Digestibility, intake, and growth performance of egg protein replaced with vegetable protein in weaning food

Abstract

Egg protein (EP) may help to decrease the risk of protein energy malnutrition (PEM) in infants. A 28 d research trial was conducted to examine the effect of different levels of EP on water intake, feed intake, nutrient digestibility, efficiency parameters, and anthropometric parameters in Wistar albino weaned pups. Weaning food was prepared by adding various EP in the diet (non-isonitrogenous and isocaloric) of 72 male and female rats to determine the best level for growth/development. Pups were randomly allotted to various concentrations of diet such as WF₀ (soybean protein), WF₁ (14% of EP), WF₂ (16% of EP), and WF₃ (18% of EP). Dry matter intake (DMI) was significant in pups fed with WF₂ (22.66 ± 0.27 g/kg on 0 d and 27.43 ± 0.32 g/kg on 28 d) and WF₃ (23.46 ± 0.28 g/kg on 0 d and 28.88 ± 0.33 g/kg on 28 d) diets as compared to the WF₀ (19.30 ± 0.24 g/kg on 0 d and 26.76 ± 0.31 g/kg on 28 d) and WF₁ (21.90 ± 0.26 g/kg on 0 d and 27.08 ± 0.32 g/kg on 28 day) diets. The crude fiber (CF) intake was significantly (P ≤ 0.05) reduced by increasing the level of EP in the diets of pups, while crude protein (CP) intake was increased with an increase in the EP levels. A similar trend was observed in ether extract (EE) intake. The DMI, CP, and EE digestibility were highest in pups fed the WF₃ diet. Likewise, a trend was noticed in the protein efficiency ratio, while the highest feed consumption ratio and consumption index were observed in pups fed a control diet. The weight gain and body length were significantly (P ≤ 0.05) increased in the pups fed with the different concentrations of EP. The WF₃ diet showed better growth after the best digestibility of the nutrients in pups and could therefore be recommended as an infant weaning food.

Keywords:

• EP
• Wistar albino weaned pups
• feed and water intake
• nutrient digestibility
• weight and length of body

1. Introduction

Protein-energy malnutrition (PEM) is a threat to the growth and development of infants and growing up children (González-Torres et al., 2014), it is a range of pathological and clinical conditions caused by a lack of adequate intake of dietary protein, calories, vitamins, and minerals. PEM leads to clinical syndromes like malaria, edema of the legs and feet, marasmus, tuberculosis, kwashiorkor, stress response to acute injury or chronic inflammation, diarrhea, electrolyte disorders, anemia, and systemic infections (Grover & Ee, 2009). In most countries, infant formula feed consists of cereals which are not a good source of protein because of the imbalance in the ratio of energy and protein in the diet of infants which may lead to PEM in infants (Ahmed et al., 2020; Semba, 2016). However, cereals that are low in protein cannot fulfill the amino acid requirement of supporting growth and development in infants. Cereal grains are deficient in a few essential amino acids such as lysine and tryptophan. Deficiency of lysine and tryptophan limits protein synthesis and causes weakness, weight loss, infantile anorexia nervosa, growth retardation, irritability, and a lack of digestive enzymes, and hormones in infants (Begum, 2008; World Health Organization WHO, 2003). Another critical factor is the presence of anti-nutrients like protease, tannins, and saponins to affect the bioavailability of the protein, phytates affect the mineral (zinc and iron) utilization, and lipase inhibitors affect the digestion of carbohydrates (Samtiya et al., 2020). Therefore, the protein contents of cereals are less, which require mixing different legumes with cereals from a nutritional point of view to fulfill the requirements of infant formula feed (Grover & Ee, 2009; Temba et al., 2016).

Combination of common cereals with legumes (soybean) which are rich in protein content and a good source of essential amino acids like lysine contents for the improvement of the nutritive value of food products (Kouakou et al., 2013; Shuluwa et al., 2021). Soy protein formulas are utilized for the feeding of infants for the accomplishment of protein as soy is a good protein source but it is not grown conventionally in Pakistan. That is why animal sources of protein in the form of eggs can be introduced in weaning food. Egg protein is recognized as an excellent source of essential amino acids and exhibits versatile functional and biological properties. According to the Digestible Indispensable Amino Acid Scores (DIAAS), animal-source (eggs) proteins have higher scores than those plant source proteins (cereals and legumes) (Mathai et al., 2017). Egg plays a part in an infant’s average protein requirement as it contains a good amount of essential amino acids (McNamara & Thesmar, 2005; Réhault-Godbert et al., 2019). Secondly, its anti-nutrient factor avidin is known to form a complex with biotin that can easily be denatured by high-pressure processing and heat treatment. Therefore, egg protein is more available as compared to plant-sourced protein (soybean). Thus, eggs are considered to be the complementary food proteins that have maximum content by providing the amino acids that lack in plant sources. At the age of 6 months, there is a need to provide complementary protein to the infants to fulfill their protein requirements. Recently, in our research work, an extruded weaning food is prepared using egg powder as an animal source, and soybean used as a plant source (Naeem et al., 2021). Since most cereal sources cannot fulfill the protein requirement of infants due to their low concentration of essential amino acids such as lysine and tryptophan and also possess some anti-nutritional factors such as saponins, tannins, phytic acid, gossypol, lectins, protease inhibitors, amylase inhibitor, and goitrogens which inhibits the availability of essential nutrients, it is therefore essential to supplement plant-based weaning foods with animal sources of quality protein such as egg protein (Amirabdollahian & Ash, 2010; Samtiya et al., 2020). The main objective of this research work is to use both animal and plant protein sources in the diet of pups for their growth. The soybean protein was replaced with egg protein (EP) (contains a more balanced amino acid profile) in the diets to estimate their impact on feed nutrient and water intake and its efficiency parameters, digestibility, and growth of pups.

2. Materials and methods

2.1. Procurement of pups

The 72 Wistar albino weaned pups (including 36 males and 36 females) of age 18 d old, each weighing 25 ± 10 g, was purchased from the National Institutes of Health, Islamabad. Punjab, Pakistan. They were kept at 25 ± 1°C and 45% to 55% relative humidity under 12-h light: 12-h dark cycle. All animals were treated according to the Principles of Laboratory Animal Care (NIH) (Sirois, 2022). The experimental procedure is authorized by the Animal Ethical Committee (Study number: 019308) of Government College University, Faisalabad, Punjab, Pakistan.

2.2. Preparation of weaning food and its chemical composition

Non-isonitrogenous and isocaloric diet was prepared using the best treatment levels of weaning foods in our recent research work of Naeem et al. (2021) In this study, 36 weaned-stage Wistar albino pups were used to examine the best level of egg powder in infant formula feed as described in the recent research work of Rahim et al. (2023). The pups were divided randomly into four major groups nine each of male and female pups and the groups were named according to diets like WF₀ (soybean protein), WF₁ (14% of EP), WF₂ (16% of EP), and WF₃ (18% of EP). In both male and female groups, each treatment was repeated three times to make experimental units. The AOAC standard methods were used for determining ash contents (Method 923.02), fat contents (Method 922.06), moisture contents (Method 62–05), protein contents (Method 954.01), and dietary fiber (Method 962.09) with minor modifications for rice flour, soybean powder, carrot powder, and egg powder in the formulated diet (AOAC, 2006). The chemical composition of formulated weaned foods is also described in our recent research work of Rahim et al. (2023). Pups were fed with an experimental diet and water ad libitum for 28 d to assess the effect of treatments on different parameters. The standard of feed intake is 5 g/100 g by weight and 10 ml/100 g of water by body weight in pups (Claassen, 1994; Martınez-Merino et al., 2013). The weekly intake of water and feed was monitored at least 4 weeks before the start of treatments to determine the amount of water drank per experimental animal.

2.3. Water intake, feed intake, and its efficiency parameters

Water and feed intake were recorded daily for 28 d. Protein efficiency ratio (PER), consumption index (CI) and feed conversion ratio (FCR) were estimated using the standard methods of AOAC (1995) with some minor modifications. The PER, CI, and FCR were calculated using the following equations 1, 2, and 3:

(1) $PER=\frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{g}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}\left(\mathrm{g}\right)}{Totalproteinconsume\left(\mathrm{g}\right)}$(1)

(2) $CI=\frac{\mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}{Averageweight\left(DuringthefeedingPeriod\right)}$(2)

(3) $FCR=\frac{\mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\left(\mathrm{g}\right)}{Bodyweightgain\left(\mathrm{g}\right)}$(3)

2.4. Nutrient digestibility analysis

The total collection method was used for measuring the digestibility of all nutrients like fat, carbohydrates, and protein according to this given equation 4:

(4) $\mathrm{N}\mathrm{D}\left(\mathrm{\%}\right)=\frac{\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{s}}{Nutrientintake}\times 100$(4)

Where,

ND = Nutrient digestibility

The previous AOAC standard methods was used for dry matter, fat, and fiber analysis of the diets as described in Section 1.2 (AOAC, 2006).

2.5. Anthropometric parameters

In this efficacy trial, height and weight of the pups were measured weekly by using standard techniques. These investigations were carried out in the morning between 9 am and 12 pm (Ijarotimi & Keshinro, 2012). Weight was measured by using a digital weighing scale (Salter, SL20348, London, UK). The length was examined by using a steel measuring tape (Length & width: 5 m × 25 mm, Ningbo, China) and calibrated to the nearest 0.1 cm. The weight and length of the animals were related using weight-for-length and length-for-age indices to determine the nutritional status.

2.6. Statistical analysis

In this research work, a completely randomized design (CRD) of the one-way analysis of variance (ANOVA) and Tukey test were applied to compare differences between PER, CI, FCR, ND, weight, and length parameters in weaned pups with level of significance (P ≤ 0.05), using Statistix Version 8.1 analytical software copyright © 1985–2005 (Steel et al., 1997).

3. Results and discussion

3.1. Water intake

In the present study, no significant differences (P ≤ 0.05) were found in the water intake of pups fed containing plant and animal source protein diets in 7 d. A similar trend was observed in its water consumption every week to 4 weeks as described in Table 1. Pups were offered a standard water intake of approximately 10 ml/day; in this trial, the amount of water intake was increased to around 3 ml compared to the standards. Finally, it was observed that the high protein-based diets non-significantly (P ≤ 0.05) increased the water requirement of pups (Table 1).

Table 1. Effect of various level of EP in weaning food of pups on water intake

Days	Parameter	Treatment				SEM	p-value
		WF0	WF1	WF2	WF3
0 to 7 days	Water intake (ml/day)	31.16 ± 0.35^A	31.70 ± 0.35^A	31.95 ± 0.36^A	32.16 ± 0.38^A	0.261	0.359^NS
8 to 14 days	Water intake (ml/day)	31.36 ± 0.35^A	31.90 ± 0.36^A	32.13 ± 0.37^A	32.40 ± 0.37^A	0.303	0.640^NS
15 to 21 days	Water intake (ml/day)	31.67 ± 0.36^A	32.10 ± 0.37^A	32.36 ± 0.37^A	32.63 ± 0.37^A	0.334	0.205^NS
22 to 28 days	Water intake (ml/day)	32.03 ± 0.37^A	32.40 ± 0.37^A	32.83 ± 0.38^A	33.10 ± 0.38^A	0.172	0.148^NS

EP = Egg protein; WF0 = Soybean protein; WF1 = 14% of EP; WF2 = 16% of EP; WF3 = 18% of EP; SEM = Standard error of the mean; ^{A to D} Means with different superscripts differ significant effect (p ≤ 0.05); ^NS = Non-significant at 0.05 level.

The increased water intake in pups might be due to greater urea production from a high protein diet for the stimulation of more water consumption to increase plasma osmolality. This result was comparable to the recent research work by Matsuoka et al. (2017) reported that the urine production in rats fed with a diet containing egg white was higher than compared to the other treatments (55.1 mL/5 days). Similar results were found by Andriamihaja et al. (2010) and Marks and Pesti (1984); there was a significant difference (P ≤ 0.05) between the average water consumption by rats fed with basel diet (39.9 ml/d) and high protein diets (45.4 ml/d), respectively. In addition, Kim (1969) contrary result found that the rat urine was increased significantly as dietary protein levels increased.

3.2. Feed intake and its efficiency parameters

The impact of different levels of egg protein (EP) in the weaning food of pups on feed intake and its efficiency parameters have been described in Table 2. In this present research work, the significance of feed intake in pups was observed in groups of WF₂ and WF₃ as compared to the control group (WF₀) that had a low protein source throughout the 28 d study trial. Furthermore, the protein efficiency ratio (PER) in pups was increased with an increase in EP levels. A similar trend was observed every week for up to 4 weeks and the amount of PER significantly (P ≤ 0.05) increased from 2.30 ± 0.13 to 3.96 ± 0.18. The concentration of feed conversion ratio (FCR) and consumption index (CI) has an inverse relationship with the weight of the pups. In this study, different levels of EP in the pup’s diet showed that the weight of pups improved significantly compared to the control group due to low FCR in pups fed the WF₃ diet. The highest FCR was observed in pups fed the WF₀ diet. However, non-significant (P ≤ 0.05) FCR was observed between WF₃ and WF₂ (0 d to 28 d). A similar trend was observed in CI. The lowest FCR and CI in pups might be due to the high level of animal protein diet as compared to the control that contains plant protein sources. A higher amount of feed intake, PCR, FCR, and CI were observed in pups fed a plant protein diet. Our results well agreed with the research work of Mori et al. (1991), as they observed that feed intake in 21 days increased by 134 g in male weaning rats when 70% of egg white in the diet was replaced with soy protein isolate. Research work by Adebiyi et al. (2021) revealed that the minimum amount of FCR and CI was considered the best in growth performance. Another research work of JIANG and J (1991) indicated that significant differences were found between body weight gain and feed consumption index of rats fed with yolk protein. In addition, Chen et al. (2019) reported that the FCR and CI in the rats fed a diet containing 132 g/kg egg white were significantly higher compared with rats fed a diet containing 16–40 g/kg egg yolk. This might be due to different protein content in egg white and egg yolk.

Table 2. Impact of different level of EP in weaning food of pups on intake, protein efficiency ratio, feed conversation ratio, and consumption index

Days	Parameter	Treatment				SEM	p-value
		WF0	WF1	WF2	WF3
0 to 7 days	Feed intake (g/day)	20.50 ± 0.24^C	23.03 ± 0.24^B	24.73 ± 0.25^A	25.53 ± 0.26^A	1.162	<0.001**
	PER	2.30 ± 0.13^B	3.52 ± 0.13^A	3.70 ± 0.14^A	3.83 ± 0.15^A	0.061	<0.001**
	FCR	4.17 ± 0.17^A	2.97 ± 0.16^B	2.82 ± 0.16^B	2.61 ± 0.17^C	0.017	0.212^NS
	CI	30.42 ± 0.32^A	14.32 ± 0.17^B	13.53 ± 0.16^C	10.23 ± 0.14^D	0.011	<0.001**
8 to 14 days	Feed intake (g/day)	21.96 ± 0.23^D	24.53 ± 0.25^C	25.99 ± 0.27^B	27.60 ± 0.29^A	1.311	<0.001**
	PER	2.35 ± 0.14^C	3.62 ± 0.15^B	3.76 ± 0.16^A	3.87 ± 0.16^A	0.021	<0.001**
	FCR	4.14 ± 0.16^A	2.67 ± 0.14^B	2.42 ± 0.13^B	2.11 ± 0.13^C	0.016	0.125^NS
	CI	30.12 ± 0.34^A	14.07 ± 0.16^B	13.18 ± 0.15^C	10.14 ± 0.14^D	0.012	<0.001**
15 to 21 days	Feed intake (g/day)	24.03 ± 0.26^C	26.01 ± 0.28^B	26.96 ± 0.29^B	28.56 ± 0.30^A	0.781	<0.001**
	PER	2.41 ± 0.15^C	3.65 ± 0.17^B	3.79 ± 0.17^B	3.90 ± 0.18^A	0.025	<0.001**
	FCR	4.11 ± 0.15^A	2.17 ± 0.12^B	2.02 ± 0.12^B	1.91 ± 0.11^C	0.016	<0.001**
	CI	29.25 ± 0.33^A	13.45 ± 0.14^B	13.05 ± 0.13^B	10.09 ± 0.10^C	0.087	<0.001**
22 to 28 days	Feed intake (g/day)	25.50 ± 0.27^C	27.23 ± 0.29^C	27.33 ± 0.29^B	29.44 ± 0.30^A	0.563	<0.001**
	PER	2.52 ± 0.16^C	3.69 ± 0.17^B	3.85 ± 0.17^A	3.96 ± 0.18^A	0.013	<0.001**
	FCR	4.06 ± 0.13^A	1.96 ± 0.12^B	1.92 ± 0.11^B	1.79 ± 0.11^C	0.021	<0.001**
	CI	28.33 ± 0.31^A	13.22 ± 0.15^B	13.03 ± 0.15^B	10.04 ± 0.12^C	0.445	<0.001**

EP = Egg protein; WF0 = Soybean protein; WF1 = 14% of EP; WF2 = 16% of EP; WF3 = 18% of EP; PER = Protein efficiency ratio; FCR = Feed conversion ratio; CI = Consumption index; SEM = Standard error of the mean; ^{A to D} Means with different superscripts differ significant effect (p ≤ 0.05); * = Significant at 0.05 level; ** = Highly significant at 0.05 level; ^NS = Non-significant at 0.05 level.

Progressive protein deficiencies in the growth of infants indicated protein energy malnutrition (PEM) (Grover & Ee, 2009). So, there is a need to use those plant and animal sources that fulfill the protein requirement of the infants in their weaning food. Secondly, most of the plant sources (cereal) are deficient in the lysine and tryptophan that have an important role in the growth of infants (Batool et al., 2015; Koo & Lasekan, 2007; Kramer et al., 2004).

3.3. Nutrient intake and their digestibility

Seventy-two Wistar albino pups were used in a 28 d growth study to estimate the impact of high-level EP on nutrient intake in weaned pups. In this study, dry matter intake (DMI) was higher (P ≤ 0.05) in pups fed the high level of EP than in those fed the plant protein diet. Similarly, the DMI in pups was increased throughout the trial (0 d − 28 d) in all treatments such as WF₀, WF1, WF₂, and WF₃. This increased intake in all diets might be due to the age of the pups after each week (Fontaine, 2012). Furthermore, a higher intake of crude fiber (CF) was observed in WF₀, while in other treatments WF₁, WF₂, and WF₃ showed a lower intake of CF in weaned pups. Following a 28 d period, CF intake was significantly reduced with increasing levels of EP. Moreover, a significant difference in crude protein (CP) intake was observed among all fed different levels of EP intake throughout the study period. The highest CP intake in pups was observed fed a WF₃ diet. The pups fed the same level of protein intake from 0 d to 28 d showed the same CP intake level. A similar trend was also observed in ether extract (EE) (Table 3).

Table 3. Effect of different level of EP in weaning food of pups on nutrient intake (g/kg)

Days	Parameter	Treatment				SEM	p-value
		WF0	WF1	WF2	WF3
0 to 7 days	DMI	19.30 ± 0.24^D	21.90 ± 0.26^C	22.66 ± 0.27^B	23.46 ± 0.28^A	0.123	<0.001**
	CF	1.11 ± 0.13^A	1.09 ± 0.13^A	0.87 ± 0.12^B	0.85 ± 0.12^B	0.012	<0.001**
	CP	2.55 ± 0.13^D	3.23 ± 0.14^C	4.03 ± 0.14^B	4.42 ± 0.14^A	0.391	<0.001**
	EE	0.32 ± 0.11^C	0.51 ± 0.11^B	0.54 ± 0.11^B	0.71 ± 0.12^A	0.007	<0.006*
8 to 14 days	DMI	22.03 ± 0.23^C	22.37 ± 0.23^C	23.24 ± 0.24^B	24.70 ± 0.25^A	0.087	<0.004*
	CF	1.15 ± 0.14^A	1.12 ± 0.14^A	0.90 ± 0.12^B	0.91 ± 0.12^B	0.015	<0.008*
	CP	2.69 ± 0.13^D	3.37 ± 0.14^C	4.12 ± 0.14^B	4.69 ± 0.15^A	0.382	<0.001**
	EE	0.34 ± 0.11^C	0.54 ± 0.11^B	0.65 ± 0.12^B	0.76 ± 0.12^A	0.016	<0.009*
15 to 21 days	DMI	23.04 ± 0.28^A	23.96 ± 0.28^B	24.06 ± 0.29^B	25.57 ± 0.30^A	0.050	<0.004**
	CF	1.16 ± 0.14^A	1.14 ± 0.14^A	0.92 ± 0.12^B	0.94 ± 0.12^B	0.009	<0.010*
	CP	2.76 ± 0.17^D	3.45 ± 0.18^C	4.21 ± 0.15^B	4.74 ± 0.15^A	0.413	<0.001**
	EE	0.38 ± 0.11^C	0.58 ± 0.11^B	0.69 ± 0.11^B	0.80 ± 0.11^A	0.019	<0.009*
22 to 28 days	DMI	26.76 ± 0.31^D	27.08 ± 0.32^C	27.43 ± 0.32^B	28.88 ± 0.33^A	0.049	<0.001**
	CF	1.17 ± 0.14^A	1.15 ± 0.14^A	0.93 ± 0.12^B	0.960.12^B	0.011	<0.003*
	CP	2.82 ± 0.15^D	3.53 ± 0.16^C	4.33 ± 0.17^B	4.85 ± 0.17^A	0.428	<0.001**
	EE	0.43 ± 0.11^C	0.63 ± 0.11^B	0.74 ± 0.12^B	0.86 ± 0.12^A	0.006	<0.008*

EP = Egg protein; WF0 = Soybean protein; WF1 = 14% of EP; WF2 = 16% of EP; WF3 = 18% of EP; DMI = Dry matter Intake; CF = Crude fiber; CP = Crude protein; EE = Ether extract; SEM = Standard error of mean; ^{A to D} Means with different superscripts differ significant effect (p ≤ 0.05); * = Significant at 0.05 level; ** = Highly significant at 0.05 level; ^NS = Non-significant at 0.05 level.

The effect of a high EP diet on nutrient digestibility has been shown in Table 4. The digestibility of DMI, CP, and EE was higher (P ≤ 0.05) in pups fed the various levels of EP diet than the plant protein diet. The nutrient digestibility was found to be (P ≤ 0.05) increased with increasing the level of EP.

Table 4. Effect of weaning foods on nutrient digestibility

Item	WF0	WF1	WF2	WF3	SEM	p-value
DM	66.48 ± 0.46^D	68.90 ± 0.48^C	69.73 ± 0.48^B	72.16 ± 0.52^A	0.0169	<0.001**
CF	47.89 ± 0.39^D	58.56 ± 0.41^B	59.28 ± 0.42^C	65.31 ± 0.45^A	0.0316	<0.001**
CP	69.89 ± 0.49^D	70.55 ± 0.51^C	72.95 ± 0.52^B	75.92 ± 0.53^A	0.0593	<0.001**
EE	79.62 ± 0.54^D	87.33 ± 0.57^C	88.77 ± 0.58^B	92.82 ± 0.59^A	0.0416	<0.001**

WF0 = Soybean protein; WF1 = 14% of EP; WF2 = 16% of EP; WF3 = 18% of EP; DM = Dry matter; CF = crude fiber; CP = crude protein; EE = ether extract; SEM = Standard error of the mean; Means with different superscripts as ^{A, B} are different significantly (P ≤ 0.05); ** = Highly significant at 0.05 level.

The DMI, CP, and EE intake in pups were increased with an increase in the concentration of EP in their diets, while the CF intake in pups was reduced with increasing the concentration of EP level in the diets. The EE was higher in pups fed the animal protein diet than the plant protein diet. Adebiyi et al. (2021) reported that the nutrient intake was not significantly affected in various weaning food treated with plant protein albino rats. Another research work by Ikujenlola (2016) indicated that the blend of cereals with animal and vegetable protein enhanced the amount of nutrient intake in rats. The nutrient digestibility was higher in pups fed with a higher concentration of EP diet (Table 3). Limited information was found on the digestibility intake in pups fed the EP diet. However, some scientists or research works showed that a high-level EP diet is a good-quality food for infants (Papanikolaou & Fulgoni, 2018).

3.4. Anthropometric parameters

In this study, physical parameters such as length and body weight were assessed weekly to check the effect of high EP diets on the pups (Table 5). It was observed that the change in length in pups was significantly (P ≤ 0.05) increased in the WF₃ diet, while a non-significant difference (P ≤ 0.05) was observed among all pups fed with different levels of protein intake at the 15 d to 21 d. At the age of 28 d, a similar measurement in length of pups was observed fed with WF₃ and WF₂ diets. The lowest body length obtained in the control group (WF₀) was 19.56 ± 0.18 cm. An increasing trend of body weight gain was observed in pups fed a diet containing EP as compared to the control diet. Furthermore, the weekly highest weight gain in pups was noticed in the WF₃ diet. The weekly lowest weight gain in pups was observed in the WF₀ diet. This trend of weight gain might be due to the biological availability of EP compared to plant protein sources.

Table 5. Effect of different level of EP in weaning food of pups on length and weight

Parameters	Days	Treatments				SEM	p-value
		WF0	WF1	WF2	WF3
Length (cm)	0 to 7 days	18.77 ± 0.17^C	19.33 ± 0.18^A	19.82 ± 0.20^B	19.91 ± 0.20^A	0.012	<0.006*
	8 to 14 days	18.83 ± 0.17^D	19.45 ± 0.18^B	19.88 ± 0.20^C	20.07 ± 0.21^A	0.016	<0.001**
	15 to 21 days	18.93 ± 0.17^A	19.55 ± 0.20^A	19.99 ± 0.20^A	20.15 ± 0.21^A	0.275	0.085^NS
	22 to 28 days	19.56 ± 0.18^D	19.74 ± 0.20^B	20.12 ± 0.21^A	20.27 ± 0.21^A	0.174	<0.004*
Body Weight gain (g)	0 to 7 days	31.01 ± 0.34^C	33.45 ± 0.37^B	35.52 ± 0.38^B	39.22 ± 0.40^A	0.307	<0.007*
	8 to 14 days	31.10 ± 0.34^C	33.55 ± 0.37^B	35.75 ± 0.38^B	39.45 ± 0.40^A	0.105	<0.004*
	15 to 21 days	31.33 ± 0.35^D	33.72 ± 0.37^C	35.94 ± 0.38^B	39.62 ± 0.40^A	0.017	<0.001**
	22 to 28 days	32.17 ± 0.36^D	34.12 ± 0.38^C	36.24 ± 0.39^B	40.22 ± 0.41^A	0.014	<0.001**
Total weight gain (g)	45 days	125.61 ± 0.62^A	133.44 ± 0.64^B	143.15 ± 0.65^C	158.11 ± 0.66^D	0.013

EP = Egg protein; WF0 = Soybean protein; WF1 = 14% of EP; WF2 = 16% of EP; WF3 = 18% of EP; SEM = Standard error of the mean; ^{A to D} Means with different superscripts differ significant effect (p ≤ 0.05); * = Significant at 0.05 level; ** = Highly significant at 0.05 level; ^NS = Non-significant at 0.05 level.

The weight gain and body length were increased (P ≤ 0.05) in the pups fed WF₁, WF₂, and WF₃ diets. The length and weight are the most important parameters to determine the effect of high protein diets on the growth of pups. However, a high correlation was found between length and body weight (Pastuszewska et al., 2008). The body weight of rats was increased from 50.9 to 88.1 g in 21-day-old pups fed egg white protein (Oliveira et al., 1999). Moreover, the egg white protein diet was a non-significant (P ≤ 0.05) effect on the body weight of rats due to the isonitrogenous diet (Matsuoka et al., 2017). This also corroborates with the findings of Chen et al. (2019) who reported that the body weight of rats fed egg white was significantly (P ≤ 0.05) higher than that of rats fed with egg yolk.

4. Conclusion

The results obtained from this study indicated the significant potential of various egg protein (EP) levels on water, feed and nutrient intake, and anthropometric parameters of the pups. Likewise, significant differences (P ≤ 0.05) in the water intake, feed intake, and PER were observed among all pups fed different levels of protein intake for 28 d. The concentration of FCR and CI was reduced by increasing the level of protein in the diet of pups. Consequently, animal protein diets yielded a significantly higher length and total body weight of pups than the plant diet having the same protein contents. We confirmed that the different concentrations of EP in the complementary diets showed better performance in the growth of pups.

Consent for publication

All authors agreed for publication of this manuscript.

Ethical approval

The experimental procedure was approved by the Animal Ethical Committee of Government College University, Faisalabad, Punjab, Pakistan.

Availability of data and material

Even though adequate data has been given in the form of tables however, all authors declare that if more data required then the data will be provided on a request basis.

Author’s contribution

Muhammad Naeem performed the methods and investigation. Prof. Dr Mahr Un Nisa conceptualized the idea and provided supervision in analysis, while Muhammad Abdul Rahim, Khunsha Khalid, Nazir Ahmad, Nukhba Khalid, Muhammad Sarwar, and Chinaza Godswill Awuchi supported for original drafting of the manuscript. Chinaza Godswill Awuchi and Muhammad Abdul Rahim validated the study.

Acknowledgments

The authors are thankful to the Library Department, Government College University Faisalabad (GCUF), and IT Department, Higher Education Commission (HEC, Islamabad) for access to journals, books, and valuable databases. The research was completed after using the available resources in the Department and University. The chemical, equipment, and glassware were supported by HEC under Pakistan funded project (HEC/R&D/NRPU/2017/9287). The authors are also grateful to Kampala International University, Kampala, Uganda, for its support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The research work was funded by Pakistan Higher Education Commission (HEC) under Pakistan funded project [HEC/R&D/NRPU/2017/9287].

Notes on contributors

Muhammad Naeem

Muhammad Naeem is a PhD candidate at Government College University Faisalabad, Pakistan and Lecturer in National Institute of Food Science and Technology University of Agricuture, Pakistan.

Muhammad Abdul Rahim

Muhammad Abdul Rahim has expertise in Spray Drying, Extrusion Technology, Microencapsulation, Lipids Chemistry, Sensory Evaluation and Food Process Engineering. Currently, Dr. Muhammad Abdul Rahim is working as an Assistant Professor in the Department of Food & Nutrition, Times Institute, Multan, Punjab, Pakistan.

Mahr Un Nisa

Prof. Dr. Mahr-Un-Nisa working as the chairperson, department of Nutritional Sciences, Government College University, Faisalabad Pakistan. I am passionate about conducting quality research and publishing my findings. I published 150+ research articles and a book along with it furnished 50+ MPhil and 10 PhD students. I received the presidential award Iziz-I-Fazeelat. I am the executive vice president of world poultry science association (women wing). I have expertise in the field of both Animal Nutrition as well as Human Nutrition

Khunsha Khalid

Dr. Khunsha Khalid, having Bachelor's degree in Medicine and Surgery. With a profound year of clinical experience under her belt, Dr. Khalid has committed herself to serving the healthcare needs of underserved communities in a rural area. Beyond her clinical practice, Dr. Khalid's journey is marked by an unwavering passion for medical research. She firmly believes that research holds the power to shape a better world than yesterday, a conviction that fuels her dedication to advancing the field of medicine.

Nazir Ahmad

Dr Nazir Ahmad has been working as associate professor and researcher since February 2013 in department of Nutritional Sciences. He holds PhD degree in Nutrition and Biotechnology processes. He has the experience of working with international scientists during his master and doctoral studies in France where he did his master in immunological response to of milk proteins from International French Institute for Agriculture (INRA), Nantes, France and PhD degree on postprandial lipemia and hepatic function from University of Lorraine, Nancy, France. His research interests are to explore the functional characteristics of nutrients and biological evaluation in health and disease.

Nukhba Khalid

Dr. Nukhba Khalid has completed her degree in Pharm D from GCUF under the Faculty of Pharmaceuticals. She has graduated with exceptional grades and remarkably excelled in academics. Working under renowned staff of Pharmaceutical sciences at GCUF, Dr. Nukhba Khalid assisted in research for Intranasal Novasomes. Primarily she is interested in researching on Nano particles for drug delivery.

Muahammad Sarwar

Dr. Muhammad Sarwar is working as Dean, post-graduate studies and research, The University of Lahore(UOL)and also as Pro-Rector, UOL, Sargodha Campus. Based- upon his brilliant career and miraculous achievement The Higher Education Commission(HEC), Islamabad, awarded him titles of HEC Distinguished National Professor and Best University Teacher Award. The Ohio State University(OSU), Columbus, USA, also awarded him the title of OSU Distinguished Alumnus-2014. He executed 16 research projects and published more than 400 publications including 6 books.

Chinaza Godswill Awuchi

Chinaza Godswill Awuchi specialises in Food and Nutrition, with skills in biochemical analyses.