1. Introduction
Early weaning of piglets is an advantageous practice as it increases sow productivity and prevents pathogenic transmission from their sows. However, the young piglets are more susceptible to various stressors, that can subsequently increase disease susceptibility and lead to economic losses. [
1,
2]. To overcome this escalating problem, it is important to get young piglets to consume more feed and gain body weight quickly as it affects days to market. The typical nutritional approach is to formulate diets by utilizing various feed ingredients with highly digestible and palatable ingredients containing functional amino acids, as well as supplemented with additives [
3]. A recent report has demonstrated that a highly digestible protein ingredient shows effects against antioxidant and anti-inflammation mechanisms, while modulation function is beneficial for gut microbiome [
4]. However, the shortage of plant-based protein resources has been the major limiting factor in driving sustainable development in the swine industry, thus, contributing to the search of an alternative feed ingredient.
Though hydrolyzed yeast (HY) derived from brewer’s yeast cells (
Saccharomyces cerevisiae) activates the cell’s rupture through specific enzymatic hydrolysis, it has been in the swine industry for many decades. It contains high amounts of digestible crude protein (41.3% of dry basis) and all essential amino acids (2.71% of lysine), including glutamic acid (4.16%) [
5]. It also represents a unique composition of nucleotides, microbial enzymes, mannan oligosaccharides (MOS), and β-glucans, which is not fully understood [
5,
6]. These components are important for RNA transcription, cell proliferation, intestinal homeostasis, and immunity that may positively affect growth performance of young animals [
7,
8]. According to Moehn et al. [
9], the incorporation of 9% yeast-derived protein led to a similar apparent and standardized ileal digestibility of amino acids as spray-dried plasma protein in young piglets. The addition of 4% dried brewer’s yeast in creep and nursery diets also promoted the growth performance of nursing and weaning piglets [
10]. Furthermore, the 4% HY-supplemented diet not only promoted the health of the animal but also activated antioxidant enzyme function and
Lactobacillus growth and reduced inflammation [
5]. However, the incorporation of a novel HY ingredient for early-weaned piglets is not fully understood. We hypothesized that the use of HY as a high digestible protein ingredient might promote animal health and feed utilization of 18-day weaned piglets. To test this hypothesis, we investigated the effect of various levels of novel HY on growth performance, apparent nutrient digestibility, gut health, and microbial fermentation in early-weaned piglets.
2. Materials and Methods
The procedures of the trial (authorization No. IACUC-KKU4/64) were reviewed and approved by the Institutional Animal Care and Use Committee of Khon Kaen University (Khon Kaen, Thailand).
2.1. Hydrolyzed Yeast Component
The HY component contains crude protein (41.84%), ether extract (2.3%), calcium (0.06%), phosphorus (0.71%), gross energy (4682 kcal/kg), β-glucan (22.43%), and MOS (15.6%).
2.2. Animal Care, Housing, and Treatment
A total of 72 mixed crossbred piglets ([Landrace × Large White] × Duroc; 5.71 ± 0.22 kg initial body weight) with the same number of males and females, weaned at 18 days, were obtained from the swine research unit of Khon Kaen University (Khon Kaen, Thailand) in two batches of 36 piglets. The healthy piglets with no symptoms of disease were assigned to three dietary treatments with six pens of four pigs (two gilts and two barrows) in each pen, containing three pens per batch in a randomized complete block design. Treatments were (i) control (CON; 18 ± 2-day weaning (
n = 24) without HY supplementation); (ii) CON + 5% of HY-supplemented diet (HY5); and (iii) CON + 10% of HY-supplemented diet (HY10). A corn–soybean meal (SBM)-based diet was prepared weekly to ensure feed quality in mash form using a horizontal feed mixer with a maximum capacity of 150 kg. The diet with no addition of HY was prepared prior to the HY-supplemented diet to avoid feed contamination among treatments. The latter was mixed at increasing levels of HY. The piglets were fed a mash diet according to a two-phase feeding program: Phase I, 1 to 14 days post-weaning; and Phase II, 15 to 28 days post-weaning (
Table 1). The experimental diets were formulated to meet or exceed the predicted requirements for weaning pigs [
11], having similar metabolizable energy, Ca, and standardized ileal amino acid content. All pigs had available access to feed and water during the study.
Each pen (1.6 × 1.6 m, with a stocking density of 0.64 m2 each) had slatted concreted floors and was equipped with a nipple drinker, polyvinyl dry feeder, and heating lamp. Unused rice straw as bedding was provided twice daily at 06:00 and 19:00 during a 14-day period to minimize temperature stress under partially controlled conditions, which conformed to the European regulations (EU Directive 2010/63/EC for animal experiment). The housing environment was controlled using mechanical ventilation to maintain a desirable temperature of 31 ± 1 °C during the 1st week, and this was gradually decreased to 29 ± 1 °C, which was maintained throughout the entire experiment. There was no occurrence of atrophic rhinitis, transmissible gastroenteritis, Aujeszky’s disease, Salmonellosis, or porcine reproductive and respiratory syndrome during the previous three years; therefore, vaccination was not administered during the study.
2.3. Growth Performance
On days 14 and 28 post-weaning, each pig’s body weight (BW), the amount of feed supplied, and feed disappearance were recorded on a pen basis at the end of each feeding phase. These values were used to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (gain to feed ratio, G:F). Growth performance criteria were summarized in each feeding program and for the overall period.
2.4. Diarrheal Occurrence
All piglets were observed daily for health parameters by two technicians. The visual assessment of fecal consistency was carried out on a pen basis and divided into 4 categories: 0 = hard feces (normal), 1 = brown and soft stool feces (normal), 2 = yellow or bloody soft formed sticky feces (diarrhea), and 3 = watery diarrhea (severe diarrhea). The diarrheal occurrence was reported as the number of diarrheal piglets/(total number of piglets x diarrhea day) × 100. Mortality was represented as the percentage of dead pigs (number of dead pig/number of initial pigs in the group).
2.5. Nutrient Digestibility
Twelve crossbred barrows (11.23 ± 0.43 kg of initial BW) were assigned to three treatments (four replicates) with a completely randomized design in individual metabolic pen (0.85 m × 1.15 m × 0.68 m), placing a tray underneath a metabolic cage for 5 days of feed adaptation to the diet and to acclimate the experiment condition. Each pen was equipped with a cup drinker near the stainless-steel feeder and concrete grate flooring, kept at a temperature of 31 ± 1 °C. Pigs were fed diets mixed with chromic oxide and ferric oxide (3 g/100 g of feed) as indigestible initial and terminal markers, respectively. Fecal sampling was performed for 5 days. Fecal samples were collected daily from the mesh bottom of each crate at 07:00 and 19:00, and sealed in plastic bags at −20 °C. These representative samples of feces and completed feed (500 g per pen) were dried to constant weight at 60 °C for 72 h; then, they were ground in a centrifugal mill to have a particle size below 0.85 mm for further analyses. The homogenous samples were detected in triplicate for crude ash (method no. 942.15) and dry matter (method no. 930.15) using a forced air drying oven at 135 °C for 2 h; crude protein (method no. 984.13; N × 6.25) was measured using the Kjeldahl method, and ether extract (method no. 920.39) using the Soxhlet apparatus as per AOAC guidelines [
12]. Nutrient digestibility was examined and calculated using the formula of Adeola [
13]. Urine was collected daily for 5 days using plastic buckets containing 50 mL of 10% H
2SO
4, covered with a glass-wool filter to eliminate unwanted matters. Each sample was subsequently diluted with 4000 mL tap water to obtain a homogenous mix prior to storage in 50 mL conical tubes for determination of nitrogen retention.
2.6. Intestinal Morphology
On day 28, pigs were deprived of feed for 12 h. Six pigs (three gilts and three barrows; n = 18) having a normal BW close to the average pen BW were removed for slaughter at a local slaughterhouse (Kasetsombun, Chaiyaphum, Thailand). Approximately 5 cm pieces of duodenum (approximately 70 cm caudal from the pyloric region of stomach) and jejunum (5 cm between stomach sphincter and ileo-cecal junction) were collected and flushed with phosphate-buffered saline and then fixed in 10% (v/v) saline solution before being delivered to the laboratory (Betagro Science Center, Pathumthani, Thailand). Intestinal tissues were cut into pieces of 5 µm thickness from paraffin blocks using a rotary microtome prior to drying in an incubator at 37 °C. The slides were subsequently deparaffinized with xylene and rehydrated with ethanol for staining with hematoxylin and eosin. Photomicrographs were detected with 40× magnification using a light microscope equipped with stereological software (Olympus Corporation, Tokyo, Japan). Six well-oriented villi with the entire villi and crypt covered in the cross-section and presented in the central lacteal were chosen to measure villous height (VH: from the villous tip to the villous–crypt junction). Crypt depth (CD) was measured from the villous–crypt junction to the villous bottom; hence, the ratio of VH to CD was calculated.
2.7. Blood Collection, Metabolic Profiles, and Immunity
On day 14, blood samples were collected from 18 pigs (one pig per pen in an equal sex), whereas the collection on day 28 was performed before euthanization. A 12 mL blood sample was obtained from individual pigs through jugular venipuncture into two sets of serum tubes coated with micronized silica particles (True Biomedical, Pathumthani, Thailand; 6 mL/tube). The collected samples were harvested for serum using a centrifugal force at 1872× g for 15 min at 4 °C and the supernatant was transferred to microcentrifuge tubes prior to being frozen (−80 °C) pending assays for metabolic profile and immune response assessment.
Serum samples were thawed at room temperature for 60 min. The serum concentrations of glucose, albumin, and blood urea nitrogen (BUN) were quantified using a colorimetric method, following the manufacturers’ instructions (Cambridge Biomedical, Cambridge, UK). Briefly, 20 μL of serum was adjusted the volume to 50 μL with glucose assay buffer (Glucose Assay Kit, Abcam, Waltham, MA, USA) or albumin assay buffer (Albumin Assay Kit, Abcam, Waltham, MA, USA) and mixed for 60 s to ensure homogeneity. These representative samples were used to quantify glucose and albumin at the absorbance of 570 and 620 nm, respectively. For BUN determination, serum was diluted in a 10-fold dilution with distilled water. Then, 50 μL of the diluted sample was placed on each microplate, and we added 75 μL of acid solution and incubated them for 30 min before determination, performed at an absorbance of 450 nm. All blood measurements of glucose, albumin, and BUN concentration were detected by spectrophotometer.
Serum immunoglobulin A (IgA) concentration was quantified using an Enzyme-Linked Immunosorbent Assay (ELISA) porcine kit (E101-102; Bethyl Laboratories, Inc., Montgomery, LA, USA). Briefly, 10 μL of diluted serum with phosphate-buffered saline with a final dilution of 1:100,000 was pipetted into each microplate, followed by the addition of 100 μL of biotinylated porcine IgA, enzyme-conjugated antibody, and color reagent, and incubated for 60 min at room temperature. The assays of interleukin-1β (IL1) and interleukin-6 (IL6) were performed using the ELISA kit (R&D System, Minneapolis, MN, USA). One hundred microliters of standard and samples were pipetted into a 96-well plate with a plate sealer, followed by incubation at room temperature for 30 min to aspirate liquid. For the IL1 assay, the samples were coated with 90 μL of chromogen TMB substrate solution and diluted hydrochloric acid as the detection reagents after incubation. For IL6 assays, the commercial kit had a detection range from 125 to 8000 pg/mL. After incubation, an aliquot of 100 μL of detection antibody and streptavidin–HRP were added as the working dilution, followed by 50 μL of 2 N H2SO4 as the stop solution. Tumor necrosis factor alpha (TNFα) was quantified using the ELISA porcine immunoassay kit (R&D System, Minneapolis, MN, USA). Fifty microliters of sample were added to each plate coated with biotinylated antibody reagent. Detection was performed using 3,3′, 5,5′ tetramethybenzidine, and a stop solution of 2 mol/L H2SO4. The absorbance for all measurements was detected at 450 nm using a spectrofluorometer in triplicate to avoid variation, with a total of 48 samples (six samples per treatment).
2.8. Microbial Fermentation
To examine microbial fermentation in pig feces, fresh fecal samples (10 g) were collected directly from each pig’s rectum after slaughtering and divided into two parts. Samples (six samples per treatment, n = 18) were pooled on a pen basis. The bacterial enumeration was detected using commercial agars—MacConkey, Salmonella–Shigella, and Lactobacillus medium II agars (Difco Laboratories, Detroit, MI, USA)—to determine Escherichia coli, Salmonella spp., and Lactobacillus spp., respectively. Each agar was prepared by suspending the dehydrated agar in 1000 mL of distilled water, boiling, and autoclaving at 121 °C for 15 min. One gram of the composite fecal sample was diluted with 9 mL of 10 g/kg buffered peptone broth (Becton Dickinson, Franklin Lakes, NJ, USA) and homogenized properly using vortex mixing for 15 min. One milliliter of diluted feces was overlaid on the specific agar (15 mL per plate) and incubated at 37 °C under aerobic conditions after solidification of the medium for 24 to 48 h to allow bacterial growth. The visible spotted colonies were counted immediately based on their morphology and color. The number of each microbial was expressed as the logarithm of colony-forming units per gram of feces, for a total of 48 samples (duplicate). Another fecal sample was snap-frozen in liquid nitrogen and immediately used to quantify short-chain fatty acids (SCFA). Briefly, feces were diluted with deionized water (1:1; w/v), mixed, and centrifuged at 1872× g for 10 min. After filtration, the supernatant fraction of 0.1 mL was added to 1.0 mL of 2-methyvaleric acid (catalog # SHBL3457) as an internal standard before injection for gas chromatography (CP-3380 GC, Varian, Inc., Walnut Creek, CA, USA). The initial temperature of the column was 170 °C and the injection temperature was held at 240 °C (run time 25 min). Hydrogen was used as a carrier gas at a flow rate of 5.0 mL/min, and the split rate was 70 mL/min. One microliter of sample was made at a split ratio of 1:10. The concentrations of SCFA were quantified from the peak area and presented as µmol per gram of feces.
2.9. Statistical Analysis
All the data were analyzed using the general linear model procedure of SAS (version 9.4, SAS Institute Inc., Carry, NC, USA) in a randomized complete block design. Each pen (n = 18) was an experimental unit for the growth performance assays, whereas each pig was an experimental unit for nutrient digestibility, intestinal morphology, blood components, immunity, and microbial fermentation. The orthogonal polynomial contrasts were tested for linear and quadratic effects in response to the dosage of HY. The statistical significance for a tendency was detected at p > 0.05 to p < 0.10. Results were reported as the least square mean and pooled standard error of the mean.