group, and resulted in FCR similar to those birds fed the PC diet. Diet effect was significant (p<.0001) for performance parameters, proportional liver and abdominal fat weights, serum triglycerides (TG) and uric acid (UA) and litter moisture and nitrogen contents; with no Gly level effect (linear and quadratic, p > .05). The sex effect was significant for performance parameters, abdominal fat, and litters’ moisture and nitrogen contents. In conclusion, it is likely that additional Gly fortification to low-CP diets under tropical climate may have a better effect on broiler chickens after the starter phase. Moreover, feeding Gly fortified low-CP diets may improve the FCR and reduce the nitrogen excretion.

Keywords:

• Glycine
• low-protein diet
• broiler chickens
• tropical climate

Introduction

It is well documented that high environmental temperature adversely affects the performance of broilers (Mujahid 2011). Birds under heat stress exhibited many behavioural (Mahmoud et al. 2015), physiological (Najafi et al. 2015) and metabolic changes (Mujahid et al. 2005; Mujahid et al. 2007; Mohamed et al. 2012). Crude protein (CP) contributes to more metabolic heat production due to its higher heat increment compared with those of fat and carbohydrates (Musharaf & Latshaw 1999). Thus, low-CP, balanced essential amino acids (AA) supplemented diets have been suggested as a nutritional mean to reduce the negative impact of heat stress (Waldroup 1982), increase protein efficiency ratio (PER) (Aletor et al. 2000) and decrease the amount of nitrogen (N) emission to the environment (Bregendahl et al. 2002). However, low-CP diets may negatively affect weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) of broiler chickens (Yang et al. 2015). Our earlier study (Awad et al. 2014a) showed that by adopting the ideal AA profile, dietary protein level during early age (1–21 days) can be reduced up to 19.2% without any adverse effect on performance of broilers under tropical climate. Additionally, we found that among the non-essential AA, only glycine (Gly) could potentially improve inferior performance in broilers fed low-CP diet during the first three weeks of age (Awad et al. 2015).

Although Gly is classically considered as a non-essential AA for animals, Graber and Baker (1973) classified Gly as semi-essential for young broiler chicks. Gly is contributing with other AA in several crucial metabolic functions such as synthesis of creatine, haem, glutathione, bile acids, nucleic acids and uric acid (UA) synthesis (Corzo et al. 2004). According to NRC (1994) young broilers (0-3 wk) need 1.25% total Gly + Ser and 1.14% during the grower period (3–6 weeks). However, it is evident that a much higher Gly + Se level is required when broilers fed low-CP, essential AA-supplemented diets (Corzo et al. 2004; Dean et al. 2006; Ospina-Rojas et al. 2013). Corzo et al. (2004) suggested a total Gly + Ser level of 1.76% for male broilers fed 18% CP from day 7 to 20. However, Dean et al. (2006) recommended a concentration of 2.32% total Gly + Ser when female broiler chicks fed 16% CP from day 0 to 17. Furthermore, Ospina-Rojas et al. (2013) recommended 2.08% total Gly + Ser in a low-CP diet (19% CP) for male broilers from 0 to 3 weeks of age. A recent study from our laboratory (Awad et al. 2015) showed that providing a level of 2.03% total Gly + Ser to a 16.2% CP diet failed to support optimal growth performance in male broiler chicks kept under tropical climate. Beside the environmental factor, two more possibilities may explain the inconsistency between our previous result and others. The first is the possibility of higher Gly + Ser requirements under tropical climate. The high environmental temperature may profoundly affect the AA requirements of broilers (Balnave 2004) through a reduction in AA digestibility (Soleimani et al. 2010). The use of male broiler chicks in our previous study (Awad et al. 2015) might be a second possibility for not achieving a standard growth performance. Male broilers are estimated to have higher AA requirements than females (Baker 2003). Moreover, by adopting a low-CP 4-phase feeding programme, Hernández et al. (2012) noted adverse effect on the performance of male broilers but not females. Thus, this study was conducted to investigate the influence of additional Gly fortification to low-CP, essential AA-supplemented diets on growth performance, carcass yield, relative weights of abdominal fat, giblets, selected serum metabolites and litter characteristics of male and female broilers under tropical climate.

Materials and methods

Birds, housing and management

The study was conducted following the guidelines of the Research Policy on Animal Ethics of the Universiti Putra Malaysia. Day-old Cobb 500 male (n = 400) and female (n = 400) broiler chicks were obtained from a local hatchery. On arrival, the chicks were weighed and randomly allocated according to sex to 50 floor pens (1.5 × 1.5 m) in a conventional open-sided house. Each pen was equipped with one tube feeder, one automated bell drinker and bedded with wood shaving as litter material. During brooding period, whenever needed pens were heated with electric pin bulbs and the sides of the house were partially covered to prevent draft. In-house temperature and relative humidity were recorded three times a day (Table 1). Birds were given ad libitum access to feed (mash form) and water. Each pen was equipped with a 60-watt scroll bulb to maintain a continuous light during the night.

Table 1. In-house environmental temperature (°C) and relative humidity (%) during the experimental period.

	8 am		2 pm		8 pm
Age (week)	Temperature	Humidity	Temperature	Humidity	Temperature	Humidity	Rained days
1	26	71	36	45	30	61	None
2	26	74	35	49	30	62	2
3	25	85	35	59	30	71	4
4	26	77	36	50	30	67	2
5	26	88	36	60	30	71	4
6	26	89	36	63	29	78	6

Treatments and diet formulation

The study consisted of 10 treatments (2 sexes ×5 diets). Each treatment (subgroup) had five replicate pens with 16 birds per pen. The ingredient and nutrient compositions of the experimental diets are shown in Tables 2 and 3. Corn and soybean meal were used as intact protein sources for diet formulation. Prior to diet formulation, representative samples of corn and soybean were taken to determine their CP and AA contents. Based on the analytical values of these ingredients five diets were formulated for two feeding periods (1 to 21 d, starter and 22 to 42 d, grower): (i) a positive control (PC) diet with 22.2% and 19.5% CP during starter and grower periods, respectively; (ii) a negative control (NC) diet with 16.2% and 13.5% CP during starter and grower periods, respectively, and fortified with Gly to meet the NRC (1994) Gly + Ser recommendations; (iii) NC + Gly(I) with 17.1% and 14.3% CP during starter and grower periods, and fortified with Gly to get 2.02% and 1.78% total Gly + Ser (positive control concentrations) during starter and grower periods, respectively; (iv) NC + Gly(II) with 17.4% and 14.5% CP during starter and grower periods, respectively, and fortified with Gly to obtain 2.22% and 1.96% total Gly + Ser levels during starter and grower periods, respectively; (v) NC + Gly(III) with 17.6% CP starter period and 14.7% CP during grower period, and fortified with Gly to achieve 2.42% and 2.13% total Gly + Ser concentrations during starter and grower periods, respectively. Diets were formulated to meet or exceed the NRC (1994) recommendations as well as meeting the ideal AA profiles that suggested by Baker (1997). To meet this ideal profile, low-CP diets were set to contain 1.14 and 0.97% digestible lysine during starter and grower periods, respectively. Formulated diets were analysed for their CP and AA contents as well (Table 3).

Table 2. Ingredient composition (as- fed basis) of the experimental starter (1–21 d) and grower (22–42 d) diets.

	Positive control		Negative control (NC)		NC + gly(I)^a		NC + gly(II)^b		NC + gly(III)^c
Ingredient, %	Starter	Grower	Starter	Grower	Starter	Grower	Starter	Grower	Starter	Grower
Corn	50.32	58.70	67.00	74.23	67.00	74.23	67.00	74.23	67.00	74.23
Soybean meal	39.02	31.68	16.31	7.52	16.31	7.52	16.31	7.52	16.31	7.52
Palm oil	6.67	6.15	4.54	4.50	4.40	4.38	4.36	4.34	4.32	4.31
Dicalcium phosphate	1.62	1.18	1.86	1.44	1.86	1.44	1.86	1.44	1.86	1.44
Limestone	1.22	1.31	1.24	1.33	1.24	1.33	1.24	1.33	1.24	1.33
Sodium chloride	0.50	0.42	0.50	0.42	0.50	0.42	0.50	0.42	0.50	0.42
Sand	0.00	0.00	4.93	6.24	4.30	5.72	4.14	5.58	3.98	5.43
Vitamin premix^d	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Mineral premix^e	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
l-Lysine.HCL	0.15	0.11	0.79	0.81	0.79	0.81	0.79	0.81	0.79	0.81
l-Arginine	0.00	0.00	0.43	0.52	0.43	0.52	0.43	0.52	0.43	0.52
dl-Methionine	0.20	0.15	0.43	0.40	0.43	0.40	0.43	0.40	0.43	0.40
l-Threonine	0.00	0.00	0.33	0.38	0.33	0.38	0.33	0.38	0.33	0.38
l-Valine	0.00	0.00	0.28	0.34	0.28	0.34	0.28	0.34	0.28	0.34
l-Isoleucine	0.00	0.00	0.28	0.36	0.28	0.36	0.28	0.36	0.28	0.36
l-Phenylalanine	0.00	0.00	0.34	0.51	0.34	0.51	0.34	0.51	0.34	0.51
l-Histidine	0.00	0.00	0.08	0.10	0.08	0.10	0.08	0.10	0.08	0.10
l-Tryptophan	0.00	0.00	0.07	0.10	0.07	0.10	0.07	0.10	0.07	0.10
l-Leucine	0.00	0.00	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15
l-Glycine	0.00	0.00	0.06	0.27	0.83	0.91	1.03	1.09	1.23	1.27
Choline chloride	0.00	0.00	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08

^a Negative control diet + supplemental glycine to obtain 2.02 and 1.78% total glycine + serine in the starter and the grower diets, respectively.

^b Negative control diet + supplemental glycine to obtain 2.22 and 1.96% total glycine + serine in the starter and the grower diets, respectively.

^c Negative control diet + supplemental glycine to obtain 2.42 and 2.14% total glycine + serine in the starter and the grower diets, respectively.

^d Supplied per kilogram of the diet: vitamin A (retinyl acetate), 8000 IU; vitamin D3 (cholecalciferol), 1000 IU; vitamin E (dl-α-tocopherol), 30 IU; vitamin K3 (menadione dimethylpyrimidinol, 2.5 mg; vitamin B1, 2 mg; vitamin B2, 5 mg; vitamin B6, 2 mg; vitamin B12, 0.01 mg; niacin, 30 mg; d-biotin, 0.045 mg; vitamin C, 50 mg; d-pantothenate, 8 mg, folic acid, 0.5 mg.

^e Supplied per kilogram of the diet: Mn, 70 mg; Fe, 35 mg; Zn, 70 mg; Cu, 8 mg; I, 1 mg, Se, 0.25 mg; Co, 0.2 mg.

Table 3. Nutrient composition (as- fed basis) of the experimental starter (1–21 d) and grower (22–42 d) diets.

	Positive control		Negative control (NC)		NC + gly(I)^a		NC + gly(II)^b		NC + gly(III)^c
Nutrient	Starter	Grower	Starter	Grower	Starter	Grower	Starter	Grower	Starter	Grower
ME, Kcal kg^−1	3130	3200	3130	3200	3131	3200	3130	3200	3130	3200
Crude protein, %	22.2 (22.3)	19.5 (19.4)	16.2 (16.2)	13.5 (13.2)	17.1 (16.8)	14.3 (14.2)	17.4 (17.0)	14.5 (14.2)	17.6 (17.3)	14.7 (14.5)
Calcium, %	0.94	0.86	0.94	0.86	0.94	0.86	0.94	0.86	0.94	0.86
Available phosphorus, %	0.45	0.35	0.45	0.35	0.45	0.35	0.45	0.35	0.45	0.35
Total amino acids^d, %
Lys	1.22 (1.22)	1.03 (1.06)	1.21 (1.18)	1.03 (0.91)	1.21 (1.20)	1.03 (0.88)	1.21 (1.12)	1.03 (0.88)	1.21 (1.11)	1.03 (0.89)
Arg	1.51 (1.39)	1.31 (1.28)	1.27 (1.23)	1.10 (1.09)	1.27 (1.27)	1.10 (1.08)	1.27 (1.27)	1.10 (1.07)	1.27 (1.26)	1.10 (1.06)
Met + Cys	0.90 (0.88)	0.79 (0.77)	0.90 (0.90)	0.78 (0.75)	0.90 (0.90)	0.78 (0.75)	0.90 (0.90)	0.78 (0.73)	0.90 (0.89)	0.78 (0.77)
Thr	0.86 (0.83)	0.76 (0.76)	0.83 (0.85)	0.74 (0.73)	0.83 (0.83)	0.74 (0.72)	0.83 (0.84)	0.74 (0.72)	0.83 (0.84)	0.74 (0.70)
Val	1.11 (0.96)	0.98 (0.91)	0.94 (0.93)	0.83 (0.83)	0.94 (0.94)	0.83 (0.80)	0.94 (0.92)	0.83 (0.80)	0.94 (0.93)	0.83 (0.79)
Ile	0.95 (0.82)	0.83 (0.76)	0.81 (0.79)	0.73 (0.72)	0.81 (0.80)	0.73 (0.69)	0.81 (0.78)	0.73 (0.69)	0.81 (0.78)	0.73 (0.71)
Phe + Tyr	1.73 (1.53)	1.51 (1.46)	1.34 (1.33)	1.22 (1.23)	1.34 (1.36)	1.22 (1.17)	1.34 (1.36)	1.22 (1.19)	1.34 (1.36)	1.22 (1.27)
His	0.61 (0.55)	0.54 (0.53)	0.45 (0.42)	0.38 (0.37)	0.45 (0.43)	0.38 (0.37)	0.45 (0.43)	0.38 (0.37)	0.45 (0.43)	0.38 (0.37)
Trp	0.26	0.22	0.20	0.18	0.20	0.18	0.20	0.18	0.20	0.18
Leu	1.82 (1.65)	1.65 (1.57)	1.35 (1.36)	1.11 (1.12)	1.35 (1.35)	1.11 (1.10)	1.35 (1.34)	1.11 (1.12)	1.35 (1.33)	1.11 (1.13)
Gly + Ser	2.02 (1.92)	1.78 (1.74)	1.25 (1.24)	1.14 (1.22)	2.02 (2.03)	1.78 (1.76)	2.22 (2.14)	1.96 (1.96)	2.42 (2.34)	2.13 (2.03)

^a Negative control diet + supplemental glycine to obtain 2.02 and 1.78% total glycine + serine in the starter and the grower diets, respectively.

^b Negative control diet + supplemental glycine to obtain 2.22 and 1.96% total glycine + serine in the starter and the grower diets, respectively.

^c Negative control diet + supplemental glycine to obtain 2.42 and 2.14% total glycine + serine in the starter and the grower diets, respectively.

^d Values in parenthesis represent analysed crude protein and amino acids.

Protein, amino acids and dry matter analysis

Crude protein (ingredients, experimental diets and litter samples) and amino acid contents (ingredients and experimental diets) were determined according to the procedure of AOAC (1990) and using high-performance liquid chromatography as described previously (Awad et al. 2014b), respectively. Litter samples were dried in a drying oven at 105 °C for 24 h to determine the dry matter following the AOAC (1990) procedure.

Performance measurements

The body weights of birds in each pen was recorded at 1, 21 and 42 d of age and average WG from 1 to 21 d and 1 to 42 d were calculated as mean body weights on day 21 – mean body weights on day 1, and mean body weights on day 42 – mean body weights on day 21, respectively. In addition, FI for the two feeding periods (starter and grower) was recorded as well, and FCR was calculated as feed/gain. Protein efficiency ratio was calculated as weight gain/protein intake. Mortality was recorded on a daily basis.

Blood collection and serum metabolites analysis

At the end of the experimental period (42 d), two birds from each pen were selected randomly and slaughtered humanely according to the halal method (Ahmed et al. 2014) and blood samples (3 mL) were collected in tubes kept in ice. Later, the blood samples were centrifuged at 4000 × g at 4 °C for 20 min. Haemolysis-free serum samples were transferred to 1.5 mL microcentrifuge tubes and stored at −20 °C until further analysis. Serum concentrations of cholesterol, triglycerides (TG), and UA were determined using an automated chemistry analyser (Hitachi 902 Automatic Analyser, Hitachi, Tokyo, Japan) with commercial test kits (Roche Diagnostics, Basel, Switzerland).

Carcass yields, abdominal fat and internal organs sampling

Prior to the halal slaughter method, body weights of selected birds (10 birds per treatment) were individually recorded. The birds were slaughtered, eviscerated and the heart, liver and abdominal fat pad samples were weighed and expressed as a percentage of live body weight. After evisceration, whole breast (without skin) and whole right leg (thigh plus drumstick) cuts were apportioned by hand and measured. The weights of carcass (without head, feathers, shankes, skin, intestines and all internal organs except the lungs and kidneys) were recorded and expressed as percentage of live body weights.

Litter sampling

Litter samples were collected on 42 d as described by Atapattu et al. (2008). Three samples from each pen were randomly taken by avoiding the area of feeder and drinker. The three samples from a pen were pooled and kept in the refrigerator for 24 h. Samples were then analysed for their moisture and N contents.

Statistical analysis

All data except mortality were subjected to ANOVA using the GLM procedure of SAS software (SAS 2005). The statistical model investigated the effect diet, sex and their interactions as the main effects. Tukey’s test was used to compare between means. Also, orthogonal polynomial contrasts were done to determine the linear and quadratic effects of increasing Gly level (excluding the positive control data). Mortality data were analysed by chi-square test. The significance is considered at p ≤ .05.

Results

There were no significant diet × sex interactions for all the parameters measured.

Diet analysis and performance

The analysed CP and AA values of our starter and grower diets were comparable to the calculated values (Table 3). Data on the effect of diet and sex on growth performance are shown in Tables 4 (starter, 1–21 d) and 5 (overall, 1–42 d). Throughout the experiment, the WG, FI, FCR and PER were significantly affected (p ≤ .001) by diet. Birds fed PC diet had consistently higher WG and FI as compared to those fed other diets (Tables 4 and 5). Fortifying NC diet with 2.02% Gly + Ser during the starter period improved FCR when compared to those fed NC. FCR of birds fed diets fortified with Gly + Ser levels higher than the NRC (1994) recommendations was similar with the PC group during the overall period. Irrespective of Gly level, feeding low-CP diets produced significantly higher PER compared to that of birds fed PC. Except for overall PER, there were neither significant linear nor quadratic relationship between level of Gly and WG, FI, FCR and PER. The main effect of sex was also significant (p < .05) for WG, FI, FCR, and PER throughout experiment. Male broilers had significantly higher WG, FI, and PER and lower FCR compared to females. Neither diet nor sex affected the mortality rate of birds (p > .05).

Table 4. Weight gain, feed intake, feed conversion ratio (FCR), protein efficiency ratio (PER), and mortality in broiler chickens as affected by the main effect of diet and sex from day 1 to 21.

Item	Weight gain, g	Feed intake, g	FCR, feed gain^−1	PER g, g^−1	Mortality, %
Diet
Positive control	846^a	1166^a	1.38^c	3.27^b	1.00
Negative control (NC)	683^c	1039^bc	1.52^a	4.06^a	1.25
NC + glycine(I)^d	742^b	1057^bc	1.43^b	4.10^a	1.00
NC + glycine(II)^e	754^b	1075^b	1.43^b	4.03^a	1.75
NC + glycine(III)^f	744^b	1050^bc	1.41^b	4.03^a	1.75
Contrast, p-values
Linear	.7822	.5851	.3006	.1080
Quadratic	.1786	.0701	.4510	.1367
Sex
Male	784^a	1112^a	1.42^b	3.93^a	1.60
Female	724^b	1042^b	1.45^a	3.87^b	1.10
SEM	21	28	0.03	0.08	ND
	Probabilities
Diet	<.0001	<.0001	<.0001	<.0001
Sex	<.0001	<.0001	.0281	.0258
Diet × Sex	.4768	.4935	.8295	.7815

^a–c Means within a column-subgroup with no common superscripts differ significantly (p < .05).

^d Negative control diet + supplemental glycine to obtain 2.02% total glycine + serine.

^e Negative control diet + supplemental glycine to obtain 2.22% total glycine + serine.

^f Negative control diet + supplemental glycine to obtain 2.42% total glycine + serine.

SEM: standard error of means; ND: not determined.

Table 5. Weight gain, feed intake, feed conversion ratio (FCR), protein efficiency ratio (PER), and mortality in broiler chickens as affected by the main effect of diet and sex from day 1 to 42.

Item	Weight gain, g	Feed intake, g	FCR, feed gain^−1	PER g, g^−1	Mortality, %
Diet
Positive control	2251^a	4073^a	1.81^b	2.72^b	7.50
Negative control (NC)	1883^c	3607^c	1.92^a	3.66^a	8.75
NC + glycine(I)^d	2035^b	3690^bc	1.81^b	3.65^a	7.00
NC + glycine(II)^e	2071^b	3750^b	1.81^b	3.60^a	6.50
NC + glycine(III)^f	2091^b	3768^b	1.80^b	3.57^a	6.25
Contrast, p-values
Linear	.0689	.1098	.5756	.0338
Quadratic	.7640	.5993	.8585	.6752
Sex
Male	2212^a	4016^a	1.82^b	3.47^a	8.00
Female	1921^b	3539^b	1.85^a	3.41^b	6.40
SEM	72	106	0.04	0.07	ND
	p-Values
Diet	<.0001	<.0001	<.0001	<.0001
Sex	<.0001	<.0001	.0245	.0070
Diet × Sex	.1063	.1621	.8022	.7546

^a–c Means within a column-subgroup with no common superscripts differ significantly (p < .05).

^d Negative control diet + supplemental glycine to obtain 2.02 and 1.78% total glycine + serine in the starter and the grower diets, respectively.

^e Negative control diet + supplemental glycine to obtain 2.22 and 1.96% total glycine + serine in the starter and the grower diets, respectively.

^f Negative control diet + supplemental glycine to obtain 2.42 and 2.14% total glycine + serine in the starter and the grower diets, respectively.

SEM: standard error of means; ND: not determined.

Carcass and relative weights of abdominal fat, heart and liver

The effects of diet and sex on carcass yields, relative weights of abdominal fat, heart and liver are presented in Table 6. Diet had no significant effect on breast, leg and carcass yields and relative weight of heart. However, feeding NC or Gly fortified NC diets resulted in significantly higher abdominal fat and relative liver weight (p < .0001). Increasing Gly level had neither linear nor quadratic relationship with carcass yields or relative weights of heart, liver, and abdominal fat. Except for abdominal fat, carcass yields and relative weights of heart and liver were not significantly affected by sex.

Table 6. Relative weight of carcass yields, internal organs, and abdominal fat as affected by the main effect of diet and sex of broiler chickens kept under tropical climate from 1 to 42 days of age.

Item	Breast, %	Leg, %	Carcass, %	Heart, %	Liver, %	Abdominal fat, %
Diet
Positive control	25.49	10.28	72.04	0.41	1.79^b	2.39^b
Negative control (NC)	24.14	10.59	71.68	0.41	2.13^a	3.02^a
NC + glycine(I)^c	25.10	10.52	72.52	0.40	2.08^a	2.86^a
NC + glycine(II)^d	25.58	10.74	72.33	0.41	2.16^a	2.86^a
NC + glycine(III)^e	24.83	10.58	72.40	0.41	2.12^a	2.87^a
Contrast, p-values
Linear	.6335	.7408	.7836	.3906	.6181	.9537
Quadratic	.2037	.2538	.7193	.6382	.3888	.9500
Sex
Male	24.85	10.61	72.47	0.42	2.10	2.54^b
Female	25.21	10.47	71.93	0.40	2.00	3.05^a
SEM	1.69	0.64	1.55	0.05	0.23	0.38
	p-Values
Diet	.0570	.2411	.4486	.8395	<.0001	<.0001
Sex	.2814	.2795	.0874	.1778	.0549	<.0001
Diet × Sex	.4980	.6715	.8414	.1595	.9380	.9835

^a,b Means within a column-subgroup with no common superscripts differ significantly (p < .05).

^c Negative control diet + supplemental glycine to obtain 2.02 and 1.78% total glycine + serine in the starter and the grower diets, respectively.

^d Negative control diet + supplemental glycine to obtain 2.22 and 1.96% total glycine + serine in the starter and the grower diets, respectively.

^e Negative control diet + supplemental glycine to obtain 2.42 and 2.14% total glycine + serine in the starter and the grower diets, respectively.

SEM: standard error of means; ND: not determined.

Serum metabolites

Data on the effect of diet and sex on serum metabolites are presented in Table 7. Diet did not affect serum cholesterol concentration. However, feeding NC or Gly fortified NC diets significantly increased serum TG concentration (p < .0001) compared to birds fed PC diet. Birds fed NC diet had the lowest (p < .0001) serum UA. Glycine fortification of NC diet to obtain a level of Gly + Ser equal to that of PC or higher resulted in a significant elevation in serum UA but the concentrations attained were still lower than the PC group. No significant linear or quadratic relationship was noted between Gly level and serum concentrations of cholesterol, TG or UA. Sex did not affect concentrations of cholesterol, TG or UA.

Table 7. Selected serum metabolites and litter moisture and nitrogen contents as affected by the main effect of diet and sex of broiler chickens kept under tropical climate from 1 to 42 days of age.

	Serum metabolites			Litter
Item	Cholesterol, mmol L^−1	Triglyceride, mmol L^−1	Uric acid, umol L^−1	Moisture, %	Nitrogen, %
Diet
Positive control	4.86	0.78^b	448^a	48.95^a	3.29^a
Negative control (NC)	3.79	1.09^a	153^c	28.35^b	2.21^b
NC + glycine(I)^d	3.98	1.12^a	328^b	29.33^b	2.36^b
NC + glycine(II)^e	4.25	1.15^a	346^b	29.58^b	2.41^b
NC + glycine(III)^f	4.27	1.14^a	336^b	28.79^b	2.44^b
Contrast, p-values
Linear	.3648	.8822	.8011	.3941	.3108
Quadratic	.6493	.8220	.6106	.3342	.8631
Sex
Male	4.41	1.01	318	34.49^a	2.65^a
Female	4.05	1.09	326	31.52^b	2.43^b
SEM	1.18	0.25	100	2.95	0.36
	p-Values
Diet	.0619	<.0001	<.0001	<.0001	<.0001
Sex	.1292	.1036	.6911	<.0001	.0038
Diet × Sex	.9937	.8349	.3857	.0857	.3384

^a–c Means within a column-subgroup with no common superscripts differ significantly (p < .05).

^d Negative control diet + supplemental glycine to obtain 2.02 and 1.78% total glycine + serine in the starter and the grower diets, respectively.

^e Negative control diet + supplemental glycine to obtain 2.22 and 1.96% total glycine + serine in the starter and the grower diets, respectively.

^f Negative control diet + supplemental glycine to obtain 2.42 and 2.14% total glycine + serine in the starter and the grower diets, respectively.

SEM: standard error of means; ND: not determined.

Litter moisture and nitrogen contents

Data on the effect of diet and sex on moisture and nitrogen contents of the litter are shown in Table 7. The moisture and N contents of the litter were significantly affected (p < .0001) by the main effect of diet. Feeding birds on Gly fortified low-CP diets markedly reduced the moisture and N contents in the litter compared to those birds fed the PC diet. No significant linear or quadratic relationship was noted between Gly level and moisture or N contents of the litter. Male broilers had significantly higher moisture (p < .0001) and nitrogen (p = .0038) compared to females.

Discussion

The present study was conducted to determine the effect of feeding low-CP diets with higher Gly + Ser Ser levels on performance, carcass yields, selected serum metabolites and litter characteristics in male and female broiler chickens kept under the hot and humid tropical climate from 1 to 42 d. Previous reports have highlighted the importance of Gly to enhance the growth performance of broiler chickens fed low-CP diets under thermoneutral condition (Corzo et al. 2005; Waldroup et al. 2005; Dean et al. 2006; Ospina-Rojas et al. 2013). Corzo et al. (2005), and Dean et al. (2006) reported equal growth performance with the addition of Gly to low-CP diets in chickens under temperate conditions. However, feeding a low-CP diet with 2.03% total Gly + Ser failed to support the growth performance to a standard level in male broilers tropical climate from 1 to 21 days of age (Awad et al. 2015). We suggested three possibilities to explain such inability to achieve optimum growth performance with feeding Gly fortified low-CP diets under tropical conditions, which are the high environmental temperature, possibility of higher Gly + Ser requirements under tropical climate and the sex effect. However, additional Gly fortification of the NC diet to obtain higher Gly + Ser levels in the present study improved, but failed to entirely restore the overall WG to equal that reported in the PC group. Nevertheless, increasing fortification of the NC diet with Gly during the starter period was eventually reduced the WG and FI when birds were fed diets with the highest Gly + Ser levels (2.44%). This suggests that failure to obtain WG equal to that of birds fed a standard diet during this period is not entirely because of the higher requirement under tropical climate. The higher WG in males is expected and can be attributed to their higher FI compared to females. However, the lack of interactions between the sex and diet indicates that the failure of Gly to support overall standard growth performance is common to both males and female broilers. Hence, the high-environmental temperature should have had a high contribution in the observed inability to obtain standard growth performance when feeding such low-CP diets under tropical climate. Birds under heat stress may lose a significant amount of water (Belay & Teeter 1993) due to the higher upper part respiratory rate (evaporative cooling) and accumulate a greater amount of UA in their blood (Kataria et al. 2008). According to Bataille et al. (2011), a decrease in the active transepithelial UA secretion in the renal proximal tubule may occur due to cellular stress. On the other hand, the activity of pathways requiring methyl group and particularly glutathione synthesis is increased under stressful condition (Wu et al. 2004; Vesco et al. 2015). These pathways are directly or indirectly dependent on glycine and its precursors (Baker 2009; Akinde 2014). Moreover, heat stress may also reduce the level of carbon dioxide in the blood, decrease potassium and elevate chloride concentrations in the body (Teeter et al. 1985), which can change the acid-base balance and affect the metabolism of the AA. Because the change in blood ions is higher in birds fed low-CP diets (Aftab et al. 2006), the high environmental temperatures may have accounted for the failure to obtain overall WG that is equal to our positive control group when fed Gly fortified low-CP diets in the present study. In the present study, the yields of breast, leg and whole carcass were not influenced by feeding the NC or NC + Gly diets. Our current findings are in agreement with previous results (Aletor et al. 2000; Namroud et al. 2008) that carcass yields were unaffected by feeding diets low in protein. This can be associated with the ideal EAA profile in our low-CP diets for protein synthesis, which can be confirmed by the higher PER in these groups. In addition, in our low-CP diets, providing Lys and total sulphur amino at levels equal to that of PC diet might partially participate in such results since Lys is mainly used for meat accretion in broiler chickens (Si et al. 2001). Feeding males or females with any of our Gly fortified low-CP diets in the present study led to the higher abdominal fat deposition. Consistent with our results, Aletor et al. (2000) and Namroud et al. (2008) reported higher fat depositions with feeding low-CP diets. This can be attributed to the higher ME: CP ratio in the low-CP diets, which in turn outcomes in an available energy excess to that required for protein deposition, and thus increases lipogenesis. Similarly, feeding Gly fortified low-CP diets increased the relative weights of the liver in this study. This could be explained by the increased lipogenesis activity when feeding such diets since the liver is the main lipogenic organ in chickens (Namroud et al. 2008). The reported higher abdominal fat in female compared to male broilers is in line with finding of Sütö et al. (1998) who found that fat deposition was significantly faster in females, because female hormones stimulate fat deposition (Rondelli et al. 2003). Feeding NC diet in this experiment decreased serum UA. Previous studies (Corzo et al. 2005; Namroud et al. 2008; Hernández et al. 2012) also reported a significant reduction in serum UA content when birds were fed low-CP diet. Since the catabolism of protein results in UA, therefore, the reported reduction in UA has been logical and can be related to the lower ingested protein and AA when feeding such low-CP diets. However, the higher fortification with Gly to our NC diet consistently increased the serum UA in both sexes. Gly is directly involved in the synthesis of UA by providing two carbons and one nitrogen atom (Mapes & Krebs 1978), which might be the reason behind the observed increase in serum UA in higher Gly levels of fortification in this study. On the contrary, feeding Gly fortified low-CP diets resulted in higher serum TG. Such results could be due to the increased lipogenic activity in the liver of birds fed on diets low in protein due to the higher energy: protein ratio (Swennen et al. 2005). Reducing emissions N to the environment is one of the reasons behind the CP reduction in broiler diets. Previously, several studies have shown that replacing CP with AA in broiler diets resulted in considerably lower excreted N (Bregendahl et al. 2002; Namroud et al. 2008). Litter N contents profoundly decreased by feeding low-CP diets, which is directly related to the lower CP level in these diets. Similarly, feeding low-CP diets in the current study, highly reduced the moisture content of the litter in both sexes. This could be attributed to the reduced water: feed consumption ratio in birds fed on low-CP diets (Alleman & Leclercq 1997). The reported higher moisture in a litter of birds fed PC diets also could be attributed to the fact that the excretion of N by-products requires additional water (Martin et al. 2005). The reported higher N and moisture in the litter of male broilers could be attributed to their higher body weights, and consequently higher feed intake, water consumption and production of excreta.

Conclusions

Although the level of Gly + Ser is critical in low-CP diets, the addition of higher levels of Gly + Ser (2.02–2.22 and 1.96–2.13% during starter and grower periods, respectively) failed to support optimal growth performance in broiler chickens raised under the tropical climate. However, Gly fortified low-CP diets improved FCR and substantially reduced the moisture and N contents of the litter.

Acknowledgements

The authors are grateful to Dr. Ahmad Mujahid for reviewing this manuscript and giving very useful comments.

Disclosure statement

The authors want to declare that they have no known conflicts of interest associated with this manuscript.

Additional information

Funding

This work was supported by the Malaysian Ministry of Higher Education under the Long-term Research Grant Scheme (No. 5526004).