Two-Stage Fermented Feather Meal-Soybean Meal Product Improves the Performance and Immunity of Lactating Sows and Piglets

Abstract

This study aimed to investigate the effects of a two-stage fermented feather meal-soybean meal product (TSFP) on the performance, clinical blood biochemistry, and immunity of sows and piglets. TSFP was fermented by Saccharomyces cerevisiae Y10 for three days in the second stage, which showed similar results to the five-day fermentation of B. coagulans (p > 0.05). Fifty hybrid sows (Duroc × KHAPS black pig) were randomly assigned into dietary supplementation groups of 2% fish meal or different levels of TSFP at 0%, 1%, 2%, or 3%. The results showed that body weight gain and feed conversion ratio of 2% and 3% TSFP groups were better than the control group and fish meal group during the gestation period (d 80–114) (p < 0.05). During the lactation period, the 3% TSFP group showed the best weaning litter weight (p < 0.05). In sows, interferon-γ and immunoglobulin G (IgG) of 2% and 3% TSFP groups were higher than the control group and fish meal group (p < 0.05). In piglets, in groups of 2% and 3% TSFP blood urea decreased (p < 0.05). The IgG of fermented groups was superior to the control group (p < 0.05). The oxidative burst of phagocytes in the 3% TSFP was higher than those of the control and fish meal groups (p < 0.05). In conclusion, TSFP supplementation exhibits the advantages of performance and immunity of lactating sows and piglets. Furthermore, adding 3% TSFP in the feed showed the best performance.

1. Introduction

Nutritional corrections for sows during late gestation and lactation periods are closely related to reproductive performance and piglet growth performance and health [1,2,3]. In Taiwan, fish meal is used as a high-quality protein source during late gestation and lactation periods of sows. Nevertheless, the use of fishmeal has the risks of easy deterioration and pollution. It would affect the performance of sows and piglets through storage life and microbial contamination [4,5]. Using microbial fermentation techniques effectively degrades anti-nutritional factors of the diet, such as glycinin, β-conglycinin of soybean meal, and so on, which can enhance its nutrient utilization to improve production performance [6,7,8]. Moreover, fermented feed supplementation in the diet of sows not only reduces the loss of body weight and backfat thickness during lactation but also increases the immunoglobulin A level in sow’s milk and promotes the mitosis potential of intestinal epithelial cells in attribution to increase the litter weight at weaning and reduce the incidence of diarrhea [9,10,11].

The crude protein (CP) of feathers is up to 85% of which the content of primary keratin is about 90%, which is enriched with disulfide bonds, hydrogen bonds, and hydrophobic properties, impacted on low solubility, and hardly decomposed by enzymes. In addition, the amino acid composition is unbalanced; thus, the amount of feather meal used in monogastric animal diets is limited [12,13,14]. The industry mainly produces hydrolyzed feather meals at high temperatures and pressures. However, the cooking process often causes amino acid loss and composition variation, which has a negative effect on digestibility and utilization in monogastric animals, therefore it limits the usage of feather meal [15,16,17]. The authors adding 2.5% or 5% TSFP to finishing pig diets could improve growth performance and regulate immunity [7]. Lee et al. [18] mixed feather meal and soybean meal at a ratio of 1:1 as fermented substrate under aerobic fermentation with Bacillus strains to produce a fermented feather meal-soybean meal product (FFSMP). A diet supplemented with 5% FFSMP can promote the growth of broilers.

Due to the slow acid production ability of L12, using Saccharomyces cerevisiae Y10 with fast acid production ability to replace L12 at the second stage of the fermented process can shorten the anaerobic fermentation time to 3 days. However, the application value of this modified TSFP in sows during late gestation and lactation has not been evaluated. This study aimed to investigate the effects of modified TSFP on performance, clinical blood biochemistry, and immunity of sows and piglets.

2. Materials and Methods

2.1. Two-Stage Fermented Product Preparation

The two-stage fermented product (TSFP) preparation and analysis referred to the method of Huang et al. [7]. Bacillus subtilis Da2, Da6, and B. amyloliquefaciens Da15, Da16, and B. subtilis var. natto N21 (N21) with strong protein decomposition capacity was used at the first-stage fermentation. Subsequently, according to the result of Chen et al. [19], B. coagulans L12 (L12) or Saccharomyces cerevisiae Y10 (Y10) with strong acid production capability was used at the second-stage fermentation. Mixed feather meal and soybean meal at a ratio of 1:1 and moisturized with additional 50% distilled water, which was used as fermentation substrate. The substrate was sterilized at 121 °C for 30 min and cooled down to 45 °C. Five Bacillus strains at 10⁶ colony-forming units per gram (CFU/g) of the substrate were premixed and inoculated at the same time to ferment aerobically at 37 °C for 2 days. Subsequently, L12 or Y10 was added at 10⁶ CFU/g of the substrate to ferment anaerobically at 28 °C for 5 or 3 days, respectively. The fermented product was then oven dried. The moisture of the final product was below 12%, and 3 batches were produced for the current study.

2.2. Animal Management and Experimental Design

Fifty hybrid sows (Duroc × KHAPS black pig), with an average parity of 3.5 ± 0.7, were selected for the experiment and were randomly assigned to dietary supplementation of 2% fish meal (positive control), and 0 (negative control), 1, 2, or 3% TSFP. Feed diets for sows were formulated with reference to the nutrient requirements recommended by NRC [1]. On the premise that crude protein and metabolizable energy were equal (gestation period CP = 12.8%; ME = 13.7 MJ/kg and lactation period CP = 17.5%; ME = 13.7 MJ/kg), the feed was provided restriction during gestation period (80–107 days, 2.3 kg per day) and were provided ad libitum during lactation period. Feed composition is shown in

Table 1. Experimental feed composition.

Items	Gestation Period					Lactating Period
	2% Fish Meal	TSFP, %				2% Fish Meal	TSFP, %
		0	1	2	3		0	1	2	3
Corn, yellow	72.23	70.70	71.04	71.5	71.95	60.81	59.13	59.63	60.1	60.59
Wheat bran	10.00	10.00	10.00	10.00	10.00	8.00	8.00	8.00	8.00	8.00
Soybean oil	2.64	2.81	2.85	2.89	2.91	2.85	3.03	3.06	3.09	3.12
Soybean meal, CP 44%	10.50	13.60	12.20	10.70	9.20	23.80	27.03	25.50	24.00	22.5
Fish meal (Peru), CP 65%	2.00	0	0	0	0	2.00	0	0	0	0
TSFP 1, CP 62%	0	0	1.00	2.00	3.00	0	0	1.00	2.00	3.00
Dicalcium phosphate	1.08	1.30	1.30	1.30	1.30	0.92	1.13	1.13	1.13	1.13
Limestone	1.00	1.06	1.06	1.06	1.06	1.02	1.08	1.08	1.08	1.09
Salt	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35
Vitamin premix 2	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Mineral premix 3	0.10	0.10	0.10	0.10	0.10	0.15	0.15	0.15	0.15	0.15
Total	100	100	100	100	100	100	100	100	100	100
Analysis
Crude protein, %	12.54	12.49	12.48	12.42	12.56	17.45	17.56	17.39	17.46	17.51
Ca, %	0.79	0.76	0.74	0.73	0.77	0.72	0.78	0.75	0.77	0.74
P, %	0.66	0.64	0.6	0.63	0.65	0.58	0.62	0.61	0.64	0.62

¹ TSFP: two-stage fermented feather meal-soybean meal product; ² Vitamin supplied the following per kilogram of diet: vitamin A, 5000 IU; vitamin D3, 1500 IU; vitamin E, 40 mg; vitamin K, 3 mg; vitamin B1, 2.6 mg; vitamin B12, 4 mg; niacin, 35 mg; pantothenic acid, 23 mg; Vitamin B2, 6 mg; Vitamin B6, 0.6 mg; Niacin, 30 mg; Pyridoxine, 1 mg; Folic acid, 0.5 mg; Biotin, 0.2 mg; ³ Mineral supplied the following per kilogram of diet: Fe (FeSO₄·7H₂O, 20.09% Fe), 217 mg; Cu (CuSO₄·5H₂O, 25.45% Cu), 125 mg; Mn (MnSO₄·H₂O, 32.49% Mn), 40 mg; Zn (ZnSO₄, 80.35% Zn), 110 mg; Se (NaSeO_3, 45.56% Se), 0.36 mg; Co (CoSO₄·H₂O, 32% Co), 0.7 mg; I (KI), 0.45 mg.

. The experimental sows were individually raised on the slatted floor and high-rise traditional housing system with natural light (2.2 m × 1 m = 2.2 m²) during gestation. The average temperature was 22–28 °C. The sows were individually housed in farrowing crates (2.2 m × 1.2 m = 2.64 m²) at 107 d gestation. All the procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Kaohsiung Animal Propagation Station, Kaohsiung, Taiwan, ROC (protocol number: IACUC-101005).

2.3. Physical and Chemical Composition of TSFP

The pH value of TSFP was measured by pH meter (pH meter, Goodly, Taiwan). A sample of TSFP was serially diluted by 0.85% NaCl, then was respectively incubated on tryptone soy agar (TSA, HIMEDIA^®, Mumbai, MH, India) at 37 °C for 24 h, on Lactobacillus MRS agar (MRSA, HIMEDIA^®, Mumbai, MH, India) at 37 °C for 24 h, or yeast extract peptone dextrose agar (YPDA, HIMEDIA^®, Mumbai, MH, India) at 28 °C for 24 h, respectively. The counts of Bacillus-like colonies, Lactobacillus-like colonies, or yeast-like colonies were counted as CFU/g. The γ-polyglutamic acid (γ-PGA) of TSFP was measured by the method of Goto and Kunioka [20]. The proximate analysis of TSFP followed the description of AOAC [21] to analyze the moisture (method 930.15), ash (method 923.03), crude protein (method 990.03), calcium (method 927.02), and phosphorus (method 935.59). The gross energy was measured with an adiabatic bomb calorimeter (model 356, Parr Instrument Company, Moline, IL, USA). The analysis of physical and chemical composition of TSFP was performed with three replicates (n = 3).

2.4. Feed Composition Analysis

The proximate analysis of feed was followed the description of AOAC [21] to analyze the crude protein (method 990.03), calcium (method 927.02), and phosphorus (method 935.59).

2.5. Reproductive Performance of Sows

The period of gestation days, the sow’s body weights at initial (80 days of gestation), 107 days of gestation, and weaning were recorded as criteria of reproductive performance. Moreover, the weight gain, feed conversion ratio during gestation, and weight loss during lactation were calculated and monitored. Additionally, average daily feed intake during gestation period and lactation period, the interval of weaning-to-estrus and weaning-to-mating, and the numbers of born and weaned piglets per litter were measured. Data were calculated with each pen as an experimental unit (n = 10).

2.6. Growth Performance of Suckling Pigs

Piglets suckled milk, and they started to intake creep feed (starter feed) after 8-day age, and piglets were fully weaned at 28 days of age. The feed was provided ad libitum. Growth performance including the mortality rate was measured. Faecal samples were collected by sterile cotton swab in order to determine Escherichia coli counts at 28 d weaning, then samples were serially diluted by 0.85% NaCl and plated on Coliform agar (Merck^®, Darmstadt, Germany), and cultivated at 37 °C for 24 h. The counts of colonies are expressed as CFU/g. The body weight of born and weaned, weaning litter weight, and the average daily feed intake of piglets were measured during lactation period. Data were calculated with sows of individual farrowing piglets in each pen as an experimental unit (n = 10).

2.7. Collection of Blood Sample

Each treatment individually selected ten sows (n = 10) and their individual farrowing male piglets (n = 10) for sampling at 28 d weaning. The blood samples were collected via the jugular vein of sows and anterior vena cava of piglets with or without an EDTA vacutainer (BD Vacutainer, Avenue Broken Bow, NE, USA), and immediately stored at 4 °C for further clinical blood biochemical analysis and immune characteristics measurements.

2.8. Clinical Blood Biochemistry

Blood samples were centrifuged (2500× g) at 4 °C for collecting plasma and stored at −20 °C until biochemical analysis. The blood biochemistry of plasma, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), blood urea (BUN), glucose (GLU), triglyceride (TG), cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-CHOL), calcium (Ca), and phosphorous (P), were measured by hematology analyzer (Express Plus, Bayer, MA, USA) according to the methods of Huang et al. [7]. Individuals of sows (n = 10) and their individual farrowing male piglets (n = 10) were seen as experimental units for blood biochemistry.

2.9. Immune Characteristics

The method was the same as described by Huang et al. [7]. Briefly, blood samples were centrifuged at 400× g for 10 min at 4 °C, then took the serum for the following measurement. Cells were diluted by RPMI-1640 medium at a ratio of 1:2 and layered onto Ficoll (Histopaque-1077, Sigma-Aldrich, St. Louis, MO, USA) for the density gradient separation. Samples were centrifuged at 450× g under room temperature for collecting peripheral blood mononuclear cells (PBMC). After a wash with cold phosphate-buffered saline (PBS), the live/dead cell counts were calculated with a hemocytometer under a microscope by using trypan blue exclusion method. After removal of the PBMCs from the samples, the red blood cells were lysed by commercial RBC-lysis buffer (BioLegend, San Diego, CA, USA) to remove red blood cells and collect granulocytes with centrifugation for the later analysis.

2.9.1. Cytokine Production

The method of cytokine measurement in whole blood was described by Edfors-Lilja et al. [22]. Heparinized whole blood was diluted to 1:50 for the detection of interferon-γ (IFN-γ) by the culture medium (Roswell Park Memorial Institute-1640; RPMI-1640) containing 50 μM 2-mercaptoethanol, 10 Mm 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), 10 U/mL penicillin and 100 μg/mL streptomycin. The diluted whole blood samples were seeded into 24-well plates and cultured in a humid incubator. The incubator was maintained at 37 °C and 5% CO₂ gas. The culture supernatant was collected after 72 h for determination of IFN-γ. The cytokine levels were determined by commercial enzyme-linked immunosorbent assay (ELISA) reagents (R&D Systems, Minneapolis, MN, USA and IFN-γ, PharMingen, San Diego, CA, USA) according to the manufacturer’s procedures. Color changes were detected at OD = 450 nm and 550 nm with a microplate reader (Multiskan Ex Microplate Reader, Thermo, Waltham, MA, USA).

2.9.2. Phagocytosis and Oxidative Burst

The method was performed as described previously in Huang et al. [7]. DioC18 labeled E. coli. phagocytosed by the granulocytes was measured as fluorescence changes by flow cytometry (Becton Dickinson FACSCaliburTM, Franklin Lakes, NJ, USA). Moreover, the granulocytes were co-incubated with unlabeled E. coli in a 37 °C incubator for 90 min, and the intracellular reactive oxygen species (ROS) was determined by adding 20, 70-dichlorofluorescin-diacetate (DCF-DA) for measuring oxidative burst. Change of intracellular fluorescence indicated cell oxidative burst was measured by flow cytometer.

2.9.3. Serum Immunoglobulin

Serum immunoglobulin including immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA) were measured by goat anti-porcine (Bethyl Laboratories, Montgomery, TX, USA). Individuals of sows and piglets (n = 10) were seen as an experimental unit for immunity characteristics.

2.10. Statistical Analysis

The continuous variable data were analyzed using the General Linear Model (GLM) procedure and used Tukey post hoc test for the paired comparison, where p < 0.05 indicated a statistically significant difference. The survival rate of piglets was analyzed using the NPAR1WAY procedure (SAS Institute, Cary, NC, USA, 2004), where p < 0.05 indicated a statistically significant difference. The polynomial contrasts were used to test the linear and quadratic effects to analyze the effects of TSFP on the reproductive performance, clinical blood biochemistry, and immune characteristics of sows and piglets, where p < 0.05 indicated a statistically significant difference.

3. Results and Discussion

3.1. Physical and Chemical Composition of Two-Stage Fermented Product

Table 2. Physical and chemical analysis of two-stage fermented product.

Items 4	TSFP 1		SEM	p-Value
	L12 2	Y10 3
First stage fermentation
pH	7.82		-	-
Bacillus-like, log CFU/g	8.52		-	-
Second stage fermentation
pH	5.68	5.71	0.11	0.8602
Bacillus-like, log CFU/g	8.16	8.35	0.14	0.3937
Lactobacillus-like, log CFU/g	8.27	-	-	-
Yeast-like, log CFU/g	-	7.63	-	-
Dry product
pH	5.75	5.68	0.07	0.5644
Bacillus-like, log CFU/g	7.56	7.59	0.12	0.8984
γ-PGA, %	4.50	4.55	0.03	0.2906
Chemical composition of dry product
Moisture, %	9.57	9.54	0.13	0.8915
Crude ash, %/DM	4.70	4.68	0.19	0.9239
Crude protein, %/DM	62.9	63.3	1.24	0.8441
Calcium, Ca %/DM	0.24	0.25	0.02	0.7226
Total Phosphorus, TP %/DM	0.56	0.55	0.02	0.6843

n = 3; ¹ TSFP: two-stage fermented feather meal-soybean meal product; ² L12: B. coagulans L12 (L12) fermented anaerobically for 5 days; ³ Y10: Saccharomyces cerevisiae Y10 (Y10) fermented anaerobically for 3 days; ⁴ CFU: colony-forming units; γ-PGA: γ-polyglutamic acid.

presents the physical and chemical composition analysis of TSFP. After the first stage fermentation for 2 days, the pH value and Bacillus-like bacteria counts were 7.82 and 8.52 log CFU/g, respectively. After the second-stage anaerobic fermentation fermented by L12 for 5 days or Y10 for 3 days, the pH value was respectively decreased to 5.68 or 5.71, and the Bacillus-like bacteria counts were decreased to 8.16 or 8.35 log CFU/g, respectively. After anaerobic fermentation by L12 for 5 days, the Lactobacillus-like counts of the product had 8.27 log CFU/g. After anaerobic fermentation by Y10 for 3 days, the yeast-like counts of the product had 7.63 log CFU/g. The oven-drying process decreased the Bacillus-like bacteria counts, but the counts of each group were above 7 log CFU/g. The chemical composition showed no significant difference among treatment groups (p > 0.05).

The fermented substrates of this experiment were rich in protein, which were decomposed by Bacillus strains and then produced alkaline by-products. The pH value was raised above 7.82 after the first-stage fermentation, which was in agreement with the results of Huang et al. [23]. Because Y10 was an efficient acid-producing strain, the pH value decreased from 7.82 to 5.71 after the second-stage fermentation for 3 days, which was similar to the result of anaerobic fermentation by L12 for 5 days (p > 0.05). This result demonstrated the merit of the strong acid-producing ability of Y10, which had resulted in 2 days reduction of fermentation time as compared with L12. Moreover, Y10 is capable of growing in a high pH value environment, while the pH value of TSFP was merely changed after drying. The product was maintained in a weak acid condition because the lactic acid produced by yeast was not volatile [24].

3.2. Performance of Sows and Piglets

Table 3. The effects of two-stage fermented product on performance of sow and their piglets during lactation period.

Items	2% Fish Meal	TSFP 1, %				SEM	p-Value	Polynomial Contrasts
		0	1	2	3			Linear	Quadratic
Sow:
Gestation period
The period of gestation, days	114	112	113	114	113	0.90	0.8224	0.7167	0.2840
Body weight (d 80), kg	220	225	220	231	230	7.10	0.6932	0.4115	0.8442
Body weight (d 107), kg	237	242	238	250	249	7.06	0.5943	0.3221	0.8667
Body weight gain (d 80–107), kg	17.2 b	17.1 b	17.7 ab	18.5 a	18.7 a	0.45	0.0450	0.0025	0.6002
Feed conversion ratio (d 80–107)	3.78 a	3.80 a	3.65 ab	3.49 b	3.45 b	0.10	0.0363	0.0021	0.5109
Lactation period
Weight at weaning, kg	220	225	220	233	233	6.93	0.5038	0.2958	0.7347
Weight loss during lactation, kg	17.1	16.5	17.8	17.1	15.8	1.32	0.8512	0.6349	0.3688
Average daily feed intake from farrowing to weaning, kg	3.52 a	3.30 b	3.47 ab	3.50 a	3.52 a	0.05	0.0159	0.0012	0.0828
Weaning-to-estrus interval, days	8.2	8.7	9.8	8.6	8.30	2.74	0.9943	0.8472	0.8016
Weaning-to-mating interval, days	16.6	19.2	18.3	16.8	14.50	4.21	0.9461	0.3858	0.8609
Piglet:
Piglet Body weight, kg/piglet
Initial weight	1.42	1.43	1.41	1.38	1.40	0.03	0.8382	0.4063	0.4771
weaning at 28 d	5.45	5.37	5.39	5.44	5.56	0.14	0.9043	0.2674	0.6753
Average daily feed intake of 8 d to weaning, g/piglet	114.2 ab	109.1 c	109.5 bc	116.5 ab	117.4 a	1.79	0.0030	<0.0001	0.8560
No. of per litter
totally born alive	11.0	10.2	10.4	10.8	11.2	0.78	0.8859	0.4082	0.9128
weaning piglets	9.10	8.50	9.00	9.20	9.90	0.54	0.4934	0.1218	0.8722
Pre-weaning mortality 2	1.90	1.70	1.40	1.60	1.30	0.43	0.8726	0.6405	1.0000
Weaning litter weight, kg/litter	49.63 b	45.66 c	48.53 bc	50.06 b	55.05 a	0.72	<0.0001	<0.0001	0.1959
Survival, %	82.9	86.5	86.6	85.6	90.1	-	0.6205	0.5206	0.5139

n = 10; ¹ TSFP: two-stage fermented feather meal-soybean meal product; ² Pre-weaning mortality: mortality count from birth to wean: born alive; ^a,b,c Means in the same row with different superscripts are significantly different (p < 0.05).

presents the effects of TSFP on the performance of sows and piglets. The body weight gain and feed conversion ratio were linearly improved with the increased dietary levels of TSFP during the gestation period (p < 0.05), and the dietary level of TSFP increased to 2% or more, it showed better improvement than the control and fish meal group (p < 0.05). The feed intake was linearly increased as the dietary level of TSFP increased during the lactation period (p < 0.05), and the 2 or 3% TSFP groups had higher feed intake than the control group (p < 0.05). The litter weight at weaning and creep feed intake of the piglets were increased linearly as the dietary level of TSFP increased (p < 0.05). When the dietary level of TSFP was up to 2% or above, it was superior to the control group (p < 0.05), while the 3% TSFP group performed the best performance (p < 0.05). There was no significant difference in E. coli number of feces among treatments (p > 0.05) (not listed).

During the gestation period, the feed was provided at a restricted level, that is to say, a daily 2.3 kg feed per sow. Nevertheless, those supplemented with 2% or 3% TSFP showed better body weight gains and feed conversion ratios (FCR) than those supplemented with fish meal and control groups (p < 0.05). Using microorganisms to convert poorly utilized macromolecules into digestible small molecules can reduce the anti-nutritional factors of substrates, thereby improving the absorption and utilization of nutrients by broilers [18,24,25]. Feathers are a significant waste entailed in the poultry industry. They are hard to be degraded by common proteases because of their structural bonds and high keratin content [26,27]. Thus, feathers are difficult to use widely in pig feeds [17]. Our previous studies have proved that the nutritional value of feather meals could be improved by two-stage fermentation technology. Huang et al. [7] used the same proteolytic strains in this experiment for a two-day aerobic fermentation in the first stage, and L12 for a five-day anaerobic fermentation in the second stage to produce TSFP. Adding 2.5% or 5% TSFP to finishing pig diets improved average daily gain (ADG) and FCR compared with the control group and fish meal group. A restricted amount of feed was provided during the gestation period in this experiment, and TSFP could improve body weight gain and FCR of sows. It proved that the co-cultivation method of keratin-degrading bacteria and proteolytic bacteria may effectively convert the macromolecular protein of substrate into easily digestible small molecules in the first stage of fermentation, thus promoting the sows’ performances.

Feeds were provided ad libitum for sows during the lactation period. Those supplemented with 1% TSFP could reach similar outcomes as the fish meal diet, while 2% and 3% TSFP groups had higher feed intakes compared to the control group (p < 0.05). Our previous study revealed that the two-stage fermented feed could improve feed intake and body weight gain of broilers. Because of using Y10 with strong carbohydrate-degrading and acid-producing capacity at the second stage of fermentation, it enhanced flavor and organic acid to increase the palatability of feed. Further, it improved the feed intake and nutrient absorption in broilers [19]. In the current study, the organic acid and aroma produced by Y10 at second-stage anaerobic fermentation could enhance palatability and improve the feed intake of sows during the lactation period.

When the TSFP supplementation was up to 2%, the creep feed intake of piglets could reach the same level as the group with fish meal. Moreover, supplemented TSFP to 2 or 3% also improved the feed intake compared with the control group (p < 0.05). The litter weight at weaning was linearly increased as the dietary level of TSFP increased, and in the group of 2% or 3% TSFP was better than the control group (p < 0.05). The health of lactating sows is closely related to the performance of piglets [10]. Previous studies have reported that adding yeast-fermented products to sow feeds benefited the performance (feed intake, body weight gain, and FCR) of piglets [28,29]. TSFP used in the current study was a mixed probiotic product, including bacteria and yeast-fermented products. Adding TFSP to sow feeds could improve the reproductive performance of sows during late gestation and lactation, and further increased the feed intake of creep feed and weaning litter weight in piglets.

3.3. Clinical Blood Biochemistry

Table 4. The effects of two-stage fermented product on clinical blood biochemistry of sow and their piglets during lactation period.

Items 2	2% Fish Meal	TSFP 1, %				SEM	p-Value	Polynomial Contrasts
		0	1	2	3			Linear	Quadratic
Sow:
ALT (U/L)	20.4 ab	21.0 a	19.2 b	18.8 b	18.7 b	0.61	0.0341	0.0104	0.1254
AST (U/L)	30.2	29.9	29.4	28.2	28.9	0.95	0.5998	0.3361	0.5116
ALP (U/L)	25.6	25.8	26.5	25.5	24.8	1.01	0.8307	0.3508	0.4286
TP (g/dL)	7.54	7.32	7.39	7.52	7.58	0.17	0.8005	0.1947	0.9948
BUN (U/L)	4.44 a	4.49 a	4.38 ab	4.26 ab	4.01 b	0.10	0.0184	0.0037	0.5586
GLU (mg/dL)	81.0	79.6	80.3	81.9	81.6	2.75	0.9341	0.4027	0.9891
TG (mg/dL)	74.2	75.2	73.2	72.2	72.0	2.45	0.8853	0.3720	0.7386
CHOL (mg/dL)	393.2	395.4	382.7	384.7	375.5	15.1	0.9470	0.6630	0.8450
HDL-CHOL (mg/dL)	115	107	107	116	118	5.36	0.2377	0.0115	0.801
Ca (mg/dL)	5.47	5.40	5.43	5.47	5.46	0.04	0.7621	0.2433	0.5474
P (mg/dL)	4.19	4.17	4.20	4.22	4.19	0.12	0.9986	0.8775	0.8124
Piglet:
ALT (U/L)	20.6 ab	21.9 a	19.6 b	19.1 b	19.1 b	0.75	0.0490	0.0120	0.1050
AST (U/L)	31.6	32.9	30.4	29.2	29.9	0.98	0.0773	0.0117	0.0627
ALP (U/L)	28.7 ab	29.7 a	28.8 ab	26.5 b	26.6 b	0.81	0.0279	0.0002	0.4041
TP (g/dL)	22.9 ab	22.6 b	24.0 ab	25.1 ab	25.4 a	0.69	0.0196	0.0004	0.2845
BUN (U/L)	7.04 a	6.99 a	6.88 ab	6.52 bc	6.50 c	0.09	<0.0001	<0.0001	0.536
GLU (mg/dL)	105	102	103	106	107	2.55	0.7598	0.0628	0.9593
TG (mg/dL)	82.2	80.1	79.2	78.2	79.0	1.18	0.1312	0.4314	0.4851
CHOL (mg/dL)	462	457	446	437	433	8.84	0.1801	0.0382	0.6630
HDL-CHOL (mg/dL)	94.4	98.5	100.8	99.2	100.4	2.43	0.3550	0.1267	0.2439
Ca (mg/dL)	6.47	6.40	6.43	6.43	6.38	0.04	0.6100	0.7023	0.2867
P (mg/dL)	4.06	3.87	3.97	4.02	3.99	0.07	0.3285	0.1165	0.2738

n = 10; ¹ TSFP: two-stage fermented feather meal-soybean meal product; ² ALT: Alanine Aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase; TP: Total protein; BUN: blood urea nitrogen; GLU: Glucose; TG: Triglycerides; CHOL: Cholesterol; HDL-CHOL: High-density lipoprotein-cholesterol; Ca: Calcium; P: Phosphorus; ^a,b,c Means in the same row with different superscripts are significantly different (p < 0.05).

presents the effects of TSFP on the clinical blood biochemistry of lactating sows and piglets. In sows, ALT and BUN levels linearly decreased as the dietary level of TSFP increased (p < 0.05). The fermented groups showed lower ALT levels than the control group (p < 0.05). Compared with the control group, the plasma BUN content of the 3% TSFP group decreased (p < 0.05). In piglets, ALT, ALP, and BUN levels linearly decreased as the dietary level of TSFP increased (p < 0.05). All the TSFP groups decreased ALT levels as compared with the control group (p < 0.05). When the TSFP supplementation was more than 2% in the pigs’ diets, it decreased ALP and BUN levels compared with the control group (p < 0.05). The TP content elevated linearly as the dietary level of TSFP increased (p < 0.05), and only the 3% TSFP group was higher than the control group (p < 0.05).

Several studies show that feeding pigs Bacillus-fermented products had liver protective effects and may prevent hepatitis occurrence and decrease ALT and AST levels [30,31]. Kim et al. [32] indicated that adding 1% Bacillus subtilis fermented product to pig feed could decrease ALT in blood as compared with the control group, which was similar to the results of the current study. ALT activity among fermented groups was significantly lower than the control group in sows and piglets, which demonstrated a beneficial effect of TSFP supplementation in sows and piglets.

TP is composed of albumin and globulin and has primary functions in nutrient transport, maintaining normal osmotic pressure, immunity, etc. BUN is a protein metabolite secreted by the kidney and discharged by urine. When animals intake large amounts of protein, disease or other factors, their BUN concentrations increase readily [33]. Shen et al. [34] observed that supplemented yeast-fermented products in feed could improve the reproductive performance of sows and had a tendency to lower serum BUN, which is similar to the results of the current study. The crude protein of each group was the same in this experiment. Increasing the dietary level of TSFP supplementation could decrease BUN content in the blood of sows (p < 0.05), which showed that TSFP might improve the utilization of protein. Supplementing 3% TSFP to lactating sow feeds could effectively increase the TP of piglets compared with the control group, and as the dietary level of TSFP was increased to 2 or 3% could effectively decrease the BUN of piglets. These results could be proved by the improvement in body weight, FCR, survival, and litter weight at weaning during gestation in Table 3. In other words, TSFP improves protein utilization in sows, increasing milk production to enhance performance in piglets.

TG is neutral fat, which primarily exists in the animal body and has little content in animal blood. CHOL, which exists in all cells, is mainly synthesized by the liver and small intestine and is presented in blood as complexes such as HDL, LDL, very low-density lipoprotein, etc. [35]. There were no significant differences in plasma levels of TG, CHOL and HDL-CHOL among groups (p > 0.05), representing that TSFP had no negative effects on the fat metabolism of pigs. According to the above results, supplementation at a maximum level of 3% TSFP to the feed showed no adverse effects on clinical blood biochemical parameters in sows and piglets and could be safely used in sow diets.

3.4. Immune Characteristics

Table 5. The effects of two-stage fermented product on immune characteristics of sow and their piglets during lactation period.

Items	2% Fish Meal	TSFP 1, %				SEM	p-Value	Polynomial Contrasts
		0	1	2	3			Linear	Quadratic
Sow
cytokine, pg/mL
IFN-γ	117 b	117 b	121 b	133 a	135 a	2.86	<0.0001	<0.0001	0.6040
mean fluorescence intensity
phagocytosis	63.3	61.3	64.5	68.2	70.0	2.51	0.1324	0.0149	0.7312
oxidative burst	168	161	164	172	175	3.79	0.0818	0.0049	0.9773
immunoglobulin, mg/mL
IgA	1.50	1.49	1.52	1.54	1.54	0.05	0.9457	0.4752	0.7666
IgM	1.83	1.80	1.82	1.86	1.85	0.05	0.8939	0.3094	0.6469
IgG	18.4 c	18.22 c	18.90 bc	21.03 ab	21.33 a	0.56	0.0002	<0.0001	0.6900
Piglet
cytokine, pg/mL
IFN-γ	109 b	107 b	117 ab	126 a	127 a	3.30	<0.0001	<0.0001	0.1023
mean fluorescence intensity
phagocytosis	80.3	79.3	79.8	86.1	85.6	3.79	0.5496	0.1809	0.9036
oxidative burst	562 b	573 b	588 b	642 ab	696 a	26.40	0.0038	0.0027	0.5044
immunoglobulin, mg/mL
IgA	1.46	1.45	1.51	1.58	1.61	0.05	0.1125	0.0121	0.8546
IgM	1.85	1.83	1.85	1.89	1.88	0.04	0.8441	0.2326	0.6966
IgG	19.7 bc	19.0 c	20.0 ab	21.3 ab	21.9 a	0.52	0.0015	<0.0001	0.6813

n = 10; ¹ TSFP: two-stage fermented feather meal-soybean meal product; ^a,b,c Means in the same row with different superscripts are significantly different (p < 0.05).

presents the effects of TSFP on the immune characteristics of lactating sows and their piglets. In sows, IFN-γ and IgG linearly enhanced as the dietary level of TSFP increased (p < 0.05), and these characteristics in the groups of 2 or 3% TSFP supplementation were higher than in control and fish meal groups (p < 0.05). In piglets, IFN-γ, IgG and the oxidative burst capacity were linearly increased as the dietary level of TSFP elevated (p < 0.05), and diets supplemented with more than 2% TSFP could enhance IFN-γ as compared with control and fish meal group (p < 0.05). The IgG of animals in fermented groups was higher than the control group (p < 0.05), and the 3% TSFP group had a higher IgG level than that of the fish meal group (p < 0.05). Similarly, the oxidative burst capacity of the 3% TSFP group was higher than the control and fish meal group (p < 0.05).

IFN-γ is an essential cytokine in Th1 helper cells mediated immunity that enhances the bactericidal effects of macrophages and is also known as a macrophage activating factor [36]. Adding 2% and 3% TSFP in sow diets could increase IFN-γ levels in the blood of lactating sows and piglets as compared with the control and fish meal group (p < 0.05). Adding 2% TSFP or more could significantly stimulate the IFN-γ secretion of sows and enhance the blood IFN-γ content of piglets by milk transportation. Nguyen et al. [37] pointed out that except for TGF-β1 and TNF-α, the concentration of other cytokines such as IL-4, IL-6, IFN-γ, IL-12, and IL-10 in colostrum/milk are associated with maternal serum concentration of sows. Moreover, it proved that maternal cytokines may transport to piglets via milk to enhance the blood cytokine concentration of piglets. This is similar to the result of the current study. Moreover, the 3% TSFP supplementation could enhance the oxidative burst capacity of sows (p < 0.10) and piglets (p < 0.05) as compared with those of the control group. This may be attributed to elevating blood IFN-γ in the TSFP group. IFN-γ could effectively activate cell-mediated immune responses such as enhancing the production of reactive oxygen species and nitric oxide synthase (NOS) activity, promoting antigen presentation, inducing autophagy in eliminating intracellular pathogens, and increasing productions of pro and inflammatory cytokines [38]. It was shown that TSFP could increase the secretion of IFN-γ of sows to increase phagocytosis and the oxidative burst capacity of phagocytic cells by preventing pathogenic microbes invasion.

IgG, which is the immunoglobulin isotype that is the most abundant in pigs, can leave the vascular system and distribute in extravascular tissue fluid with many protective functions [36]. The piglets mainly obtain immunoglobulins from colostrum and breast milk from sows for carrying out passive immune protection. The immunoglobulins in colostrum mainly come from serum, and the absorption effect of IgG is the best [39]. Besides, the serum IgG concentration of piglets is depending on the intake of colostrum, IgG concentration in colostrum can be readily absorbed prior to the intestinal tract closure time [40]. Adding 2% or 3% TSFP in sow diets could increase serum IgG concentration when compared with the control group (p < 0.05), and therefore increased serum IgG concentration of piglets (p < 0.05). The piglets started synthesizing IgG at 7-day age. The synthesized IgG content had a positive correlation with IgG content absorbed from colostrum and was related to the feed composition of sows [41]. This is similar to the result of the current study, showing that adding 2% or 3% TSFP in the diet could stimulate IgG synthesis of sows, and it would be transported to piglets via colostrum, thereby enhancing the blood IgG concentration of piglets.

4. Conclusions

TSFP showed a positive effect on the performance and immunity of sows and piglets. The 3% TSFP group showed the best weaning litter weight and adding 2 or 3% TSFP to the diet of sows and piglets improved their immune characteristics.