Effects of Yarrowia lipolytica supplementation on growth performance, intestinal health and apparent ileal digestibility of diets fed to nursery pigs

Objective The objective was to evaluate the efficacy of increasing supplementation of Yarrowia lipolytica (YL) up to 3.0% replacing 1.6% poultry fat and 0.9% blood plasma for growth performance, intestinal health and nutrient digestibility of diets fed to nursery pigs. Methods Twenty-four pigs weaned at 24 d of age (initial body weight at 7.2±0.6 kg) were allotted to three dietary treatments (n = 8) based on the randomized complete block. The diets with supplementation of YL (0.0%, 1.5%, and 3.0%, replacing poultry fat and blood plasma up to 1.6% and 0.9%, respectively) were fed for 21 d. Feed intake and body weight were recorded at d 0, 10, and 21. Fecal score was recorded at every odd day from d 3 to 19. Pigs were euthanized on d 21 to collect proximal and distal jejunal mucosa to measure intestinal health markers including tumor necrosis factor-alpha, interleukin-8, immunoglobulin A and immunoglobulin G. Ileal digesta was collected for apparent ileal digestibility (AID) of nutrients in diets. Data were analyzed using Proc Mixed of SAS. Results Supplementation of YL (1.5% and 3.0%) replacing poultry fat and blood plasma did not affect growth performance, fecal score and intestinal health. Supplementation of YL at 1.5% did not affect nutrient digestibility, whereas supplementation of YL at 3.0% reduced AID of dry matter (40.2% to 55.0%), gross energy (44.0% to 57.5%), crude protein (52.1% to 66.1%), and ether extract (50.8% to 66.9%) compared to diets without supplementation. Conclusion Yarrowia lipolytica can be supplemented at 1.5% in nursery diets, replacing 0.8% poultry fat and 0.45% blood plasma without affecting growth performance, intestinal health and nutrient digestibility. Supplementation of YL at 3.0% replacing 1.6% poultry fat and 0.9% blood plasma did not affect growth performance and intestinal health, whereas nutrient digestibility was reduced.


INTRODUCTION
Animal fats and plant oils have been used in swine diets in order to improve dietary energy digestibility [1], growth and feed efficiency [2]. Fatty acids (FA) play important roles in cell-signaling, immune function and cell membrane integrity [3]. In addition, FA can be divided into two main groups comprised of saturated FA and unsaturated FA. The quantities of FA vary greatly, depending on the sources (Table 1). Unsaturated FA improves the animal health and meat quality by enhancing intestinal barrier functions [4] and altering the pork fat composition [5], respectively.
The production of animal fats was increased by 15% from 2013 to 2020 in the USA [6]. Other than animal fats, the production of plant oils, including soybean oil, seed oils, palm oil and coconut oil, was increased by 22% from 172 to 209 million tons globally during the same period [6]. However, animal fats and plant oils have also been largely used in different industry sectors, including biodiesel and food industries [7]. For the bioenergy production, the global demand of fats and oils has been increased by 38% in the last decade [8] and the food industry requires higher fat quality than the feed industry. Therefore, fats with lower quality, such as restaurant grease and animal-vegetable blend oils are commonly used in animal production in the USA. These fat by-products could have higher peroxidation possibly with negative impacts on intestinal health and growth performance of animals [9]. Due to these concerns, alternative fat supplements have been sought, including oleaginous microorganisms [10] and insect oils [11].
Oleaginous microorganisms include yeasts, bacteria and microalgae rich in oil and these are also called single cell oils (SCO). Depending on the sources, SCO contain FAs with unique profiles different from conventional fats and oils, proteins from cell wall with unique functions and other organic compounds such as vitamins [12]. Recently SCO have been used in aquaculture to replace conventional fats without adverse effects on fish growth and fish quality [13]. Yarrowia lipolytica is one of oleaginous yeasts and generally recognized as safe in food industry [14]. The strain of Yarrowia lipolytica used in this study contains 72.7% of fat and high concentration (52.3%) of oleic acid (Table 1). Oleic acid has been shown to reduce oxidative stress and inflammatory response by activating peroxisome proliferator activated receptor in animals [15]. Previous studies showed that animal fats and plant oils are 80% digestible by weaned pigs [1]. Similar to Yarrowia lipolytica, restaurant grease has similar oleic acid content, however, its high peroxidation rate compromises its benefits [9].
In general, β-glucans, including 1,3 and 1,6 β-glucans, are the major components of the yeast cell wall (55% to 65%) [16]. The second major component from the yeast cell wall is mannoprotein (30% to 40%), which is glycoprotein mainly containing mannans on the external surface of the wall [17]. Beta-glucans and mannans are bioactive compounds from yeast cell walls with potential benefits on the development of intestinal immune system of animals [18,19] that can be similar to the role of functional compounds in blood plasma. Blood plasma has long been used in swine industry in order to improve growth performance [20] of nursery pigs by enhanced intestinal health that reduces inflammatory activation and promotes the intestinal barrier function [21]. Otherwise, β-glucans and mannans from yeast cell wall also have beneficial effects on intestinal health by mitigating the release of pro-inflammatory cytokines and preventing the colonization of pathogens on intestinal mucosa [18,19].
Therefore, the hypothesis was that Yarrowia lipolytica can be used as a fat supplement in nursery diets providing bioactive components, including oleic acid, β-glucans and mannans to enhance intestinal health, nutrient digestibility and growth performance of nursery pigs. To test the hypothesis, the objective was to evaluate the efficacy of increasing supplementation of Yarrowia lipolytica up to 3.0% replacing 1.6% poultry fat and 0.9% blood plasma for growth, intestinal health and digestibility of nutrients in diets fed to nursery pigs.

Animal care
The experimental protocol was approved by the Institutional Animal Care and Use Committee of North Carolina State University.

Animal, design and diets
Twenty-four newly weaned pigs at 24 d of age with initial body weight (BW) of 7.2±0.6 kg were allotted to three dietary treatments (n = 8) based on the randomized complete block design. Initial BW (light and heavy) and sex (gilts and barrows) were considered as blocks following Holanda et al [22]. Pigs were housed in pens individually. The dietary treatments consisted of a basal diet supplemented with Yarrowia lipolytica (CJ Bio; Fort Dodge, IA, USA) at three 2) Based on analyzed composition using gas chromatography/mass spectrometry (GC/MS) method. 3) Amino acid profile is based on Michalik et al [24]. levels (0.0%, 1.5%, and 3.0% of Yarrowia lipolytica as YL0, YL1.5, and YL3, respectively). Yarrowia lipolytica was supplemented at 1.5% and 3.0%, replacing poultry fat (0.8% and 1.6%, respectively) and blood plasma (0.45% and 0.9%, respectively) balancing metabolizable energy and standardized ileal diges tible Lys in phase 1 and phase 2 diets. The nutrient compositions of Yarrowia lipolytica and experimental diets are shown in Table 1 and 2, respectively. All experimental diets were formulated to meet or exceed the nutrient requirements suggested by NRC [23] and fed to pigs for 21 d divided into two phases: phase 1 (d 0 to 10) and phase 2 (d 10 to 21). Titanium dioxide at 0.4% was added in diets as an indigestible external marker during the last 7 d of experiment.
Growth performance and fecal score Body weight and feed intake were recorded at d 0, 10, and 21 to calculate average BW, average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). Fecal scores from every pig were recorded every odd day using a 1 to 5 scale: i) very hard and dry stool, ii) firm stool, iii) normal stool, iv) loose stool, and v) watery stool with no shape following Guo et al [25].

Sample collection and processing
After 21 d of feeding, all pigs were euthanized by the penetration of a captive bolt to the head following by exsanguination in order to collect samples. Sections of proximal (1.5 m after the pyloric duodenal junction) and distal (1.5 m before the ileocecal junction) jejunum were collected and rinsed with 0.9% saline solution. The jejunal mucosa was collected into 2 mL microcentrifuge tubes and immediately frozen in liquid nitrogen and then stored at -80°C for immune status measurements. Ileal digesta was collected into 150 mL containers placed on ice and then stored at -20°C for apparent ileal digestibility (AID). Jejunal mucosa samples (1 g) from the 2 mL microcentrifuge tubes were taken and added with 1 mL of phosphatebuffered saline solution into 5 mL polypropylene tubes. Mucosa samples were homogenized using a tissue homogenizer (Tissuemiser, Thermo Fisher Scientific Inc., Rockford, IL, USA) for 30 s on ice and transferred to a new 2 mL microcentrifuge tube for centrifugation for 15 min at 14,000×g (MiniSpin plus; Eppendorf AG, Hamburg, Germany) as described by Holanda et al [22]. The supernatant was divided into 6 sets of 0.15 mL into polypropylene tubes and stored at -80°C for further analysis.

Intestinal health makers
The concentrations of total protein, tumor necrosis factoralpha (TNF-α), interleukin-8 (IL-8), immunoglobulin A (IgA), and immunoglobulin G (IgG) in proximal and distal jejunal mucosa were analyzed by the colorimetric method and the absorbance was measured on a plate reader (Synergy HT; BioTek Instruments, Winooski, VT, USA).
The concentration of total protein was analyzed using Pierce BCA Protein Assay Kit (#23225; Thermo Fisher Scientific, USA) as described by Holanda et al [22]. Mucosa samples were diluted (1:50) to reach the working range of 20 to 2,000 μg/mL. The absorbance was measured at 562 nm. The concentration of total protein was calculated by the standard curve and used to normalize the concentrations of TNFα, IL-8, IgA, and IgG.
The concentration of TNF-α was analyzed using the Porcine TNF-α Immunoassay Kit (#PTA00; R&D Systems, Minneapolis, MN, USA) as described by Duarte et al [26]. The working range of standards was 0 to 1,500 pg/mL. The absorbance was measured at 450 nm and corrected with 570 nm. The concentration of TNF-α was calculated by the standard curve and described as pg/mg of protein.
The concentration of IL-8 was analyzed using the Porcine IL-8/CXCL8 Immunoassay Kit (#P8000; R&D Systems, USA) following Moita et al [27]. Mucosa samples were diluted (1:6) to reach the working range of standards from 0 to 4,000 pg/mL. The absorbance was measured at 450 nm and corrected with 570 nm. The concentration of IL-8 was calculated by the standard curve and described as ng/mg of protein.
The concentration of IgA was analyzed using the pig ELI-SA kit (#E101-102; Bethyl Laboratories, Montgomery, TX, USA) following Holanda et al [22]. Mucosa samples were diluted (1:1,500) to reach the working range of standards from 15.6 to 1,000 ng/mL. The absorbance was measured at 450 nm and corrected with 540 nm. The concentration of IgA was calculated by the standard curve and described as μg/mg of protein.
The concentration of IgG was analyzed using the pig ELI-SA kit (#E101-104; Bethyl Laboratories, USA) following Duarte et al [26]. Mucosa samples were diluted (1:3,600) to reach the working range of standards from 7.8 to 500 ng/mL. The absorbance was measured at 450 nm and corrected with 570 nm. The concentration of IgG was calculated by the standard curve and described as μg/mg of protein.
Apparent ileal digestibilities of DM, GE, CP, and EE were calculated using the titanium dioxide concentration in diets and digesta following Chen et al [31]. The AID was calculated with the following equation:

Statistical analysis
Data were analyzed using the MIXED procedure in SAS 9.3 (SAS Inc, Cary, NC, USA). Dietary treatments were defined as fixed effects. Initial BW and sex were considered as blocks and defined as random effects. Outliers were defined as data greater than the 75th percentile+1.5×the interquartile range and smaller than the 25th percentile-1.5×the interquartile range and removed from analysis. One pig from treatment YL1.5 was removed for analysis of AID of CP and EE due to insufficient digesta. The least squares mean comparisons with the general linear model procedure was used to calculate mean values for all treatments. The least significant difference test (PDIFF) was used for a pairwise comparison among dietary treatments. The analysis of fecal score data was performed by using Kruskal-Wallis Test with Dwass, Steel, Critchlow-Fligner method option for pairwise two-sided multiple comparisons following Guo et al [25]. Statistical significance was p<0.05 and 0.05≤p<0.10 was considered as a tendency.

RESULTS
Initial BW was 7.2±0.6 kg and did not differ among treatments. Overall ADG, ADFI, and G:F were 252±37 g, 421±52 g, and 0.60±0.04, respectively and these were not affected by the supplementation of Yarrowia lipolytica at 1.5% and 3.0% in diets (Table 3). There were no difference in fecal scores among the treatments (Figure 1)

DISCUSSION
This study showed that supplementation of Yarrowia lipolytica at a range of 1.5% and 3.0% is an effective fat supplement in nursery diets. Supplementing nursery diets with Yarrowia lipolytica at 1.5%, replacing 0.8% poultry fat and 0.45% blood plasma, did not affect the growth performance, intestinal health and nutrient digestibility. Supplementing Yarrowia li- ADFI, average daily feed intake; G:F, gain to feed ratio. 1) The dietary treatments consisted of a basal diet supplemented with Yarrowia lipolytica at 0.0%, 1.5%, and 3.0% as YL0, YL1.5, and YL3, respectively.  polytica at 3.0% replacing 1.6% poultry fat and 0.9% blood plasma did not adversely affect the growth performance of pigs, whereas it reduced the AID of DM, GE, CP, and EE. The current results are in agreement with previous studies that reported the supplementation of yeasts at 30% in fish diets [13] and at 1.6×107 colony-forming unit/g of nursery diets [32] reduced the apparent digestibility of nutrients but did not affect the overall growth performance. Conversely, Czech et al [33] reported that supplementation of 3.0% of Yarrowia lipolytica replacing soybean meal enhanced the growth performance of nursery pigs from 10 to 32 kg. A reduction of nutrient digestibility with 3.0% of Yarrowia lipolytica without affecting the growth performance observed in this study is probably due to the bioactive compounds from yeasts including oleic acid, β-glucans and mannans that play important roles on microbiota modulation and immune system [34,35]. The decreased nutrient digestibility observed in this study may be due to the complex structures from yeast cell walls that have negative effects on nutrient digestibility [22]. In this study, Yarrowia lipolytica was added to diets by replacing up to 1.6% poultry fat and 0.9% blood plasma. Considering that the Yarrowia lipolytica was not processed to lyse the cell wall prior the study, the cell wall may have partially protected the fat from digestion which may have contribute to reduce the EE digestibility [36]. Additionally, the protein in the Yarrowia lipolytica used in this study may have lower digestibility compared to blood plasma and consequently reduced the CP digestibility in feed with 3.0% Yarrowia lipolytica. The yeast cell wall are fermented by the intestinal microbiota in the large intestine to produce short-chain fatty acids (SCFA) and, consequently, release cell components in the large intestine [37]. In addition to the cell wall compounds, the undigested nutrients are fermented by the intestinal microbiota along the intestine affecting the production SCFA and consequently affecting the energy utilization [38]. The majority microbial fermentation occurs in the cecum-colon portion [39], therefore, the SCFA produced during fermentation in the large intestine was not counted in the analysis of AID, although they could affect the growth performance even with reduced AID of nutrients [40]. Yeast cell wall is known for their effects on microbiota modulation of intestinal microbiota [37]. Kiros et al [41] showed that the increased relative abundance of Prevotella, an effective producer of SCFAs, in pigs fed diets with yeast had positive correlations with growth performance by increasing energy utilization. In addition, dietary mannans and β-glucans have been related to increased relative abundance of butyrate-producing bacteria including Clostridium [35] and Faecalibacterium prausnitzii [42]. Besides the energy metabolism, butyric acid is known as an important compound for the intestinal health status [43].
It is well known that supplementing blood plasma in nursery diets improves the growth performance of pigs [20]. Besides the highly digestible protein, 20% of protein in blood plasma is IgG that inhibit the adherence of antigens in the intestinal mucosa, enhancing the immune response and, consequently improving the growth performance [44]. Yarrowia lipolytica has a high concentration of oleic acid, which possesses anti-inflammatory and anti-oxidative properties [15] and, consequently, would enhance the health and growth performance of pigs. Similar to the immune modulating function of dietary blood plasma, β-glucans and mannans present in yeast cell wall are beneficial to animal growth and health [19]. The first mechanism of β-glucan and mannans is that both can bind to the carbohydrate receptors on pathogenic bacteria preventing infection by inhibiting the adherence of pathogens to the intestinal mucosa [32]. Secondly, they can be bind by receptors on intestinal epithelial cells, including M cells, macrophages and dendritic cells and, consequently activate the innate response to release cytokines and adoptive response [45]. Following the innate response, activated intestinal epithelial cells stimulate Th-1 cells to reduce the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-8 [34]. Li et al [18] described that the uptake of β-glucans from Saccharomyces cerevisiae at 50 mg/kg in nursery increased the release of the anti-inflammatory cytokine, IL-10, to inhibit the overproduction of proinflammatory cytokines, TNF-α and IL-6 in plasma under Escherichia coli challenge. In addition, supplementing Saccharomyces cerevisiae in pigs [46] enhanced immune system by promoting the concentrations of IgA and IgG in blood and resulted in improved growth performance. However, the immune markers measured in this study were not adversely affected by supplementing Yarrowia lipolytica up to 3.0% indicating that the bioactive components from Yarrowia lipolytica may have similar effects as those expected from blood plasma [20] for maintaining animal growth and health. 1) The dietary treatments consisted of a basal diet supplemented with Yarrowia lipolytica at 0.0%, 1.5%, and 3.0% as YL0, YL1.5, and YL3, respectively. a,b Within a row, means without a common superscripts letter are significant different (p < 0.05). A,B Within a row, means without a common superscripts letter are tended to be different (p < 0.10).