Effects of fermented cottonseed meal on growth performance, serum biochemical parameters, immune functions, antioxidative abilities, and cecal microflora in broilers

ABSTRACT

The study was to investigate the effects of fermented cottonseed meal (FCSM) on growth performance, serum biochemical parameters, immune functions, antioxidative abilities, and cecal microflora in broilers. The cottonseed meal (CSM) was fermented with Bacillus subtilis ST-141 and Saccharomycetes N5. Hundred and twenty-eight one-day-old male Arbor Acres broilers were randomly divided into four groups: a corn-soybean meal (SBM) basal diet, and three experimental diets (CSM, FCSM-1, and FCSM-2) in which the control diet was supplemented with CSM or FCSM to partially replace SBM. Results showed that the fermentation process can effectively decrease free gossypol level, and increase acid-soluble protein level in CSM. The levels of immunoglobulin, biochemical parameters, antioxidative abilities in serum or liver tissue, and Lactobacilli and total anaerobic bacteria counts in ceca digesta of birds fed FCSM-1 were improved compared with birds fed CSM or FCSM-2 on days 21 and 42. So FCSM can be effectively applied in broiler diets.

KEYWORDS:

• Fermented cottonseed meal
• immune function
• serum biochemical parameter
• antioxidative function
• cecal microflora
• broiler

Introduction

Soybean meal (SBM) is generally recognized as an effective and high-quality protein feedstuff (Azarm & Lee, 2014; Kim, 2014; Taliercio, Loveless, & Turano, 2014). However, the supply of high-quality protein feedstuff (such as SBM and fish meal) becomes deficit, and the cost of SBM is increasing and fluctuant, especially in China. China is very rich in cottonseed meal (CSM) and has been considered as an alternative to SBM for a long time. Although CSM is an inexpensive potential source of protein with high protein content, (Nie et al., 2015), its nutrient bioavailability in poultry diets is low due to the presence of anti-nutritional factors, such as free gossypol (FG), cycloproponoic fatty acids, and crude fiber (Tang et al., 2012), which may cause negative effects on growth, reproductive performance, and organ abnormalities (Nie et al., 2015; S. ŚWIĄTKIEWICZ, 2016).

Microbial solid-state fermentation is an effective way to reduce anti-nutritional factors and improve nutritional value of CSM (Alshelmani, Loh, Foo, Sazili, & Lau, 2016; Bortolaia, Damborg, & Guardabassi, 2014; Sun et al., 2015b). Tang et al. (2012) reported that fermentation with Bacillus subtilis BJ-1 can reduce FG level in CSM and dietary inclusion of 12% FCSM can improve growth performance and immunity. In addition, recent studies also showed that replacing SBM with CSM fermented by B. subtilis BJ-1 improved growth performance and intestinal morphology, increased the numbers of beneficial bacteria and the activity of digestive enzymes in the intestinal tract in broilers (Sun et al., 2013a, 2013b). Xiong, Wang, Miao, Meng, and Wu (2016) suggested that fermentation of CSM with Candida utilis markedly decreased free and total gossypol contents, and there was no adverse effect on growth performance when SBM is replaced by FCSM at 50% proportion (155 g/kg) in broilers’ diet. However, there is a big variation in the nutritional value of FCSM fermented by different microorganisms (Zhang, Xu, Sun, & Yang, 2006), which leads to the inconsistent effects in animal. Besides, there is little research on FCSM application in poultry diets.

Therefore, the objectives of this study were to investigate the nutritional value of FCSM fermented by B. subtilis ST-141 and Saccharomycetes N5, and to determine the effects of FCSM (two levels), compared with SBM and CSM, on growth performance, serum biochemical parameters, immunoglobulin level, antioxidant functions, and cecal microflora in broilers.

Materials and methods

Fermentation process of CSM

The strains of B. subtilis ST-141 and Saccharomycetes N5 were preserved by Microbiological research group, Academy of State Administration of Grain (Beijing, China). For the fermentation of CSM, a basal substrate containing 95% CSM, 2% glucose, 2% wheat bran, and 1% ammonium sulfate (w:w:w:w) was mixed with water in a ratio 1:0.5 (w:w). Then, the basal substrate was inoculated with B. subtilis ST-141 (9 logs cfu/mL, 2:10, v/w) and Saccharomycetes N5 (0.5:100, w/w). The mixture was fermented in a stainless steel fermentation tank (diameter: 1360 mm, height: 1360 mm) for 48 h at 30°C. After fermentation, the FCSM samples were dried using the fluidized bed.

Chemical analysis

CSM and FCSM samples were ground to pass through a 1-mm mesh screen for further chemical analysis. The dry matter (DM) content of CSM and FCSM was analyzed by drying the samples at 105°C for 5 h (AOAC, 1999), crude protein (CP) by the automatic Kieldahl apparatus (AOAC, 1999; KjeltecTM 8400, FOSS, China), gross energy (GE) by an adiabatic bomb calorimeter (AOCS, 2009; method Ba 7b-96; Parr Instruments, Moline, IL, USA), crude fiber by Fibertec™ 2010 (AOCS, 2009; method Ba 6a-05; FOSS, China), crude ash by incineration at 550°C for 5 h in a muffle furnace (AOAC, 1999). The concentrations of calcium (method Ca, 15b-87) and phosphorus (method P 12b-92) were analyzed by AOCS (2009). The content of acid-soluble protein was determined by the trichloroacetic acid method (Zhang & Shen, 2013). Amino acid content was analyzed using the amino acid analyzer (Hitachi L-8800, Japan). The value of pH was determined by a pH meter (Ohaus Starter 2100, America). FG contents were determined according to the method of the American Oil Chemists Society (AOCS, 2009; method Ba 7b-96). Saccharomycetes and B. subtilis were analyzed according to the method of ISO 4833 (2013).

Experimental design and bird management

Broilers care and handling were in compliance with the Animal Ethics Committee Guidelines of Academy of State Administration of Grain (Beijing, China). A total of 128 one-day-old male Arbor Acres broilers were obtained from a commercial hatchery (Huadu Broiler Breeding Farms, Beijing, China), and the initial body weight was 41.90 ± 0.22 g. The birds were randomly divided into four groups with eight replicates of four birds each. The control birds were fed with a corn-SBM basal diets, and three experimental diets were supplemented with 8.9% of CSM, 8.9% of FCSM or 17.9% of FCSM in starter phase (days 1–21), and supplemented with 7.5% of CSM, 7.5% of FCSM, or 15.1% of FCSM in the grower phase (days 22–42), respectively. The four groups were labeled as SBM, CSM, FCSM-1, and FCSM-2. Experimental diets (Table 1), in mash form, were formulated to meet nutrient requirements of Chinese feeding standard of chicken (NY/T 33-2004), and all experimental diets had the same nutrient level. Feeding trial consisted of feeding starter (days 1–21) and grower (days 22–42) diets.

Table 1. Ingredient composition and nutrient content of experimental diets containing fermented cottonseed meal.

Items	Starter phase (days 1–21)				Grower phase (days 22–42)
	SBM	CSM	FCSM-1	FCSM-2	SBM	CSM	FCSM-1	FCSM-2
Ingredients (% as-fed)
Corn	52.01	51.9	52.38	52.65	64.27	63.62	63.55	62.76
Soybean meal	40.73	31.15	30.59	20.45	30.15	22.63	22.63	15.09
Fermented cottonseed meal	0	0	8.9	17.9	0	0	7.5	15.1
Cottonseed meal	0	8.9	0	0	0	7.5	0	0
Soybean oil	2.63	3.13	3.16	3.7	1.64	2.07	2.1	2.55
Dicalcium phosphate	1.86	1.79	1.79	1.71	1.28	1.21	1.21	1.15
Limestone	1.2	1.26	1.26	1.33	1.32	1.38	1.38	1.43
Salt	0.45	0.45	0.45	0.45	0.32	0.32	0.32	0.32
Choline chloride (50%)	0.26	0.26	0.26	0.26	0.2	0.2	0.2	0.2
Vitamin premix^a	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Mineral premix^b	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
L-Lysine•HCl (98%)	0.303	0.524	0.531	0.749	0.29	0.473	0.477	0.664
DL-methionine (98%)	0.287	0.31	0.338	0.389	0.257	0.277	0.3	0.343
Threonine (98%)	0.05	0.106	0.121	0.192	0.053	0.1	0.113	0.173
Total	100	100	100	100	100	100	100	100
Calculated chemical composition
Crude protein (%)^c	23.6	23.0	23.4	23.7	20.1	20.3	20.5	20.7
ME (kcal/kg)	3000	3000	3000	3000	3100	3100	3100	3100
Calcium (%)	1.0	1.0	1.0	1.0	0.9	0.9	0.9	0.9
Total phosphorus(%)	0.7	0.72	0.63	0.55	0.58	0.6	0.52	0.46
Non-phytate P (%)	0.45	0.45	0.45	0.45	0.35	0.35	0.35	0.35
Methionine	0.50	0.50	0.50	0.50	0.40	0.40	0.40	0.40
Lysine (%)	1.15	1.15	1.15	1.15	1.00	1.00	1.00	1.00
Met + Cys (%)	0.94	0.94	0.94	0.94	0.82	0.82	0.82	0.82

Note: SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^aVitamin premix provided the following per kilogram of diet; vitamin A, 9500 IU; vitamin D₃, 3800 IU; vitamin K₃, 2.65 mg; vitamin B12, 0.025 mg; thiamine, 2 mg; vitamin B₂, 6 mg; vitamin E, 30 IU; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; nicotinic acid, 50 mg.

^bThe mineral premix provided the following per kg of diet: Cu, 8 mg; Zn, 75 mg; Fe, 80 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg.

^cCrude protein level is analyzed value.

The birds were housed in cages with wire mesh floor in an environmentally controlled room. Environmental temperature in the room was maintained at 32–35°C in the first week and then gradually reduced to 22°C until the end of the trial. The stocking density was 560 cm²/bird. Feed and water were provided ad libitum. The broilers were vaccinated with ND-IB vaccine at day 7, and IBD vaccine at day 14.

Growth performance

On days 21 and 42, body weight and feed intake were recorded on a cage basis after a 12-h fasting. ADG, ADFI, and FCR were calculated during the starter (days 1–21), finisher (days 21–42), and overall period (days 1–42). The mortality was recorded during the whole feeding trial.

Serum biochemical parameters

On days 21 and 42, one bird was randomly selected from each cage, and serum samples were taken from the wing vein. Then the blood samples were centrifuged at 3000 g for 15  min at 4°C, and the serum was harvested and stored at −20°C. The levels of serum IgA, IgM, and IgG were measured by chicken-specific ELISA kits (Uscn Life Science Inc., Wuhan, China). The ELISA procedures were done as described by the manufacturer’s protocol.

The levels of serum TP (cat#: A045-2), ALB (cat#: A028-2), TG (cat#: A110-1), BUN (cat#: C013-2), aspartate aminotransferase (AST, cat#:C010-2), and alanine aminotransferase (ALT, cat#: C009-2) were measured by the commercially available colorimetric diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The level of MDA (cat#: A003-1), and the activities of T-AOC (cat#:A015), GSH-Px (cat#: A005) and T-SOD (cat#: A001-3) in the serum were measured by commercially available colorimetric diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Antioxidative abilities of liver tissue

On days 21 and 42, after collecting serum samples, the birds were euthanized by jugular bleeding. Liver tissues were isolated and immediately put into liquid nitrogen, then the liver tissues were minced and homogenized (10%, w/v) in physiological saline water at 4°C, then centrifuged at 3500 g for 15  min at 4°C, and the supernatant was collected and stored in −20°C for further analysis. The level of MDA (cat#: A003-1), and the activities of T-AOC (cat#: A015), GSH-Px (cat#: A005) and T-SOD (cat#: A001-3) in liver tissue were measured by commercially available colorimetric diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein content of the liver tissue was measured by Coomassie Brilliant Blue G-250 reagent (cat#: A045-2) using bovine serum albumin as a standard. The measured procedures were done as described by the manufacturer’s protocol.

Cecal microbiota

The cecal contents were aseptically removed and put in sterilized tubes, and stored at 4°C for subsequent enumeration of microbial population. The cecal digesta samples were diluted 10-fold with sterilized physiological saline and vortexed for 5 min. The number of Lactobacilli was quantified on MRS agar as described by Chiang et al. (2010) and total anaerobes were cultured on Wilkins-Chalgren agar (Mountzouris et al., 2010). All digesta samples were determined in triplicate, and the best replicates from the resulting dilutions in 30–300 colonies were used to calculate the target microbial populations. The results were expressed as log¹⁰ colony-forming units per gram of digesta for statistical analysis. All agars were obtained from Hopebiol, Bio-technology Co., Ltd (Qingdao, China).

Statistical analysis

The statistical analysis was performed with SPSS 17.0 software for Windows. Data are presented as the mean ± standard deviation. Differences between the mean values for individual groups were assessed by one-way ANOVA and Student–Newman–Keuls tests. A significance level was defined when P-value was < .05.

Results

Chemical composition of CSM and FCSM

Fermentation of CSM with B. subtilis ST-141 and Saccharomycetes N5 dramatically reduced the FG level (from 820 to 346 mg/kg) and dramatically increased the content of acid-soluble protein (from 3.59% to 5.63%). Comparatively, FCSM had lower crude fiber level (8.5% vs. 7.97%) and pH value (6.45 vs. 5.61) than CSM. Moreover, FCSM (wet basis) also had greater concentrations of Saccharomycetes and B. subtilis than CSM. But the contents of other nutrient compositions were similar between FCSM and CSM (Table 2).

Table 2. Chemical composition of cottonseed meal and fermented cottonseed meal used (%, air-dry basis)^a.

Component	CSM	FCSM
Dry matter	91.92	90.2
Crude protein	49.8	51
Gross energy (MJ/kg)	17.85	17.6
Crude fiber	8.5	7.97
Ash	6.83	7.16
Calcium	0.14	0.12
Total phosphorus	1.08	1.07
Acid-soluble protein	3.59	5.63
Amino acids
Aspartate	3.15	2.55
Threonine	1.75	2.49
Serine	1.85	2.18
Glutamate	9.2	8.82
Glycine	1.91	1.9
Alanine	1.79	1.89
Cystine	0.97	0.73
Valine	2.05	1.93
Methionine	0.53	0.43
Isoleucine	1.41	1.38
Leucine	2.83	2.75
Tyrosine	1.17	1.27
Phenylalanine	2.58	2.3
Lysine	1.94	1.93
Histidine	1.53	1.1
Arginine	5.67	5.51
pH	6.45	5.61
Free gossypol (mg/kg)	820	346
Bacillus subtilis (cfu/g)^b	ND	3 × 10^8
Saccharomycetes (cfu/g)^b	ND	2 × 10^8

Note: CSM, cottonseed meal; FCSM, fermented cottonseed meal. ND, not determined.

^aAnalytical values, means of triplicate analyses.

^bBacillus subtilis and Saccharomycetes counts were determined in wet basis before drying.

Growth performance

The effect of FCSM on growth performance is presented in Table 3. From days 1–21, there were no significant effects on ADG, ADFI, and FCR among all the groups (P > .05). Over the whole experimental period (days 1–42), there was no significant effect on ADG among SBM, CSM, FCSM-1 groups, but ADG was significantly decreased in birds fed FCSM-2 diet (P < .05). Compared with the SBM group, FCR was significantly increased in birds fed CSM, or FCSM-2 diet (P < .05), but FCR had no significant difference between CSM and FCSM-2 group (P > .05). From days 22–42, FCR was significant in birds fed FCSM-2 diet (P < .05).

Table 3. Effect of fermented cottonseed meal on the growth performance¹.

Items	SBM	CSM	FCSM-1	FCSM-2	P-value
Days 1–21
ADG (g)	32.7 ± 1.32	31.6 ± 1.47	32.8 ± 1.00	32.8 ± 3.33	.592
ADFI (g)	45.5 ± 1.43	44.4 ± 2.01	46.1 ± 1.83	44.6 ± 4.71	.577
FCR	1.40 ± 0.04	1.41 ± 0.06	1.40 ± 0.04	1.36 ± 0.10	.535
Days 22–42
ADG (g)	75.2 ± 3.66	75.6 ± 2.09	76.3 ± 4.19	70.9 ± 4.42	.093
ADFI (g)	140 ± 11.6	150 ± 5.10	148 ± 9.63	151 ± 7.38	.143
FCR	1.87 ± 0.15^a	1.99 ± 0.05^a	1.93 ± 0.07^a	2.13 ± 0.09^b	.002
Days 1–42
ADG (g)	53.8 ± 2.29^b	53.6 ± 1.28^b	54.4 ± 2.52^b	50.6 ± 2.48^a	.043
ADFI (g)	91.2 ± 6.02	95.4 ± 3.15	94.8 ± 5.67	92.4 ± 6.29	.457
FCR	1.70 ± 0.09^a	1.78 ± 0.05^b	1.74 ± 0.04^ab	1.82 ± 0.09^b	.030

Note: ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio; SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^a,bMeans in the same row with no common superscripts differ significantly (P < .05).

¹Data were means of eight cages of birds.

Serum immunoglobulin level

The effect of FCSM on serum immunoglobulin level is presented in Table 4. On day 21, no significant difference was observed in serum IgA level among all groups. Compared with the other groups, the serum IgM level of birds in the FCSM-1 group was significantly increased on days 21 and 42 (P < .05). The levels of serum IgM and IgG of birds fed FCSM-1 diet were greater than that in the CSM group (P < .05). Compared with the SBM group, the serum IgM level of birds fed CSM diet was significantly decreased (P < .05), but the serum IgM level of birds between SBM and FCSM-2 groups had no significant difference (P > .05) on day 42.

Table 4. Effect of fermented cottonseed meal on serum immunoglobulin level (mg/mL)¹.

Items	SBM	CSM	FCSM-1	FCSM-2	P-value
Day 21
IgA	0.21 ± 0.02	0.19 ± 0.01	0.22 ± 0.09	0.20 ± 0.06	.767
IgM	1.56 ± 0.35^a	1.33 ± 0.28^a	2.40 ± 0.40^b	1.67 ± 0.35^a	.003
IgG	7.02 ± 1.16^ab	5.75 ± 0.87^a	7.98 ± 0.67^b	6.84 ± 1.20^ab	.036
Day 42
IgA	0.23 ± 0.03	0.21 ± 0.22	0.23 ± 0.03	0.22 ± 0.01	.739
IgM	1.70 ± 0.09^b	1.48 ± 0.17^a	2.56 ± 0.19^c	1.73 ± 0.16^b	<.001
IgG	7.39 ± 1.12	6.82 ± 0.75	8.05 ± 1.76	7.31 ± 1.64	.503

Note: ²IgA, immunoglobulin A; IgM, immunoglobulin M; IgG, immunoglobulin G; SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^a,b,cMeans in the same row with no common superscripts differ significantly (P < .05).

¹Data were means of eight birds per treatment.

Serum biochemical parameters

The effect of FCSM on serum biochemical parameters is presented in Table 5. Compared with other groups, the levels of serum TP, ALB, and BUN were significantly decreased on days 21 and 42 (P < .05), and the level of serum TG was significantly increased on day 42 (P < .05) in birds fed CSM diet. The levels of serum TP, ALB, and BUN on day 21 and the level of serum TG on day 42 of birds in SBM, FCSM-1, and FCSM-2 groups had no significant differences (P > .05). There was no significant effect on serum AST and ALT levels of birds among all the groups.

Table 5. Effect of fermented cottonseed meal on serum biochemical parameters¹.

Items	SBM	CSM	FCSM-1	FCSM-2	P-value
Day 21
TP (g/L)	27.8 ± 3.86^b	15.9 ± 7.50^a	28.7 ± 6.31^b	24.6 ± 2.05^b	.004
ALB (g/L)	12.0 ± 0.40^b	9.40 ± 0.83^a	12.5 ± 0.96^b	12.0 ± 0.16^b	<.001
BUN (mmol/L)	1.53 ± 0.25^a	2.64 ± 0.49^b	1.41 ± 0.17^a	1.62 ± 0.21^a	<.001
TG (mmol/L)	0.37 ± 0.12	0.42 ± 0.07	0.37 ± 0.17	0.39 ± 0.10	.891
AST (U/L)	114 ± 14.9	123 ± 19.1	108 ± 12.6	114 ± 0.10	.568
ALT (U/L)	2.59 ± 0.41	3.32 ± 1.72	2.22 ± 0.91	2.42 ± 0.27	.305
Day 42
TP (g/L)	35.8 ± 2.65^b	26.1 ± 3.55^a	37.0 ± 8.04^b	35.2 ± 5.44^b	.001
ALB (g/L)	14.0 ± 1.38^b	11.0 ± 1.10^a	14.6 ± 1.40^b	13.8 ± 1.06^b	<.001
BUN (mmol/L)	1.60 ± 0.25^a	2.84 ± 0.31^b	1.49 ± 0.47^a	1.61 ± 0.31^a	<.001
TG (mmol/L)	0.25 ± 0.08^a	0.33 ± 0.04^b	0.24 ± 0.04^a	0.26 ± 0.05^a	.022
AST (U/L)	34.3 ± 11.6	42.5 ± 12.9	31.6 ± 9.31	33.5 ± 8.67	.281
ALT (U/L)	1.88 ± 0.63	2.41 ± 0.71	1.55 ± 0.45	1.83 ± 1.09	.34

Note: TP, total protein; ALB, albumin; BUN, blood urea nitrogen; TG, triglycerides; AST, aspartate aminotransferase; ALT, alanine aminotransferase; SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^a,bMeans in the same row with no common superscripts differ significantly (P < .05).

¹Data were means of eight birds per treatment.

Serum antioxidative ability

The effect of FCSM on serum antioxidative ability is shown in Table 6. The activities of serum GSH-Px and T-SOD in birds fed FCSM-1 diets were greater than that in the CSM group on day 21 (P < .05). The level of serum MDA was lower and the activities of serum T-AOC and GSH-Px in birds fed FCSM-1 diets were greater than that in the CSM group on day 42 (P < .05). Compared with the SBM group, birds fed CSM diets had lower serum T-SOD and T-AOC activities on day 21 (P < .05), and had higher MDA level on day 42 (P < .05). The activities of serum T-AOC, GSH-Px, and T-SOD on days 21 and 42, and the level of MDA on day 42 had no significant differences (P > .05) among SBM, FCSM-1, and FCSM-2 groups. Besides, there were no significant differences (P > .05) in the levels of serum T-AOC and MDA on day 21 and the activity of serum T-SOD activity on day 42 among all the groups.

Table 6. Effect of fermented cottonseed meal on serum antioxidative capacity¹.

Items	SBM	CSM	FCSM-1	FCSM-2	P-value
Day 21
T-AOC (U/mL)	4.40 ± 0.78	4.19 ± 0.92	5.04 ± 0.73	4.46 ± 0.91	.403
GSH-Px (U)	1666 ± 125^ab	1564 ± 114^a	1809 ± 57.7^b	1636 ± 123^ab	.047
T-SOD (U/mL)	142 ± 9.15^b	138 ± 8.57^a	157 ± 6.43^b	144 ± 2.48^b	.003
MDA (nmol/mL)	3.57 ± 0.61	3.79 ± 0.18	3.53 ± 0.38	3.53 ± 0.21	.75
Day 42
T-AOC (U/mL)	9.10 ± 0.47^b	7.99 ± 0.76^a	9.44 ± 0.73^b	8.54 ± 0.37^ab	.026
GSH-Px (U)	1328 ± 64.9^ab	1207 ± 159^a	1520 ± 157^b	1281 ± 199^a	.029
T-SOD (U/mL)	186 ± 38.7	176 ± 35.9	197 ± 19.1	193 ± 12.9	.692
MDA (nmol/mL)	2.31 ± 0.59^a	3.33 ± 0.51^b	2.25 ± 0.22^a	2.41 ± 0.45^a	.002

Note: T-AOC, total antioxidant capacity; GSH-Px, glutathione peroxidase; T-SOD, total superoxide dismutase; MDA, malondialdehyde; SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^a,bMeans in the same row with no common superscripts differ significantly (P < .05).

¹Data were means of eight birds per treatment.

Antioxidative ability in liver tissue

The effect of FCSM on liver antioxidative ability is presented in Table 7. Compared with the CSM group, the birds in the FCSM-1 group had greater T-AOC and GSH-Px activities, and lower MDA level in the liver tissue on day 21 (P < .05), and had greater T-AOC and T-SOD activities in the liver tissue on day 42 (P < .05). Compared with the FCSM-2 group, the birds in the FCSM-1 group had greater GSH-Px activity on day 21 (P < .05), and had greater T-AOC and T-SOD activities in the liver tissue on day 42 (P < .05). The activities of T-AOC and GSH-Px and the level of MDA on day 21, and the activities of T-AOC and T-SOD on day 42 had no significant differences between SBM and CSM groups (P > .05). Besides, there were no significant differences (P > .05) in T-SOD activity on day 21 and in GSH-Px activity and MDA level on day 42 among all the groups.

Table 7. Effect of fermented cottonseed meal on antioxidative capacity of liver tissue¹.

Items	SBM	CSM	FCSM-1	FCSM-2	P-value
Day 21
T-AOC (U/mg protein)	1.85 ± 0.12^ab	1.67 ± 0.10^a	2.02 ± 0.26^b	1.83 ± 0.22^ab	<.001
GSH-Px (U/mg protein)	1294 ± 97.2^a	1255 ± 119^a	1430 ± 157^b	1292 ± 78.6^a	.045
T-SOD (U/mg protein)	331 ± 19.0	322 ± 9.14	344 ± 15.7	337 ± 11.1	.096
MDA (nmol/mg protein)	3.31 ± 0.43^bc	3.73 ± 0.77^c	2.40 ± 0.86^a	2.77 ± 0.64^ab	.017
Day 42
T-AOC (U/mg protein)	1.71 ± 0.11^a	1.61 ± 0.15^a	2.17 ± 0.14^b	1.73 ± 0.09^a	<.001
GSH-Px (U/mg protein)	1295 ± 256	1116 ± 105	1366 ± 164	1254 ± 123	.09
T-SOD (U/mg protein)	356 ± 3.65^a	339 ± 12.6^a	365 ± 16.2^b	352 ± 16.2^a	.028
MDA (nmol/mg protein)	3.28 ± 0.75	3.78 ± 0.50	2.75 ± 0.38	3.01 ± 0.88	.116

Note: T-AOC, total antioxidant capacity; GSH-Px, glutathione peroxidase; T-SOD, total superoxide dismutase; MDA, malondialdehyde; SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^a,bMeans in the same row with no common superscripts differ significantly (P < .05).

¹Data were means of eight birds per treatment.

Cecal microbiota

The effect of FCSM on cecal microflora population is presented in Table 8. Birds in the FCSM-1 group had greater Lactobacilli counts in ceca digesta than other groups on day 21 (P < .05), and birds in the CSM group had lower total anaerobic bacteria counts on day 42 (P < .05) and had lower Lactobacilli counts in ceca digest on days 21 and 42 (P < .05). Compared with the FCSM-2 group, the birds in the FCSM-1 group had greater Lactobacilli counts in ceca digesta on day 21 (P < .05).

Table 8. Effect of fermented cottonseed meal on cecal microflora population (log cfu/g)¹.

Items	SBM	CSM	FCSM-1	FCSM-2	P-value
Day 21
Total anaerobic bacteria	8.06 ± 0.44	7.81 ± 0.46	8.32 ± 0.44	8.02 ± 0.50	.325
Lactobacilli	7.85 ± 0.33^b	7.37 ± 0.36^a	8.30 ± 0.32^c	7.82 ± 0.32^b	.001
Day 42
Total anaerobic bacteria	8.66 ± 0.37^b	8.15 ± 0.55^a	8.92 ± 0.47^b	8.69 ± 0.14^b	.026
Lactobacilli	8.14 ± 0.50^ab	7.61 ± 0.07^a	8.42 ± 0.46^b	8.12 ± 0.50^ab	.038

Note: SBM, soybean meal; CSM, cottonseed meal; FCSM, fermented cottonseed meal.

^a,bMeans in the same row with no common superscripts differ significantly (P < .05).

¹Data were means of eight birds per treatment.

Discussion

The current study showed that CSM fermented by B. subtilis ST-141 and Saccharomycetes N5 had lower FG level, crude fiber level, pH value and had higher acid-soluble protein content compared with CSM. Similarly, Sun et al. (2015a) reported that fermentation of CSM with B. subtilis BJ-1 significantly reduced FG level and increased CP level. Nie et al. (2015) reported that CSM fermented by Candida tropicalis and C. tropicalis plus Saccharomyces cerevisiae significantly improved CP and crude fat levels. Gadelha, Fonseca, Oloris, Melo, and Sotoblanco (2014) reported that FG can significantly inhibit growth performance and increase mortality in broilers. But the negative effect of FG is related to additional level. He et al. (2015) reported that 75% SBM could be replaced by low-gossypol CSM (21.76 mg/kg) without affecting the poultry growth performance. Other report also demonstrated that compared with the control group (0% CSM), 6% CSM (74.4 mg/kg FG) had no negative effects on ADFI and laying rate from 40 to 49 weeks in laying hens (Yuan et al., 2014). The current result showed between days 22–42 and 1–42, ADG and ADFI had no significant difference among SBM, CSM (65.6 mg/kg FG), and FCSM-1 (27.7 mg/kg FG) groups, but FCSM-2 group (55.3 mg/kg FG) significantly decreased ADG and increased FCR. So it is interesting to note that though FG level in FCSM-2 diets was lower than CSM diets, it had negative effect on growth performance. The reasons may be associated with the other anti-nutritional factors such as malvalic and sterculic acid (He et al., 2015), which may contribute to performance depression. Besides, it has been proved that lysine and arginine had antagonism effect. The ration of arginine and lysine in FCSM-2 diet was higher, which could influence the digestibility of amino acid and may relate to performance depression.

The microbial fermentation process can produce many beneficial substances, such as small-size peptides, exoenzymes, vitamins, organic acids, which can promote the immunity of animals (Johnson, 2013; Zhao, Schieber, & Gänzle, 2016). In the present study, dietary supplementation of FCSM increased serum IgM and IgG levels compared with SBM groups. Tang et al. (2012) reported that birds fed FCSM diets exhibited increased serum IgM, IgG level, and complement C4 level on day 42 compared with birds fed SBM diet. Moreover, the microorganisms used for CSM fermentation is focused on probiotics, such as Lactobacillus, B. subtilis, Yeast, or Fungi, which can positively affect the modulation either innate or acquired immunity, or both (Amerah et al., 2013; Bai et al., 2013). The live microbes in FCSM may act as probiotics to enhance humoral immune response of broiler (Latesh, Singh, & Manoj, 2013; Stašová et al., 2015). Besides, although the live microbes in the FCSM was dead through the drying process, heat-killed microbes also can generate beneficial biological responses to stimulate the gastrointestinal immune function (Adams, 2010).

Blood biochemical parameters can indicate the metabolism process of nutrients (Hu et al., 2015). The current study showed the broilers fed FCSM-1 diets had higher serum and liver antioxidative abilities compared with bird fed CSM diet. Previous studies have shown that fermented soy products and fermented camel milk had high levels of antioxidant activity along with the ability to scavenge free radicals in vitro (Soleymanzadeh, Mirdamadi, & Kianirad, 2016; Xu, Du, & Xu, 2015). So fermented feed might alleviate the tissue lipid peroxidation, enhance antioxidant capacity and decrease oxidative damage (Hu et al., 2015). The enhancement of antioxidative capacity in FCSM may be attributed to many active peptides derived from the CSM protein hydrolysates that can scavenge the free radicals. Gao, Cao, and Li (2010) demonstrated that cottonseed protein hydrolyzed with Neutrase contains peptides with strong antioxidant activity. In addition, previous study indicated that the cottonseed peptides prepared by the fermentation process displayed a dose-dependent effect on the free radical-scavenging activity, hydroxyl radical activity, metal-chelating ability, and reducing power (Sun et al., 2015b).

Intestinal microbial populations play an important role in maintaining intestinal health of broilers. Lactic acid bacteria belong to aboriginal microbe in the gastrointestinal tract of broilers (Mataharo, Reséndizsandoval, & Hernández, 2014; Prado-Rebolledo et al., 2016). Jose, Bunt, and Hussain (2015) indicated that Lactobacilli can inhibit the growth of putrefactive and pathogenic bacteria. The current study showed that broilers fed FCSM diets significantly increased Lactobacilli counts in ceca digesta compared with the CSM diet. The effect of FCSM on the cecal microflora composition is most likely due to the existence of Lactobacilli (live or dead cells) after fermentation. A balanced microbial population would support a healthy intestinal tract functions (Ohland & Jobin 2015). Sun et al. (2013a) reported that dietary inclusion of FCSM (210 mg/kg FG) did not affect the intestinal microbial diversity, but increased Lactobacilli counts. Chiang et al. (2010) demonstrated that broilers fed fermented rapeseed meal had greater Lactobacilli counts in ceca digesta. In the present study, the broilers fed FCSM-1 diets significantly increased the number of total anaerobic bacteria in ceca digesta compared with CSM diet on day 42, which is consistent with the result of Sun et al. (2013b).

Conclusions

In summary, the current study indicated that solid-state fermentation with B. subtilis ST-141 and Saccharomycetes N5 significantly reduced FG level and improved the nutritional value of CSM. FCSM could improve growth performance, increase serum immunoglobulin level and antioxidative abilities, and balance cecal microflora in broilers, but higher FCSM inclusion level had negative effects on growth performance. Therefore, FCSM could be a promising alternative protein source in broiler feed.

Acknowledgements

We also thank Microbiological research group, Academy of State Administration of Grain, P. R. China, for the supply of the fermented cottonseed meal.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Yongwei Wang is a Ph. D in Animal nutrition and feed science. His research interests are protein feedstuff development. probiotics development and application, nutrition and immune regulation. Presently, he is working in Academy of State Administration of Grain, Beijing, China.

Qingqing Deng is a master student in Animal nutrition and feed science. Her research interests are protein feedstuff development and application.

Dan Song is a master in Animal nutrition and feed science. Her research interests are probiotics development and application, nutrition and immune regulation in poultry. Presently, she is working in Academy of State Administration of Grain, Beijing, China.

Weiwei Wang is a Ph. D in Animal nutrition and feed science. Her research interests are probiotics development and application, nutrition and immune regulation in swine. Presently, she is working in Academy of State Administration of Grain, Beijing, China.

Hang Zhou is a master in Animal nutrition and feed science. His research interests are protein feedstuff development. probiotics development and application, nutrition and immune regulation. Presently, he is working in Academy of State Administration of Grain, Beijing, China.

Li Wang is a master in Animal nutrition and feed science. Her research interests are probiotics development and application, nutrition and immune regulation in ruminant animals. Presently, she is working in Academy of State Administration of Grain, Beijing, China.

Aike Li is a Ph. D and professor in Animal nutrition and feed science. His research interests are protein feedstuff development. probiotics development and application, cereal and oil nutrition. Presently, he is working in Academy of State Administration of Grain, Beijing, China.

Additional information

Funding

This study was supported by the Modern agricultural industry technology system, Peking poultry innovation team [grant number BAIC04-2017].