The Influence of Wheat Germ Expeller on Performance and Selected Parameters of Carbohydrate, Lipid, and Protein Metabolism in Blood Serum for Broilers

Abstract

The effect of replacing (5, 10, and 15%) wheat middlings in the feed of broiler chickens (EX5, EX10, EX15) with wheat germ expeller (WGE) on their parameters of carbohydrate, lipid, and protein metabolism was examined. Thirty-two randomly chosen broilers on day 43 were slaughtered, and their blood and liver were sampled. The concentration of glucose, triglycerides, total cholesterol, and protein and their fractions were tested in the blood serum. In livers, total protein and fat contents were analyzed. It has been found that replacing wheat middlings with 10% and 15% of expeller results in (p ≤ 0.05) a lower final body weight than CT. A higher feed, fat, protein, and methionine intake was noted in groups EX5-EX15 compared to CT. No alterations were found in the protein and fat content in the livers and the blood lipid profile of chickens. Changes in the protein metabolism of broilers indicate the need to research. WGE does not interfere with the metabolism of carbohydrates and lipids. However, WGE did not contribute to obtaining production benefits.

1. Introduction

It is imperative nowadays to look for solutions in poultry feeding that would involve introducing components that may improve poultry welfare and resistance to diseases and ensure and improve the health of raw poultry meat. In May 2020, the European Commission presented the “From Farm to Fork” strategy as one of the key initiatives of the European Green Deal. The priority of this action is food security, based on, among other things, animal welfare [1]. In contrast, industrial food production generates many by-products which may be managed, e.g., by using them as components of animal feedstuffs, which limits the costs of their utilization and, at the same time, does not burden the environment [2]. By-products include a wide range of feedstuffs obtained from, among others, cereal grain and oilseed cleaning, milling, or extraction [3,4].

Wheat germ (WG), a significant by-product of the cereal industry, accounts for 2.5 to 3.8% of the total grain weight [5]. Wheat germ is widely recognized as a nutritious raw material for incorporation into food product formulations or as a food in its own right. Wheat germ, containing about 8–14% oil (average 10%), is mainly used in food, medical and cosmetic industries as a source of oil [6]. Wheat germ, also used in animal feeding, is characterized by a high content of vitamins (e.g., B1 and B2), mineral components (potassium, magnesium, calcium, zinc, manganese), polyunsaturated fatty acids (PUFA), dietary fiber, bioactive compounds, e.g., carotenoids, tocopherols, tocotrienols, phenolics, phytosterols. Moreover, in comparison to whole grains, wheat germ contains fewer antinutrients—raffinose and lectins [5,7]. However, these bioactive compounds are prone to oxidation and degradation under the conditions used for conventional extraction and refining methods [8]. Also, wheat germ has a poor shelf life due to the presence of unsaturated fatty acids and oxidative and hydrolytic enzymes, making the product highly susceptible to rancidity. Another by-product used in broiler feeding is a defatted wheat germ (DWG). It is a by-product of wheat oil extraction. It includes over 30% protein of high nutritional value, with a well-balanced amino acid profile, as it includes 17 amino acids, especially the essential amino acids (EAA), such as lysine (Lys), methionine (Met) and threonine (Thr) [9,10]. In food research conducted on poultry in the 1960s, raw or sterilized wheat germ and wheat germ flour were already used as feed compounds. It follows from the literature [11] that using these components resulted in reduced body weight of birds, increased excretion of fat, and pancreatic hypertrophy among the chickens fed a high amount of wheat germ additive (33.5 and 38.5%). Due to the abovementioned risks, these supplements were replaced by wheat germ oil [12] or a fermented wheat germ extract [13]. Commission Regulation (EU) [14] allows using wheat germ expeller in feedstuffs, which is a product of oil manufacture, obtained by pressing wheat germ (Triticum aestivum L., Triticum durum Desf. and other cultivars of wheat) and de-husked spelled (Triticum spelta L., Triticum dicoccum Schrank, Triticum monococcum L.), to which parts of the endosperm and taste may still adhere (item 1.11.13). In animal feeding, using by-products provided by various local agricultural and food industries, especially the oil industry, is preferred and more economically justified than using imported components [15,16].

In the available literature, few studies exist on the use of wheat germ expeller in broiler chickens’ nutrition. They are mainly concerned with the energy and nutritional value of the obtained meat [11]. No studies have been found on this additive’s effect on the chickens’ metabolism. Therefore, the study aimed to (1) determine the influence of wheat germ expeller on the performance of Ross-308 chicken after adding to the feed (by replacing wheatmeal) 5%, 10%, and 15% wheat germ expeller, (2) evaluate the influence of wheat germ cake on selected parameters of carbohydrate, lipid, and protein metabolism in their blood serum.

2. Materials and Methods

2.1. Ethics Statement

Advisory Team approved the experiment for the Welfare of Animals in The Faculty of Biology and Animal Science of Wrocław University of Environmental and Life Sciences (Decision no. 1/2019). The study protocol did not require the approval of the Ethics Committee. The chickens were maintained according to European Union and Ethical Commission regulations [17].

2.2. Experimental Design and Diets

The dietary experiment (pilot study) was conducted on 112 Ross-308 broiler chickens from the Złotoryja Hatchery in Poland (PL 16096409, Ferma Tarnica 1, 41-156 Gracze). Male one-day-old chicks were vaccinated with Poulvac IB Primer (Zoetis Polska Sp. z o.o., Warszawa, Poland) against infectious bronchitis and with Nobilis ND C2 (Intervet International BV, AN Boxmeer, The Netherlands) against Newcastle disease virus. They were randomly allocated into 28 battery cages (4 birds per cage), constituting four groups (7 replications per group). The maximum bird density was lower than 16 kg/m² by the requirements of the European Council Directive 2007/43/EC [18]. During the experiment’s first day, the room temperature was 32–33 °C and was systematically lowered to 20–22 °C on the last day. Moreover, an intermittent light program was used, with a repeated light and dark cycle of 18 and 6 h, respectively (with the exclusion of the first two days of the grow-out, when the chicks had access to light 24 h/24 h), according to the requirements included in the information brochure concerning Ross-308 chicken grow-out [19].

During the experiment, the chickens had constant access to water. Chickens from the control group (CT—Control Treatment) were fed standard, isoenergetic, and isoprotein mixes ad libitum: starter—from day 1 to day 10, grower—from day 11 to day 25, and finisher –from day 26 to 43rd day of life; the feeds were based on wheat, corn, and post-extraction soya middlings. All diets were served in a loose form, and their formulation was adjusted to the feeding requirements of Ross-308 chickens [20]. In contrast, chickens from all three experimental groups, EX5, EX10, and EX15, were fed where wheat middlings was replaced with 5, 10 and 15% of wheat germ expeller, respectively. The dietary ingredients used in the experiment are presented in

Table 1. Dietary ingredients content, metabolizable energy (ME) value (MJ), and essential nutrients of experimental diets (g/kg of feed).

Ingredients	Starter (1–10 d)				Grower (11–25 d)				Finisher (26–43 d)
	CT	EX5	EX10	EX15	CT	EX5	EX10	EX15	CT	EX5	EX10	EX15
Ground maize	403.0	432.9	459.9	486.7	443.7	470.6	496.5	523.4	446.5	472.4	499.3	526.2
Ground wheat	150	100	50	0	150	100	50	0	150	100	50	0
Soybean meal	358	326	296	266	304	274	245	215	303	274	244	214
Rapeseed oil	48	50	53	56	60	63	66	69	65	68	71	74
Wheat germ expeller	0	50	100	150	0	50	100	150	0	50	100	150
Sodium bicarbonate	5.40	5.40	5.32	5.32	5.77	5.74	5.72	5.69	5.77	5.75	5.72	5.68
Monocalcium phosphate	13.7	13.7	13.6	13.6	12.9	12.8	12.8	12.7	12.0	12.0	11.9	11.9
Limestone	13.7	13.8	14.0	14.2	14.1	14.3	14.5	14.7	12.6	12.9	13.1	13.3
L-Lysine	2.14	2.21	2.24	2.27	2.86	2.89	2.89	2.92	0.84	0.86	0.87	0.91
DL-Methionine	2.77	2.61	2.45	2.29	2.73	2.57	2.40	2.25	1.66	1.50	1.33	1.17
L-Threonine	0.71	0.87	1.01	1.15	1.49	1.62	1.75	1.90	0.07	0.21	0.34	0.48
Premix 0.25%	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50	2.50
ME [MJ/kg of feed] value and essential nutrients of experimental diets [g/kg of feed]
ME	12.50	12.49	12.50	12.50	12.98	12.99	12.99	12.99	13.19	13.19	13.19	13.19
Dry matter	892	899	905	910	893	901	907	912	893	901	906	911
Crude protein	221	220	220	220	200	200	200	200	200	200	200	200
Crude fiber	28.3	27.2	26.2	25.1	27.1	26.1	25.0	24.0	27.1	26.1	25.0	24.0
Ca	9.45	9.40	9.40	9.40	9.20	9.21	9.20	9.20	8.50	8.51	8.50	8.50
P available	4.30	4.30	4.30	4.30	4.00	4.00	4.00	4.00	3.80	3.80	3.80	3.80
Na	1.60	1.61	1.60	1.60	1.70	1.70	1.70	1.70	1.70	1.70	1.70	1.70
Lysine (total)	12.00	12.00	12.00	12.00	11.51	11.51	11.50	11.50	9.50	9.52	9.50	9.51
Methionine (total)	5.50	5.50	5.50	5.50	5.20	5.20	5.20	5.21	4.30	4.30	4.30	4.30
Threonine (total)	8.01	8.00	8.00	8.00	8.01	8.00	8.00	8.01	6.60	6.61	6.60	6.60

Abbreviations: CT, Control Treatment; EX5, Experimental diet with 5% wheat germ expeller; EX10, Experimental diet with 10% wheat germ expeller; EX15, Experimental diet with 15% wheat germ expeller. The content of additives added in the premix (0.25%) in 1 kg of the mixture: Vitamin A—8100 UI.; D3—3000 UI.; E—22.50 UI.; K3—1.25 mg; B1—1.25 mg. B2—4.60 mg; B6—2.20 mg; B12—0.02 mg; PP—20.0 mg; choline chloride—184.25 mg; calcium D-pantothenate—6.50 mg; folic acid—0.34 mg; biotin—0.10 mg; betaine; hydrochloride—41.89 mg; Cu—10 mg; Fe—30 mg; Zn—60 mg; Mn—70.40 mg; J—0.75 mg; Se—0.20 mg; Substances improving digestibility: Endo-1.4.beta-xyl—300 U; Subtilisin—4.000 U; Alpha-amyl—400 U; Endo-1.4-beta-xylanase—1.525 U. Endo-1.3(4)-beta-glucanase—190 U; 6-phytase—500 FTU.

. Metabolic energy was calculated for feed components based on the results of chemical analyses, before formulating diet recipes, per the guidelines of the Polish Nutritional Recommendations and Nutritional Value of Feeds for Poultry [21].

The body weights of the birds were measured on the first day and on days when the complete feed mix was switched into the grower feed (day 11), the finisher feed (day 26), and the last day of the experiment (day 43). It was measured using an electronic weigh made by Radwag (Poland) to the nearest 0.1 g.

Twelve hours before the end of the experiment, the chickens were laid off the feed and provided only with water. On the last day of the investigation, eight birds from each group were randomly selected and slaughtered, according to the procedures specified in Annex IV of the Directive 2010/63/EU [22] of the European Parliament and the Council [1].

During bleeding, the blood was collected into polyprenol collection tubes with a coagulation activator made by Sarstedt. After the clot was centrifuged (4 °C, at 3500 r.p.m., for 20 min.) in a centrifuge (MPW-350R, MPW Med. Instruments, Warszawa, Poland), the blood serum underwent the biochemical analysis on the same say. The retracted livers were frozen and stocked at −18 °C until chemical determinations were done.

2.3. Chemical Analysis

Before the experiment, an analysis of the basic chemical structure of the wheat germ expeller (

Table 2. Chemical composition of wheat germ expeller (Mean, SD).

Items	Unit	Wheat Germ Expeller
Gross energy	MJ kg−1 of feed	14.14
Crude protein	%	35.6 ± 2.5
Crude fat	%	6.0 ± 0.5
Carbohydrates	%	27.8
Crude ash	%	4.75 ± 0.20
Water and volatiles	%	11.6 ± 0.8
Total fiber	%	2.8 ± 0.3
Nonessential amino acid	g kg−1 of feeds
Alanine		17.05
Arginine		17.61
Aspartic acid		22.53
Glutamic acid		38.81
Glycine		16.20
Proline		11.65
Serine		12.49
Tyrosine		10.37
Total NEAA		146.71
Essential amino acid
Cysteine		5.45
Histidine		12.29
Isoleucine		3.70
Leucine		13.60
Lysine		15.20
Methionine		7.31
Phenylalanine		10.14
Threonine		8.36
Tryptophan		2.23
Valine		5.25
Total EAA		83.53
EAA:NEAA		0.57

) and the feeds used in the study (

Table 3. Chemical composition of the experimental diets (applies to the finisher’s diet).

Items	Unit	CT	EX5	EX10	EX15
Gross energy	MJ kg−1 DM	19.71	19.80	19.70	19.82
Dry matter	%	90.59	90.66	91.51	91.71
Crude protein	%	23.38	23.46	23.14	22.98
Ether extract	%	6.06	6.09	6.67	7.15
Crude ash	%	5.31	4.84	5.91	5.72
Nitrogen free extractives	%	52.77	53.13	52.58	53.28
Crude fiber	%	3.07	3.14	3.21	2.63
Crude Protein: Energy ratio		1.19	1.18	1.17	1.16
Nonessential amino acid	g kg−1 DM
Alanine		11.82	11.39	11.72	11.73
Arginine		13.58	12.67	13.21	13.03
Aspartic acid		24.72	23.71	22.92	22.20
Glutamic acid		49.71	46.07	43.32	41.35
Glycine		10.52	9.87	9.88	9.70
Proline		15.08	14.03	13.52	12.75
Serine		12.52	12.39	11.93	11.52
Tyrosine		6.90	6.51	6.16	5.83
Total NEAA		144.9	136.6	132.7	128.1
Essential amino acid
Cysteine		4.20	4.32	4.0	3.93
Histidine		7.17	6.68	6.71	6.58
Isoleucine		8.37	7.26	7.36	6.65
Leucine		19.79	18.31	18.38	17.64
Lysine		13.83	12.91	13.36	13.06
Methionine		3.66	4.34	4.24	4.21
Phenylalanine		11.78	10.49	10.32	9.87
Threonine		10.29	10.10	10.06	9.82
Tryptophan		2.34	2.26	2.21	2.14
Valine		9.36	8.12	8.59	8.03
Total EAA		90.8	84.8	85.2	81.9
EAA:NEAA		0.62	0.62	0.64	0.64

Abbreviations: CT, Control Treatment; EX5, Experimental diet with 5% wheat germ expeller; EX10, Experimental diet with 10% wheat germ expeller; EX15, Experimental diet with 15% wheat germ expeller.

) was conducted. The chemical composition of feed components was analyzed following the requirements of the Association of Official Analytical Chemists [23].

The following contents were assayed: gross energy, measured in calories with the use of calorimeter KL-10 (Precyzja-Bit PPHU, Sp. z.o.o., Bydgoszcz, Poland); moisture (%), measured by the oven-drying of 2 g samples at 105 °C for 24 h to a constant weight in a Pol-Eco SLN 115 Eco SUP-4M laboratory dryer (POL-EKO, Wodzisław Śląski, Poland) (950.46B, p. 39.1.02); total nitrogen, measured with the Kjeldahl method converted (a conversion factor 6.25) into an amount of crude protein (%) on the Kjeltec 2300 Foss Tecator distiller (Häganäs, Sweden) (992.15, p. 39.1.16); and crude fat content (%), measured by petroleum ether extraction using a Büchi Extraction System B-811 (Büchi, Flawil, Switzerland) (960.39 (a), p. 39.1.05). The ash (total mineral content%) was determined by incineration at 650 °C for 10 h in an FCE 7SHM muffle furnace Czylok (Jastrzębie Zdrój, Poland) (920.153, p. 39.1.09). The crude fiber was assessed using the van Soesta method in ANAKOM 200 fiber Analyzer (978.10). Carbohydrates contents in feeds were evaluated from the difference between dry matter and the sum of other fixed components.

The value of 18 amino acids in feeds was also established (Table 3). The amino acid profile without Tryptophan (Trp) was determined with the use of an Amino Acids Analyzer AAA 400 (INGOS, Praga, Czech Republic) according to AOAC, 994.12 protocol [24]. The Trp content measurement was performed using HALO DB-20 double beam spectrophotometer (Dynamica Scientific Ltd., 4 Bain Square, Kirkton Campus, Livingston, UK) at λ = 590 nm, according to AOAC 988.15 method [24]. The Essential amino acid (EAA) was constituted by adding the analyzed values of Cys, Met, Thr, Val, Ile, Leu, Tyr, Phe, Lys, and His. The nonessential amino acid (NEAA) was calculated by adding Asp, Ser, Glu, Gly, Ala, Arg, and Pro.

Biotests from BioSystems were used to determine selected metabolic parameters in the blood of broiler chickens: glucose (no. cat. 11803), total protein (no. cat. 115720), triacylglycerols (no. cat. 11828), total cholesterol (no. cat. 11805), HDL fraction -cholesterol (HDL-C) (no cat. 11648), LDL-cholesterol fraction (no cat. 11579).

Glucose concentration in blood serum was determined using the Shimadzu UV-1900i spectrophotometer (Shimadzu Corporation, Kyoto, Japan). In terms of protein metabolism parameters, the concentration of total protein (TP) was determined using the biuret method in an automatic biochemical analyzer A15 by BioSystems (Barcelona, Spain) and protein fractions—prealbumins, albumins: α1-, α2- β1- β2- and γ-globulins using the capillary electrophoresis (no cat. 2223) in the Maxi Kit Minicap Proteine 6 analyzer by Sebia (Lisses, France). Each protein fraction was expressed as relative concentrations (%) according to the obtained optical density. The fractions’ absolute concentrations (g/L) were consequently quantified from the total serum protein concentrations. The albumin to globulins (A/G) ratio was calculated by dividing the sum of pre-albumin and albumin by the sum of globulin fractions.

Among the parameters of lipid metabolism in the serum of chickens, the following concentrations were determined: triacylglycerols (TG) and total cholesterol (TC) by the enzymatic method, HDL-cholesterol fraction (HDL-C) by the precipitation method, and LDL-cholesterol fraction by the direct way (LDL-C). The percentage content of lipoprotein fractions: pre-β-lipoproteins (VLDL-C) was determined in the serum of chickens, using the electrophoretic separation method on the Paragon Electrophoresis System HYDRAGEL Lipo K20 gel (no cat. 3007) by Sebia, and the reading was done in a Beckman Appraise densitometer.

2.4. Indices

The Feed Conversion Ratio (FCR) factor was calculated following the formula:

FCR % = total feed intake (g)/final body weight (g);

The Protein Efficiency Ratio (PER) was calculated following the formula:

PER = weight gain (g)/weight of protein consumed (g) [25].

Moreover, the indicators of the Atherogenic Index of Plasma in the blood serum lipid profile were also established [26,27]:

• TC/HDL-C (Coronary Risk Index),
• LDL-C/HDL-C,
• TG/HDL-C,
• (TC—HDL-C)/HDL-C.

2.5. Statistical Analysis

The results obtained were verified for normality distribution with the Shapiro-Wilk Test and homogeneity variation with Laven’s test. The findings were log-transformed to attain or approach a normal distribution, and subsequently, a one-way analysis of variance (ANOVA) was made. Statistically significant differences between the averages of the groups were calculated using Tukey’s multiple comparisons test, on the level of significance p ≤ 0.05 and p ≤ 0.01, with the use of Statistica^® 13.1 software [28]. The tables show arithmetic means and standard error of the mean (SEM). All data are reported as means of 2 parallel measurements.

3. Results and Discussion

In recent years, there has been an increased interest among poultry producers in alternative feed components that could be used in feed mixes used in intensive farming. This interest stems from expectations to minimize animal husbandry’s negative environmental impact and reduce the cost of importing feed components [29]. One of the components in the production of complete feed mixes for poultry could be wheat germ expeller, a by-product of the oil industry.

Based on conducted research (

Table 4. Feed intake and body weight gain in male 43-day-old Ross-308 broilers (Mean, SEM, n =32).

Item	CT	EX5	EX10	EX15	SEM	p-Value
Initial body live weight (g)	36.6	37.1	36.8	37.1	0.359	0.932
Final body live weight (g)	2514 a	2379	2270 b	2264 b	35.8	0.042
Daily feed intake (g)	4258 B	4580 A	4461	4382	41.0	0.032
Feed consumption (g/100 g of BW)	169.7 B	194.1 A	197.1 A	193.9 A	0.004	0.001
Body weight gain (g)	2477 a	2342	2233 b	2227 b	35.8	0.041
Body weight gain (g/100 of feed)	58.2 A	51.2 B	50.1 B	50.8 B	0.854	0.001
Energy intake (kJ/100 g of BW)	32.5 Bb	36.3 Ba	41.2 A	34.7 B	0.720	0.001
Protein intake (g/100 g of BW)	39.7 Bb	45.5 A	45.6 A	44.6 a	0.686	0.001
Fat intake (g/100 g of BW)	10.3 B	11.8 ADb	13.1 Aa	13.9 AC	0.284	0.001
Carbohydrate intake (g/100 g BW)	115.3	109.2	110.0	108.3	1.38	0.278
Protein Efficiency Ratio (PER)	2.49 Aa	2.18 B	2.16 B	2.21 b	0.036	0.001
Feed Conversion Ratio (FCR)	1.70 B	1.94 A	1.97 A	1.94 A	0.030	0.001

Abbreviations: CT, Control Treatment; EX5, Experimental diet with 5% wheat germ expeller; EX10, Experimental diet with 10% wheat germ expeller; EX15, Experimental diet with 15% wheat germ expeller; SEM, standard error of the mean; BW: body weight. Means within a row followed by different superscript letters differ significantly ^A–D p ≤ 0.01; ^{a, b} p ≤ 0.05.

), it has been concluded that replacing wheat middling in the 10% (EX10) and the 15% mix (EX15) with wheat germ expeller had a significant (p ≤ 0.05) impact on the final reduction in the chickens’ body weight, in comparison to the control group CT. However, compared to the experimental groups, chickens from the CT group were characterized by a significantly (p ≤ 0.01) lowest feed intake per 100 g of body weight. This stems from a difference in feed components and their use, which is indicated by significant (p ≤ 0.01) differences in FCR indicators between the examined groups of animals. Chickens in the CT group were characterized by a lower (p ≤ 0.01) value of this indicator by 0.24 and 0.27% compared to the broilers in the experimental groups. The value of the FCR indicator in the CT is comparable to the one established by Livingston et al. [30] in males of this chicken breed (1.704%).

One of the factors affecting the amount of feed taken in by animals is its energy value [31]. The feed with an addition of 10% of wheat germ expeller EX10 had the highest energy value (Table 3), therefore in this group of chickens, the highest energy intake was determined (Table 4) as per 100 g of body weight (BW), in comparison to the CT group. Similarly, in group EX10, the highest (p ≤ 0.01) protein intake was determined. However, the highest (p ≤ 0.01) intake of fat was determined in chickens that were fed the feed with 15% of wheat germ expeller EX15, which undoubtedly resulted from its higher content (7.15%) in the feed (Table 3). It has been established that an increase in fat content in the feed not only provides a higher amount of energy but also improves the palatability of the feed and slows down the passage through the gastrointestinal tract enabling better absorption of the nutrients and energy [32].

It should be acknowledged that the highest (p ≤ 0.01; p ≤ 0.05;) body weight increase, both in absolute value and calculated per 100 g of consumed feed, was achieved by chickens in the CT group, compared to the EX10 and EX15 groups. Broilers from the CT group used their feed and its protein most effectively (even though they consumed the least amount of feed), which is indicated by the significantly (p ≤ 0.01) highest values of the indicator PER, in comparison to the experimental groups (Table 4). A lower feed intake in group CT could result from its larger protein content (Table 3), which is the most filling. Similarly to mammals, in response to the presence of amino acids (especially lysine), cholecystokinin (CCK) is released in the small intestine of birds, which functions anorexigenically on the neuron populations in the arcuate nucleus of the hypothalamus, where co-expression of proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) takes place [33]. Other gastrointestinal hormones, such as Glucagon-like peptide-1, peptide YY or ghrelin, could have influenced a decrease in feed consumption by the chickens [34,35]. Moreover, better utilization of the total protein and amino acids that it contains by the broilers from the CT group may guarantee a more effective production and foster environmental protection by decreasing the amount of excreted nitrogenous compounds [36].

The lowest (p ≤ 0.01) consumption of total protein by broilers in group CT (Table 4) affected the amount of amino acids consumed (

Table 5. Calculated average daily intake of amino acids by male 43-day-old Ross-308 broilers (Mean, SEM, n = 32).

Amino Acids Intake (g/100 g of BW)	CT	EX5	EX10	EX15	SEM	p-Value
Nonessential amino acid
Alanine	20.1 B	22.1	23.1 A	22.7 A	0.336	0.001
Arginine	23.0 B	24.6	26.0 A	25.3	0.352	0.001
Aspartic acid	41.9	46.0	45.3	43.0	0.605	0.063
Glutamic acid	84.4	89.4 a	86.2	80.2 b	1.187	0.042
Glycine	17.9	19.2	19.5	18.8	0.249	0.112
Proline	25.6	27.2	26.7	24.7	0.359	0.053
Serine	21.2 Bb	24.0 A	23.5 a	22.3	0.338	0.009
Tyrosine	11.7	12.6 a	12.1	11.3 b	0.169	0.031
Total NEAA	245.8	265.2	262.3	248.3	3.40	0.115
Essential amino acid
Cysteine	7.13 Bb	8.38 A	7.89 a	7.62	0.125	0.012
Histidine	12.2	13.0	13.2	12.8	0.167	0.144
Isoleucine	14.2 a	14.1	14.5 a	12.9 b	0.198	0.013
Leucine	33.6	33.5	136.2	34.2	0.456	0.165
Lysine	23.5 b	25.1	26.3 a	25.3	0.348	0.025
Methionine	6.21 B	8.42 A	8.36 A	8.16 A	0.191	0.001
Phenylalanine	20.0	20.4	20.3	19.1	0.253	0.307
Threonine	17.5 Bb	19.6 a	19.8 A	19.0	0.282	0.006
Tryptophan	3.97	4.39	4.36	4.15	0.059	0.036
Valine	15.9	15.8	16.9	15.6	0.210	0.097
Total EAA	154.1	164.6	168.0	158.9	2.15	0.106

Abbreviations: CT, Control Treatment; EX5, Experimental diet with 5% wheat germ expeller; EX10, Experimental diet with 10% wheat germ expeller; EX15, Experimental diet with 15% wheat germ expeller; SEM, standard error of the mean; BW: body weight. Means within a row followed by different superscript letters differ significantly ^{A, B} p ≤ 0.01; ^{a, b} p ≤ 0.05.

). An analysis of consumption of the NEAA included in the feed mixes indicates that the broilers fed with a feed containing wheat middling (CT) consumed the smallest (p ≤ 0.01) amount of Ala (compared to EX10 and EX15 groups), Arg (compared to EX10 group) and Ser (compared to EX5 and EX10 groups). The broilers fed with the feed mixture that included 15% of wheat germ expeller (EX15) consumed less (p ≤ 0.05) Glu and Tyr than those from group EX5. When it comes to EAA, the chickens from the group CT consumed less (p ≤ 0.01; p ≤ 0.05) Cys and Thr compared to EX5 and EX10 groups, whereas consumption of Met was the lowest (p ≤ 0.01) compared to all the experimental groups (Table 5). However, Ile’s lowest (p ≤ 0.05) intake was determined in the broilers from group EX15, compared to the groups EX10 and CT. Yet, no significant differences between the groups of examined chickens were established regarding the total consumption of NEAA and EAA.

An analysis of selected biochemical parameters in the broilers’ blood allows the identification of metabolic changes resulting from many endogenous and exogenous factors, e.g., genetic type, farming conditions, season, sex, and age [37]. It has been shown that the concentration of total protein and total cholesterol in broiler chickens of the same breed (Isa Vedette) increased with age (1–7 weeks) but did not differ significantly depending on sex. The concentration of triacylglycerol in the serum of chickens was related to age in birds of both sexes, but this was not observed in the concentration of glucose [38].

In commercial farming conditions, where broiler chickens are fed standard feed mixes, the husbandry line and age of the birds seem to be the key factors that affect the intensity of the body metabolism, which is reflected in, among other things, changes in the value of blood parameters. Many factors of the so-called environmental stress may, even if it operates at a low intensity but a duration long enough to affect the animal body, lead to an imbalance in its body homeostasis [39].

The ingredients of the feed mix may be one of such non-specific factors. Metabolic changes resulting from such stressors are adapted in the body. An apparent adaptive reaction in a body is evident by glucose and lipids in the blood, which largely depends on an efficient liver operation.

In the study, the glucose concentration values in the blood serum were not significantly different between groups (

Table 6. Fat content in the liver and selected parameters carbohydrate-lipid metabolism in the blood serum of male 43-day-old Ross-308 broilers (Mean, SEM, n = 32).

Trait	Unit	CT	EX5	EX10	EX15	SEM	p-Value
Fat Liver	%	5.86	5.66	5.0	4.09	0.266	0.051
Glucose (GL)	mMol/L	12.3	14.0	15.0	15.4 9	0.518	0.151
Triacylglycerol (TG)	mMol/L	1.67	0.95	0.73	1.19	0.274	0.819
Total cholesterol (TC)	mMol/L	4.6	4.5	4.9	4.7	0.156	0.666
HDL-C	mMol/L	1.99	1.9	2.5	2.38	0.103	0.091
LDL-C	mMol/L	2.10	2.10	2.42	2.34	0.055	0.078
Non-HDL-C	mMol/L	2.64	2.55	2.37	2.30	0.156	0.814
VLDL-C	%	22.88	24.0	20.0	20.25	1.043	0.788
TC/HDL-C		2.35	2.33	2.08	2.07	0.088	0.467
LDL-C/HDL-C		1.05	1.12	1.03	1.05	0.036	0.781
TG/HDL-C		0.83	0.50	0.32	0.56	0.135	0.660
HDL-C/TC		0.43	0.45	0.52	0.51	0.020	0.373

Abbreviations: CT, Control Treatment; EX5, Experimental diet with 5% wheat germ expeller; EX10, Experimental diet with 10% wheat germ expeller; EX15, Experimental diet with 15% wheat germ expeller; SEM, standard error of the mean.

). At the same time, the values of glucose concentration were typical for males of Ross-308 chickens on day 42 of their breeding [30,38]. Similar values of glucose concentration in the blood of studied chickens, independently of the composition of the feed mixes consumed by the chickens, might have resulted from a lack of statistically confirmed differences in carbohydrates consumed in the feed (Table 4) but also from the correct glucostatic function of the liver [40].

In general, fats consumed in the diet deliver fatty acids with the blood to the liver, which undergo esterification and synthesis of lipoproteins, and then released into the bloodstream [40]. It was therefore expected that the increased fat intake in the feed (Table 4) of broilers fed with complete feed mixtures with a varied content of wheat germ cake (EX5-EX15) would contribute to an increase in the concentration of lipoproteins in the blood of chickens. All the more so because in contrast to mammals, the synthesis of fat in birds is greater in the hepatic tissue and very limited in the adipose tissue [41]. Yet, such a result was not observed, as no significant differences between the fat contents in the livers of chicken, nor in the concentration of lipids and lipoproteins in blood serum was determined (Table 6), irrespective of the ingredients used in feed. Concentrations of triglycerides, total cholesterol and its fractions LDL-C, HDL-C, and the percentage of VLDL-C marked in the blood serum of examined broilers were not significantly different and stayed within the ranges considered to be correct for the males of the Ross-308 breed [38].

A smaller consumption of protein by the broilers fed the diets with a varied percentage of wheat germ expeller (Table 4) did not result in changes in its contents both in the liver as well as the total protein concentration in the blood serum, in comparison to the CT group (

Table 7. Protein content in the liver and total protein and its fractions in the blood serum of male 43-day-old Ross-308 broilers (Mean, SEM, n = 32).

Trait	Unit	CT	EX5	EX10	EX15	SEM	p-Value
Protein liver	%	20.2	19.5	20.4	20.5	0.20	0.291
Total protein	g/L	31.5	30.2	29.1	29.5	0.40	0.189
Pre-albumin	g/L %	2.23 7.13	1.81 6.06	1.90 6.60	1.86 4.96	0.14 0.40	0.186 0.186
Albumin	g/L %	16.2 51.5	14.4 48.2	13.2 45.6	17.7 48.4	0.83 1.12	0.120 0.328
α1-globulin	g/L %	1.51 4.76	1.61 5.36	1.83 6.38	1.46 4.16	0.11 0.37	0.743 0.269
α2-globulin	g/L %	3.93 12.4	2.66 9.03	1.79 6.04	3.33 8.34	0.35 0.89	0.148 0.138
β1-globulin	g/L %	2.90 9.10	3.45 11.2	3.88 13.2	4.09 11.5	0.28 0.76	0.577 0.482
β2-globulin	g/L %	1.74 Bb 5.48 Bb	3.05 a 9.93 a	3.60 A 12.3 A	3.05 a 9.27 a	0.21 0.70	0.003 0.001
γ-globulin	g/L %	3.02 9.59	3.18 10.4	2.95 b 9.96	4.80 a 13.4	0.26 0.56	0.033 0.075
A/G		1.43	1.26	1.15	1.11	0.05	0.165

Abbreviations: CT, Control Treatment; EX5, Experimental diet with 5% wheat germ expeller; EX10, Experimental diet with 10% wheat germ expeller; EX15, Experimental diet with 15% wheat germ expeller; SEM, standard error of the mean. Means within a row followed by different superscript letters differ significantly ^{A, B} p ≤ 0.01; ^{a, b} p ≤ 0.05.

). The lack of these differences indicates a proper function of the liver in terms of amino acid metabolism [42].

The relation between individual protein fractions in serum reflects birds’ functional, metabolic, and health status. Their concentration in the blood serum depends on, among other things, the genetic type, husbandry conditions, season, sex, and age of the chickens [43,44]. The plasma proteins are sensitive to nutritional influences, but the changes are often subtle and difficult to detect and interpret. Plasma proteins play an important role in keeping the body homeostasis, as they significantly affect the regulation and maintenance of the correct values of colloid osmotic pressure. They are a source of free amino acids in glucose synthesis in gluconeogenesis, participate in the transport of mineral elements and hormones, and in the synthesis of enzymes and antibodies of the immune system [42,45]. The determined concentrations of total protein are similar to the findings published by other authors concerning male Ross-308 chickens on day 42 with growth [30,46].

As birds with seemingly correct contents of total protein may exhibit irregularities in concentrations of its fractions, protein electrophoresis in blood serum is used as an effective diagnostic tool [47]. In the conducted research, no significant changes in the concentration and percentage of albumins were established (Table 7) in the blood serum of broilers from various groups. It might result from the fact that it is a source of amino acids that participate in the synthesis of tissue proteins, especially in the fast somatic growth of broilers in the final stage with growth. The determined concentrations of albumins in the blood serum of broilers are similar to the findings reported by other authors [44,46], who examined the biochemical profile of blood in male Ross-308 chickens on day 42 of feeding.

No significant differences among chickens from various groups were noted when it came to the concentration and percentage of fractions α1-, α2, and β1-globulin in the blood serum, and the values determined in this research are similar to those concerning broilers with the same genotype noted by other authors [43,44,46]. Similar concentrations of α1-globulin, α2-globulin, and β1-globulin in the blood serum of chickens from various groups (Table 7) were confirmed by a lack of significant differences in the concentration of HDL-cholesterol fraction, LDL-cholesterol, and percentage of VLDL-cholesterol (Table 6), which are the components of these globulins [47].

However, a significant impact was determined by adding wheat germ expeller to the feed on the concentration of β2-globulin fraction in the blood serum of chickens in groups EX5-EX15, compared to CT (Table 7). High concentrations of β2-globulins may be related to hypercholesterolemia. Still, in the conducted research, no significant differences were found in the concentration of total cholesterol and its fractions in the blood serum of chickens from the studied groups (Table 6). Determined values of lipoproteins TG, TC, LDL-C, HDL-C, and VLDL-C in the blood serum of examined groups were similar to those found in the birds of the same breed and sex (on day 42 of breeding) by other authors [38,44]. However, in our opinion, an observed increase of β2-globulin fractions in the blood serum of chickens does not have to indicate dysproteinaemia [45] because no significant differences between the groups of animals in the value of Albumin/Globulin (A/G) factor were noted.

The chicken is a suitable animal model for the study of atherosclerosis since it presents lipoprotein levels similar to humans and develops spontaneous and induced atherosclerosis mainly under high cholesterol diets, vascular injury, or infections similar to human atherosclerosis [48]. Determined values of atherogenic factors in the lipid profile of the blood (Table 6) were not significantly different between the studied groups of chickens, which is a result of a similar concentration of lipoproteins in blood serum. However, β2-globulin fractions, which are transferrin, ferritin, and hemopexin, are related to the metabolism of iron [48]. An increase in this fraction’s concentration in the chickens’ blood may indicate an increased demand for iron, a substrate of hemoglobin synthesis. In chickens, during the period of their intense growth, the total number of red blood cells is increased as a result of intense erythropoiesis, which is a response of the body to a very short (28–35 days) life span of the bird, its high body temperature and an intense pace of metabolism [43]. Chemical compounds occurring in the β2-globulin fraction are, among others, plasminogen, angiostatin, β2-microglobulin, C3 complement, C4 complement, and C-reactive protein. Nevertheless, establishing whether the concentration of these compounds is increased in the blood of the chickens would demand further research.

The conducted research established a significant (p ≤ 0.05) increase in the concentration of γ-globulin fraction in the blood of chickens from group EX15, compared to group EX10. The γ-globulin fraction predominantly comprises immunoglobulins of various classes (IgG, IgA, IgM, IgD, and IgE), produced by adaptive immune system cells, activated B cells, and plasma cells in response to the exposure to antigens [47]. An increase in this fraction’s concentration in chickens fed a mix with 15% wheat germ expeller might suggest immune-mediated disorders, yet establishing that would need further research.

Available literature does not offer any findings related to the influence of feeding broiler chickens or other husbandry animals with a feed that includes wheat germ expeller on the parameters of carbohydrate, lipid, and protein metabolism in blood serum. Therefore, it is not easy to compare the results of our research with the findings of other scientists. Research findings published by other authors [49,50] concern the influence of feeding broilers with feeds that included rapeseed expeller and palm kernel expeller (which are the by-products of the oil industry) on standardized ileal digestibility (SID) of amino acids (AA) and nitrogen-corrected apparent metabolizable energy, or the state of gut microbiota. It should be noted that the components above have a different chemical composition and have a different nutritional value than the wheat germ expeller used in this study. It should also be highlighted that this research should be continued because the ingredients of feeds affect not only the biochemical indicators in the blood of broilers but also the quality of their meat.

Goluch et al. [11] showed that the use of various additives of germ expeller (5%, 10%, and 15%) did not significantly affect the energy value of the muscles, but it did affect their nutritional value, which resulted from the composition of the feed and the amount of its consumption. The chicken breast muscles had lower energy values and contained more moisture, crude protein, P, Mg, Fe, Cu, and Mn. In contrast, with a higher energy value and lower water content, thigh muscles contained more crude fat, crude ash, Ca, and Zn. According to the authors, a 5% addition of germ expeller would be optimal because without worsening the basic chemical composition of the muscles, it increased the content of P, Na, and Ca in them, compared to the CT group, although with slightly less effective indicators of rearing chickens.

4. Conclusions

Using 10% and 15% wheat germ expeller as an alternative feed ingredient significantly impacted the lower daily growth of broilers, and simultaneously encouraged its higher intake, resulting in a lower final body weight of the birds. Changes found in biochemical parameters of protein metabolism in broilers indicate a need for further research on the effectiveness of using wheat germ expeller in feeding. The lack of changes in the values of selected parameters of carbohydrate-lipid metabolism in the blood of chickens allows us to conclude that the components contained in the wheat germ expeller added to the feed do not adversely affect the disturbance of homeostasis in this respect. However, wheat germ expeller did not contribute to obtaining production benefits.