Introduction

Chicken meat is a good source of protein, but it also contains vitamins and minerals such as vitamin B. It prevents cataracts and is also used to boost immunity, reduce fatigue, regulate digestion, and strengthen the nervous system. Various breeding programmes and researchers' studies on the genetics of modern broilers can account for this increase in bird body weight gain. Furthermore, high-quality feed formulation and strategies were the primary reasons for raising poultry farming throughout the process (Sadeghi and Tabiedian 2005). In previous years, broiler strains lacked the potential for rapid growth that modern strains have. Factors involved in this type of productivity in poultry farming include high dietary crude protein and amino acid concentrations in their feed (Esonu et al. 2006). The higher cost of feedstuff and excretion of nitrogen into the environment is the main concern for nutritionists facing poultry farming. Feed cost is the major hurdle in farming, it required about 65–70% of the total cost. Protein covers 15% of total feed cost (Aggrey et al. 2010).

Crude protein (CP) is a vital component of diet presents in high levels as well most costly ingredient. The amount of CP decides the nitrogen excretion by a bird in environment. Corn or sorghum in cereal grains are used for energy purpose and these ingredients have low CP. Further, high price of soybean and canola meal (high CP) limit their use in broilers diet. Recent researches on feed efficiency introduced synthetic amino acids, which may reduce protein usage in a diet regarding the feed and cost. Essential amino acids are those that body cannot synthesis itself and should take from outside sources to fulfill the body requirements (Quadros et al. 2009). Broiler’s body cannot synthesize Thr, so, it is considered as the third limiting amino acid after lysine and methionine. Addition of Thr in diet had the same effect on the bird’s growth as a diet offered with high crude protein (Abdaljaleel et al. 2019). Among other amino acids, threonine functions as the growth of birds as well used as precursors of L-lysine and serine in the body. It is also used in the synthesis of many proteins that promote gastrointestinal mucus production activity and performed its role in immune responses of broilers. Excessive or imbalanced amount of protein is used to increase the dietary requirement of threonine, which used as a precursor for glycine in uric acid formation (Zhang and Kim 2014). Broilers demand glycine or serine for proper body functioning (Kheiri and Alibeyghi 2017). Threonine metabolism is classified as amino acid metabolism, and it consists of several steps, including protein synthesis and degradation, nitrogen excretion in the form of uric acid in an amino acid corporation and amino acid conversion into fat, energy, glucose, protein, CO₂ and water. It is also involved in the formation of non-protein derivatives (Baker et al. 2002). Protein synthesis required the addition of limiting amino acid Thr so alternatively; Thr catabolism covers and participates in many other metabolism processes like (glycine, acetyl CO enzyme –A and pyruvate). Use of limiting amino acids in low protein broilers diet may improve the growth performance and biochemical Parameters. Therefore, the purpose of this research was to evaluate the effects of super dosing of threonine in low protein diet on the growth performance (body weight gain, feed intake, feed conversion ratio, carcass yield), biochemical parameters (total cholesterol, total protein, blood glucose triglycerides, VLDL, HDL and LDL) and for liver enzyme activity (ALT, AST and ALP) and economics efficiency.

Materials and Methods

Trial location

The experimental trial was conducted at R&D house at Institute of Animal and Dairy Sciences, University of Agriculture, Faisalabad. The research trial continued for 35 days.

Shed management

The brooder was started in the house 24 h before the arrival of chicks to make sure that the temperature in the brooding area was uniform. Immediately after arrival, the chicks were examined for their physical health and were put in the brooding area. The birds were reared in standard management conditions like floor space, light, ventilation, temperature and humidity. On the daily basis, all drinkers and feeders were washed to avoid contamination and fungus. A matte with limestone powder was used at the entrance of the shed throughout the research trial.

Vaccination schedule

All the experimental birds were treated with vaccination against Newcastle disease + infectious bronchitis at day 3, infectious bursal disease at day 13 and 20 and Newcastle disease at day 25.

Experimental birds

One hundred and fifty-day-old broiler chicks (Hubbard’s) were divided into three treatments including T₁ (control group), T₂ (CP 1% less and Thr 10% extra) and T₃ (CP 2% less and Thr 20% extra) (Table 1). Iso-caloric diets were formulated according to two phases as a starter (1–21 days) and finisher (22–35 days) (Table 2 and 3). Treatments were replicated into 5 subgroups having 10 chicks in each. After the arrival of chicks were facilitated with sugar solution (1 kg sugar/5L) for flushing purpose. On day 1^st temperature was kept at 95°F and further reduced at 5°F within every week until the 75^oF. Feed and water were provided ad labium 24 h.

Data recording

Growth performance: On arrival, broiler chickens were weighed using a digital weighing balance which uses as an initial weight for the starter phase. On day 22^nd, all birds within a pen were weighed again, which use as an initial weight for finisher phase. Body weight of broilers was recorded at the end of each consequent week. Weekly BWG was calculated from the data on body weight of birds. Data were recorded on feed intake and BWG for the determination of birds’ efficiency of each replicate on weekly basis. It was calculated as follows:

Feed Intake (g/bird) = [Feed offered (g)-Feed Refusal (g)]/no. of birds

“FCR = Feed Intake (g)/Weight gain (g)”

Carcass characteristics: At the end of the experiment, two birds/pen were randomly selected. Birds were weighed individually and processed to get data on carcass response. After processing feathers detached, evisceration was done in order to obtain carcass weight including internal organs (liver, heart, spleen and gizzard), breast meat weight and thigh meat weight and abdominal fat of the birds were calculated through this relationship:

“Dressing percentage = Carcass weight (g)/Live weight (g) × 100”

“Breast meat yield = Breast meat weight (g)/Live weight (g) × 100”

“Thigh meat yield = Thigh meat weight (g)/Live weight (g) × 100”

“Relative Organs weight = Organ Weight (g)/Live weight (g)× 100”

Biochemical parameters: At the end of the experimental trail, two birds/pen was selected for blood sampling. Blood sampling was done from selected broiler chickens for evaluation of blood glucose and total protein, liver enzyme activity (ALT, AST and ALP) and serum biochemistry parameters (triglycerides, cholesterol, HDL, LDL and VLDL) (Shoaib et al. 2021).

Economics

Cost of production per live weight was recorded on the basis of feed cost and live bird weight.

Table 1: Experimental treatments

Treatments	Starter Phase (1-21 days)	Finisher Phase (22-35 days)
T1	CP 21.5% Thr normal	CP 20% Thr normal
T2	CP 1% less (20.5%) + Thr 10%	CP 1% less (19%) + Thr 10%
T3	CP 2% less (19.5%) + Thr 20%	CP 2% less (18%) + Thr 20%

Table 2: Ingredients composition of experimental diets

Ingredients	Starter Diet			Finisher Diet
Ingredients	T₁	T₂	T₃		T₁	T₂	T₃
Maize grain	53.00	53.65	55.10		56.15	56.07	56.01
Rice tips	2.00	3.00	3.00		6.10	6.85	8.70
Rice polishing	2.00	2.00	2.00		2.00	2.00	2.00
Soybean meal 44%	27.60	27.60	27.60		23.20	23.20	23.00
Fish Meal	4.00	2.50	1.00		2.40	1.20	0.55
Poultry by-product meal	2.50	1.70	0.90		3.00	1.90	0.55
Canola meal	0.91	0.91	0.91		0	0	0
Molasses	2.00	2.00	2.50		1.50	2.50	2.50
Oil	3.00	3.00	3.00		3.00	3.00	3.00
Lime stone	0.50	0.50	0.50		0.50	0.50	0.50
DCP	1.50	1.90	2.00		1.40	1.80	1.90
L-Lysine sulphate, 55%	0.55	0.67	0.79		0.38	0.50	0.61
DL-Methionine, 99%	0.20	0.22	0.24		0.13	0.15	0.17
L-Threonine, 98%	0.14	0.25	0.36		0.14	0.23	0.41
Nutrimin^*	0.05	0.05	0.05		0.05	0.05	0.05
Vitalink^**	0.05	0.05	0.05		0.05	0.05	0.05
Total	100.00	100.00	100.00		100.00	100.00	100.00

“Each kg of Vitalin* supplied: vitamin A 20000 KIU; vitamin D₃ 5400 KIU; vitamin E 48000 mg; vitamin K₃ 4000 mg; vitamin B₁ 4000 mg; vitamin B₂ 9000 mg; vitamin B₆ 7600 mg; vitamin B₁₂ 20 mg; niacin 60000 mg; pantothenic acid 20000 mg; folic acid 1600 mg; biotin 200 mg

Each Kg of Nutrimin^** supplied: Iron 10000 mg; zinc 120000 mg; manganese 140000 mg; copper 12000 mg; iodine 1800 mg; cobalt 400 mg and selenium 360 mg”

Table 3: Nutrients composition of experimental diets

Nutrients	Starter Diet			Finisher Diet
Nutrients	T₁	T₂	T₃		T₁	T₂	T₃
Crude protein	21.5	20.5	19.5		20.00	19.00	18.00
ME (kcal/kg)	3030	3030	3030		3100	3100	3100
Ether extract	6.32	6.12	5.94		6.36	6.12	5.89
Crude fiber	3.27	3.08	2.91		3.11	2.86	2.52
Ash	3.53	3.10	2.71		3.00	2.66	2.32
Dig. Lysine	1.28	1.28	1.28		1.05	1.05	1.05
Dig. Methionine	0.51	0.51	0.51		0.41	0.41	0.41
Dig. Threonine	0.81	0.89	0.97		0.72	0.79	0.95
Calcium	0.99	0.97	0.88		0.88	0.87	0.80
Phosphorus, available	0.46	0.45	0.40		0.40	0.39	0.35

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

Statistical analysis

Data collected for each treatment group was analyzed through the Analysis of Variance technique under Completely Randomized Design and comparison of means were made by Tukey’s test (Steel et al. 1997).

Results

Growth performance

Feed intake: During starter phase, feed intake was higher (P < 0.05) in birds of T1 (Control) treatment while it was lower (P < 0.05) in birds of T2 treatment (1% less CP + 10% higher Thr). During finisher phase, it was not similar (P > 0.05) in all treatments. Overall, lower (P < 0.05) feed intake was recorded in birds of treatment T2 (1% less CP + 10% higher Thr) than other treatments (Table 4).

Body weight gain: During starter phase, body weight gain was higher (P < 0.05) in birds of treatment T1 and T2 (1% less CP + 10% higher Thr), while lower (P < 0.05) weight gain was recorded in birds of treatment T3 (2% less CP + 20% higher Thr). During finisher phase, weight gain was higher (P < 0.05) in birds of treatment T1 (control) than others. Overall, higher (P < 0.05) body weight gain was recorded in birds of treatment T1 and T2 (1% less CP + 10% higher Thr), and lower (P < 0.05) body weight was seen in birds of treatment T3 (2% less CP + 20% higher Thr) (Table 5).

Feed conversion ratio: Improved (P < 0.05) FCR was recorded in birds of treatment T1 and T2 (1% less CP + 10% higher Thr) and poor (P < 0.05) FCR was observed in birds of treatment T3 (2% less CP + 20% higher Thr) during starter, finisher and overall period (Table 6).

Carcass characteristics

Table 4: Feed intake of broilers with diets containing various levels of CP and threonine

Treatments	FI (g/ bird)
Treatments	1–21 days	22–35 days	1–35 days
T₁	1248.20^a	1903.62	3151.82^a
T₂	1230.44^b	1896.90	3127.34^b
T₃	1244.32^ab	1905.56	3149.88^a
SEM	4.34	6.68	5.15
P-value	0.032	0.640	0.010

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

P > 0.05 (non-significant), P < 0.05 (significant)

Table 5: Weight gain of broilers reared on diets containing various levels of CP and threonine

Treatments	WG (g/ bird)
Treatments	1–21 days	22–35 days	1–35 days
T1	839.54^a	1029.24^a	1868.78^a
T₂	836.56^a	1025.70^ab	1862.26^a
T₃	803.71^b	1014.59^b	1818.30^b
SEM	2.97	3.12	2.97
P-value	0.0001	0.016	0.0001

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

P > 0.05 (non-significant), P < 0.05 (significant)

Table 6: Feed conversion ratio of broilers reared on diets containing various levels of CP and threonine

Treatments	Feed conversion ratio
Treatments	1–21 days	22–35 days	1–35 days
T1	1.49^b	1.85^b	1.69^b
T₂	1.47^b	1.85^b	1.68^b
T₃	1.55^a	1.88^a	1.73^a
SEM	0.006	0.007	0.004
P	0.0001	0.016	0.0001

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

P > 0.05 (non-significant), P < 0.05 (significant)

Dressing percentage and chest yield were higher (P < 0.05) in birds of treatment T1 (control) and they were lower (P < 0.05) in treatment T3 (2% less CP + 20% higher thr). Leg, heart, gizzard, liver, spleen and abdominal fat percentage were not affected (P > 0.05) by increasing level of threonine in low protein broilers diet (Table 7).

Biochemical parameters

At the end of experimental trail, two birds/pen was selected for blood sampling. Blood parameters (glucose and total protein), liver enzyme activity (ALT, AST and ALP) and serum biochemistry parameters (triglycerides, cholesterol, HDL, LDL and VLDL) were not affected by increasing level of threonine in low protein broilers diet (Table 8).

Economics efficiency

Cost of production per live weight was lower in birds of T2 (1% less CP + 10% higher Thr) and it was higher in T3 (2% less CP + 20% higher Thr) (Table 9).

Discussion

Super dosing of Thr (10 and 20%) with low protein (1 to 2%) had increased feed intake in broilers. This might be due to that Thr improve the gut health and reduce passage rate resulting in increased feed intake. These results of feed intake were in agreement with Mejia et al. (2012) who reported that feed intake was higher when birds fed a diet containing 0.77% threonine during the experimental period day (35–49). Nasr and Kheiri (2011) demonstrated that chicks fed on a diet containing 1.2% Thr had higher feed intake during the overall period day (0–42). Panda et al. (2011) reported that feed intake was increased with an elevated level of Thr up to 1.2% during the starter phase day (1–21). Zaghari et al. (2011) demonstrated that threonine at 0.8 and 0.9% had higher feed intake than other dietary treatments during the starter phase day (1–21). Sterling et al. (2003) recorded results in an increase in feed intake of broilers fed diets with increasing Thr at day (9–18). Results are not in line with the findings of Ahmad et al. (2020) who reported that 110 and 120% Thr had lower feed intake. Wijtten et al. (2004) concluded that different levels of Thr (0.92, 1.04, 1.17, 1.32 and 1.43%) had no effect on feed intake during the finisher phase day (14–34) in commercial broilers. Vieira et al. (2004) concluded that feed intake was similar in birds fed different ratios of Thr and methionine days (14–35).

Super dosing of Thr (10 and 20%) extra with low protein (1 and 2%) less had increased weight gain of broilers. This is because Thr improve the gut health and villus surface area resulting in improved nutrient absorption and body weight gain. The results are similar with Ahmad et al. (2020) who reported that Thr (10 and 20%) resulted in a greater growth performance than the control group. Ishii et al. (2019) observed that addition on lysine + Thr in broilers diet had increased body weight gain than control diet. Zarrin- Kavyani et al. (2018) reported that addition of 110% Thr in broilers diet had higher weight gain than 100 and 120% during grower phase. Wils-Plotz et al. (2013) observed that feed intake and weight gain were significantly increased at 0.54% threonine supplementation in those birds, which were not facilitated to that fed 0.17% threonine. The broilers gained 127.8 and 95.4 g more weight at an equal level of feed intake at pre- and post-inoculation. Rezaeipour et al. (2012) concluded that the highest FCR and weight gain were found at 7.6 g/kg in birds. Similar results were also obtained by Wijtten et al. (2004) observed that BWG was increased in birds fed a high level of threonine (1.45%) during the trial (day 14–34^th) in commercial broilers. In contrast, Helal et al. (2020) reported that different levels of Thr (100, 150 and 200%) had no effect on body weight gain.

Table 7: Carcass characteristics of broilers reared on diets containing various levels of CP and threonine

Treatments	Carcass characteristics (%)
Treatments	Dressing	Chest	Leg	Relative heart	Relative gizzard	Relative liver	Relative spleen	Relative abdominal fat
T₁	60.39^a	26.39^a	22.15	0.52	1.12	2.42	0.16	2.17
T₂	60.37^ab	26.06^ab	21.73	0.53	1.15	2.56	0.16	2.19
T₃	58.97^b	25.19^b	21.74	0.52	1.15	2.44	0.17	2.24
SEM	0.376	0.246	0.204	0.0310	0.038	0.079	0.006	0.029
P	0.032	0.013	0.289	0.942	0.809	0.443	0.515	0.240

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

P > 0.05 (non-significant), P < 0.05 (significant)

Table 8: Blood parameters of broilers reared with diets containing various levels of CP and threonine

Treatments	Blood Parameters		Serum biochemistry parameters					Liver enzyme activity
Treatments	Sugar (mg/dL)	Total Protein (g/dL)	Triglycerides (mg/dL)	Cholesterol (mg/dL)	HDL (mg/dL)	LDL (mg/dL)	VLDL (mg/dL)	ALT (IU/L)	AST (IU/L)	ALP (IU/L)
T₁	290.80	4.65	99.55	129.28	75.76	39.44	22.08	4.498	165.74	223.00
T₂	302.52	4.58	106.45	126.05	81.59	39.06	21.19	4.448	163.35	229.70
T₃	307.74	4.52	108.19	129.08	80.92	38.47	22.15	4.434	162.39	249.50
SEM	8.17	0.05	6.86	6.47	4.54	4.16	1.41	0.14	2.26	13.90
P	0.356	0.176	0.652	0.925	0.622	0.986	0.868	0.944	0.575	0.403

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

P > 0.05 (non-significant), P < 0.05 (significant)

Table 9: Economics of broilers reared with diets containing various levels of CP and threonine

Treatments	Day old bird cost	Total feed cost (per bird)	Miscellaneous	Production cost/bird	Av. body weight (g)	Production cost (per kg)
T₁	25	187.55^a	20	232.55^a	1913.86^a	121.51^b
T₂	25	184.73^b	20	229.73^b	1907.18^a	120.46^c
T₃	25	184.93^b	20	229.93^b	1863.64^b	123.38^a
SEM	-	0.297	-	0.297	2.98	0.22
P	-	0.0001	-	0.0001	0.0001	0.0001

T1= (Control group)

T₂= (1% less CP and 10% higher threonine)

T₃= (2% less CP and 20% higher threonine)

P > 0.05 (non-significant), P < 0.05 (significant)

Super dosing of Thr (10%) extra with low protein (1%) less had improved FCR. Improved FCR in 10% higher Thr may be associated with improved nitrogen retention. Results are similar with the findings of Ahmad et al. (2020) who reported that 110 and 120% Thr had improved FCR. Rasheed et al. (2018) reported that birds fed diet containing 10% higher Thr had improved FCR than control and 20% higher Thr. Zarrin-Kavyani et al. (2018) reported that addition of 110% Thr in broilers diet had improved FCR than 100 and 120% during starter phase. Ishii et al. (2019) observed that addition on lysine + Thr in broilers diet had improved FCR than control diet. Dozier et al. (2008) used different Thr levels (0.53, 0.63 and 0.73%) from 42 to 56 days. Highest weight gain and better FCR was recorded in broilers at 0.73 and 0.63% threonine, respectively. Results are not in line with Helal et al. (2020) who reported that different levels of Thr (100, 150 and 200%) had no effect on FCR. Zaghari et al. (2011) who concluded that the lower FCR ratio was recorded at 0.74% threonine level in the diet. Ayasan et al. (2009) demonstrated that at 0.86% Thr level lower the FCR in broilers during the finisher phase (22–42).

Dressing percentage and breast yield of broilers were higher in broilers of group T1 (control) and it was lower in group T3. This might be due to that Thr addition increased the abdominal fat resulting in reduced breast yield. Super dosing of Thr (10 and 20%) with low protein (1 and 2%) had no effects on thigh meat, liver, heart, spleen, gizzard weight and abdominal fat of broilers. Results obtained for carcass yield are agreed with Helal et al. (2020) who observed that Thr at 100% had higher dressing percentage, while relative organs weight were not affected by different Thr levels. Nasr and Kheiri (2011) concluded that birds fed a diet containing 1.3 percent Thr had higher carcass weight and lower fat deposition in broilers over the course of a day (0–42). Ghahri et al. (2010) discovered that birds fed three different levels of Thr (0.9, 0.10 and 1.1 percent) had a significant dressing percentage, breast, and thigh yield (21–42). The results contradict the findings of Chen et al. (2017) who found that adding Thr at 1 g/kg improved spleen weight and Thr at 3 g/kg improved thymus weight.

Blood parameters (glucose and total protein), liver enzyme activity (ALT, AST and ALP) and serum biochemistry parameters (triglycerides, cholesterol, HDL, LDL and VLDL) were not affected by increasing level of threonine in low protein broilers diet. This might due to variation is data leads to non-significant results. Results are in line with the findings of Mehdipour et al. (2020) who showed that different levels of Thr had no effect on blood hematology parameters. Helal et al. (2020) reported that different levels of Thr (100, 150 and 200%) had no effect on albumin and triglycerides concentration. These results were not supported by widely published work of Al-Hayani (2017) experimented to check the effects of graded levels of threonine (Thr, 300, 600 and 900 mg/kg) on blood parameters in broilers. The concentration of Thr 900 mg/kg showed a significant increase on (ALT, AST, ALP) and on total glucose levels as well.

Conclusion

Based on these findings, it can be concluded that lowering CP by 1% and increasing threonine by 10% improved growth performance, breast yield and economics efficiency than lowering CP by 2% and increasing threonine by 20% without any negative effect of liver enzyme activity and biochemical parameters.

Acknowledgments

The authors acknowledge Dr. Mahboob Ali Hamid, Assistant Professor, Institute of Animal and Dairy Science, University of Agriculture, Faisalabad for English grammar corrections and for improving the overall presentation of results

Author Contribution

FW, AJ and MAJ did experimental work and manuscript writing; MS and AM performed data analysis; KR and NR designed the experiment; MS and FM prepared the manuscript

Conflict of Interest

The authors declare no conflict of interest

Data Availability

Data is available

Ethical Approval

All procedures performed in studies were in accordance with the ethical committee of University of Agriculture, Faisalabad, Pakistan

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