Improved quality of cottonseed meal: effect of cottonseed protein isolate on growth performance, nutrient digestibility, and intestinal health in growing pigs

Abstract

Cottonseed meal (CSM) is abundant in proteins that have the potential to substitute for conventionally utilized protein supplements for animals. However, the presence of anti-nutritional factors in CSM, particularly free gossypol, has limited its application. This study evaluated the nutritional value of a cottonseed protein isolate (CPI) derived from CSM using an alkaline extraction and acid precipitation process and explored its effect on intestinal health in growing pigs. 32 Duroc-Landrace-Yorkshire castrated male pigs (initial body weight 19 ± 2 kg) were divided into 4 treatment groups: nitrogen-free diet, corn–soybean meal diet (CSD), cottonseed meal diet (CSMD, where CSM replaced 35% of the nitrogen in CSD), CPI diet (CPID, where CPI replaced 35% of the nitrogen in CSD). Our study revealed that, as compared to the CSM, the CPI exhibited significantly higher crude protein content and lower levels of crude fiber, neutral detergent fiber, acid detergent fiber, and free gossypol (P < 0.01). Interestingly, CPI feeding significantly decreased the ratio of gain to feed (G:F) in growing pigs (P = 0.012). Moreover, CPI also showed an improved apparent and true digestibility of protein, as well as enhanced nitrogen utilization in growing pigs (P < 0.05). The metabolizable energy of CPI was significantly higher than that of CSM (P < 0.01). Additionally, CPI showed higher apparent ileal digestibility and standardized ileal digestibility for amino acids such as arginine, aspartic acid, glutamic acid, and serine (P < 0.05). Importantly, CPI feeding improved the intestinal health in pigs as indicated by increases in villus height and digestive enzyme activities (P < 0.05), as well as increases in the production of short-chain fatty acid and beneficial microbiota (0.05 ≤ P < 0.10). The results not only showed an improved quality of CPI as compared to the CSM but also indicated a beneficial effect of CPI on growth performance and intestinal health in growing pigs. These attributes should make it an attractive candidate to substitute for conventionally utilized protein supplements like soybean meal.

This study highlights the potential of cottonseed protein isolate (CPI) as a sustainable alternative to soybean meal in animal feed. CPI enhances growth performance, nutrient digestibility, and intestinal health in growing pigs, offering an eco-friendly solution for livestock production.

1 Introduction

Soybean meal (SBM) is one of the conventionally utilized protein supplements in the animal nutrition and feed industry due to its high protein content and well-balanced amino acid profile (Deng and Kim, 2024). However, overdependence on SBM may result in a series of challenges including supply instability (Parisi et al., 2020), environmental concerns (Nijdam et al., 2012; Van Zanten et al., 2018), and competition with human food resources (Mottet et al., 2017). For instance, fluctuations in soybean production caused by climate change and market dynamics may contribute to instability in price and availability. Moreover, large-scale cultivation of soybeans exacerbates environmental issues, such as deforestation, soil degradation, and the overuse of water resources, raising concerns about the long-term sustainability of SBM as a primary protein source. Therefore, the development of various substitutes for SBM has garnered considerable research interest worldwide.

Cottonseed meal (CSM) is an abundant resource, with global cottonseed production projected to reach 41.50 million metric tons in the 2023 to 2024 season (USDA, 2024). The CSM is rich in crude protein (CP; Nagalakshmi et al., 2007), and various indispensable amino acids (Li et al., 2011). Additionally, it is more cost-effective than SBM, making it a promising plant-based protein feed alternative with significant development potential (Lestingi, 2024). However, the presence of anti-nutritional factors, particularly gossypol, limits CSM’s widespread use in animal nutrition (Yu et al., 1996; Gadelha et al., 2014). Free gossypol is a toxic binaphthyl compound (Lin et al., 2023), and studies have reported that free gossypol exhibits cytotoxic, neurotoxic, and hepatotoxic properties (Tang et al., 2017; Zhu et al., 2021; Cao and Sethumadhavan, 2023). Excessive or prolonged intake of gossypol can accumulate in the body, causing poisoning, disrupting immune and reproductive functions, and, in severe cases, leading to death (Gadelha et al., 2014). Additionally, lignin and other indigestible fibers in CSM can negatively impact feed conversion efficiency and hinder animal growth performance (Jazi et al., 2017). Consequently, the widespread use of CSM is restricted by these anti-nutritional factors, particularly at higher inclusion rates, which can adversely affect animal health and growth.

To enhance the use of CSM in animal nutrition, recent research has focused on improving processing methods to reduce anti-nutritional compounds and enhance protein utilization (Xu et al., 2022; Zhang et al., 2022). Physical and chemical pretreatments have been explored to lower free gossypol levels in CSM, but both approaches have limitations. Physical methods often fail to fully remove anti-nutritional factors and can be costly, while chemical treatments may leave residual chemicals that affect feed palatability (Saxena et al., 2012; Pelitire et al., 2014; Rathore et al., 2020). Biological methods, such as genetic breeding and microbial fermentation, have also been explored. However, genetic breeding requires long development cycles, and the success of new varieties is often hindered by regional differences in soil and environmental conditions, limiting large-scale application (Zhang and Wedegaertner, 2021; Wen et al., 2023). Microbial fermentation, although effective in reducing anti-nutritional factors (Zhang et al., 2006, 2022), is complex and requires strict control over fermentation conditions, with mixed results in free gossypol removal (Zhang et al., 2024). In contrast, the alkaline extraction and acid precipitation method is a well-established and cost-effective protein isolation technique (Hou et al., 2017; Zhang et al., 2020; Hadidi et al., 2023). This method dissolves CSM proteins in an alkaline solution, followed by pH adjustment to precipitate the protein at its isoelectric point, resulting in a high-purity protein isolate after drying. The alkaline extraction and acid precipitation process is advantageous because it effectively reduces anti-nutritional factors like gossypol and crude fiber (CF) while maintaining a high protein yield, and it does not involve the use of potentially harmful residual chemicals. Moreover, it is simpler and more scalable, making it a practical solution for large-scale feed production.

In this study, CSM was used as the raw material to produce cottonseed protein isolate (CPI) through an alkaline extraction and acid precipitation process. The nutritional value of CPI in growing pigs was evaluated, with the goal of mitigating the inherent limitations of untreated CSM. This study aims to demonstrate the potential of CPI as a high-quality, cost-effective alternative to SBM in animal nutrition.

Materials and Methods

Animal ethics statement

All experimental procedures in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (No.20200915). The experiment was conducted at the Animal Experiment Center of Sichuan Agricultural University in strict accordance with institutional guidelines for the care and use of laboratory animals.

Preparation of CPI

Cottonseed meal (45.04% protein, 90.29% dry matter (DM), ground to pass through a 40-mesh sieve) was provided by Xinjiang Taikun Protein Industry Co., Ltd. The large-scale preparation of CPI was conducted as follows: 10 kg of cottonseed meal (accurate to 0.01 kg) was weighed and placed into a temperature-controlled mixing tank, followed by the addition of 140 kg of water heated to 60 °C. During stirring, an appropriate amount of 30% NaOH solution was added to the extraction mixture to adjust the pH to 10. Alkaline extraction was carried out for 2 h under optimal conditions. After the extraction, the mixture was filtered using 120-mesh gauze, and the filtrate was collected. While stirring, an appropriate amount of 30% HCl solution was gradually added to the filtrate to adjust the pH to 4.6, followed by acid precipitation for more than 3 h to allow complete precipitation of the CPI. The precipitate was then filtered using 120-mesh gauze, collected, and air-dried at 55 °C to obtain the CPI sample.

Experimental diets

The experiment was conducted using a completely randomized design, selecting 32 Duroc-Landrace-Yorkshire crossbred castrated male pigs with an initial body weight of 19 ± 2 kg. The pigs were randomly assigned to 4 treatments, each with 8 replicates and one pig per replicate. The diets administered were a nitrogen-free diet, a corn–soybean meal diet (CSD), a cottonseed meal diet (CSMD, where CSM replaced 35% of the nitrogen in CSD), and a CPI diet (CPID, where CPI replaced 35% of the nitrogen in CSD). The average daily gain (ADG), average daily feed intake, and the ratio of gain to feed (G:F) were calculated. Each diet included 0.4% chromium. The feed formulations and nutritional levels are presented in Table 1.

Table 1.

Composition and nutritional level of experimental diets (fed basis, %)

Ingredients	NFD	CSD	CSMD	CPID
Corn starch	84.70	—	10.31	12.55
Corn	—	65.30	57.80	59.28
Soybean meal	—	22.40	11.08	10.38
CSM	—	—	11.4	—
CPI	—	—	—	7.84
Cellulose	4.00	—	—	—
Soy oil	2.00	3.60	0.77	1.13
Sugar	5.00	5.00	5.00	5.00
Choline chloride	0.15	0.15	0.15	0.15
CaCO3	0.42	0.70	0.90	0.70
CaHPO4	2.28	1.40	1.15	1.50
NaCl	0.30	0.30	0.10	0.10
L-Lys	—	0.30	0.49	0.52
DL-Met	—	0.10	0.10	0.10
L-Thr	—	0.10	0.10	0.10
K2CO3	0.40	—	—	—
MgO	0.10	—	—	—
Vitamin premix1	0.05	0.05	0.05	0.05
Mineral premix2	0.20	0.20	0.20	0.20
Cr2O3	0.40	0.40	0.40	0.40
Total	100	100	100	100
Nutrient level3
CP	0.30	15.35	15.35	15.36
Ca	0.70	0.68	0.72	0.64
Total phosphorus	0.47	0.56	0.63	0.57
Lys	—	1.09	1.13	1.14
Met	—	0.30	0.29	0.31
DE (MJ/Kg)	—	14.33	14.35	14.55

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet; NFD, nitrogen-free diet.

¹The premix provides following per kg diet: VA 8,000 IU; VD₃ 1,500 IU; VE 20.0 IU; VK₃ 2.0 mg; VB₁ 1.5 mg; VB₂ 4.0 mg; VB₆ 1.5 mg; VB₁₂ 20.0 μg; nicotinic acid 30.0 mg; D-pantothenic acid 15.0 mg; folic acid 0.6 mg; biotin 0.1 mg.

²The premix provided following per kg diet: Fe (FeSO4·H₂O) 60.0 mg; Cu (CuSO₄·5H₂O) 4.0 mg; Zn (ZnSO₄·H₂O) 60.0 mg; Mn (MnSO₄·H₂O) 2.0 mg; I (KI) 0.14 mg; Se (Na₂SeO₃) 0.2 mg.

³The nutrients levels of diets were analyzed.

Feeding and management

The experiment was conducted at the research and teaching practice base of Sichuan Agricultural University. The trial period lasted 14 d, including a 3-d adaptation phase, a 7-d pre-feeding phase, and a 4-d digestion and metabolism trial phase. Thirty-two pigs were individually housed in metabolic cages. During the adaptation phase, all pigs were fed a CSD with ad libitum access to water. After the adaptation phase, pigs were weighed and grouped. During the pre-feeding phase, pigs were fed their respective diets at 4% of their body weight. In the digestion and metabolism trial phase, the feed intake for each pig was set at the average intake during the pre-feeding phase. The ambient temperature in the pens was maintained between 25 °C and 30 °C. Pigs were fed 3 times a day at 8:00, 14:30, and 19:30 hours.

Sample collection and processing

Feed Samples: After the diets were prepared, 200 g of each diet was randomly collected per group, placed in ziplock bags, labeled, and stored at −20 °C.

Fecal Samples: All feces were collected during the digestion trial (from 21:00 on day 10 to 21:00 on day 14). After collection, feces were immediately weighed and recorded. A 10% portion of the fresh fecal weight was sampled, with 10% sulfuric acid solution added to fix nitrogen and a few drops of toluene for preservation. The mixture was stored at −20 °C. After the digestion and metabolism trial ended, feces from each pig were pooled for the 4-d period, with 20% of the pooled sample freeze-dried, ground, and stored at −20 °C.

Urine Samples: Urine was filtered through 8 layers of gauze, and 10% of the total volume was sampled. Fifty milliliters of 10% sulfuric acid solution was added to fix nitrogen, and the samples were stored at −20 °C. At the end of the trial, the 4-d pooled urine samples from each pig were filtered through 8 layers of gauze and stored at −20 °C for the determination of urinary energy and nitrogen content.

Serum Samples: On the morning of day 15, after a 10-h fasting period, 10 mL of blood was collected from the anterior vena cava of each pig. The blood samples were left to stand at room temperature for 30 min, followed by centrifugation at 3,500 r/min for 15 min to separate the serum. The serum was then aliquoted, labeled, and stored at −20 °C for subsequent analysis of blood biochemical parameters.

Tissue, digesta, and Mucosal Samples: After blood collection on day 15, pigs were fed at 20-min intervals, and 2.5 h post-feeding, they were anesthetized with chlorpromazine hydrochloride and slaughtered. The abdominal cavity was opened, and the duodenum, jejunum, ileum, cecum, and colon were isolated and collected. A 2- to 3-cm segment from the middle of the duodenum, jejunum, and ileum was fixed in 4% paraformaldehyde for the analysis of intestinal morphology. The remaining intestinal sections were cut longitudinally and rinsed with saline. Mucosal samples were scraped off using sterile glass slides and stored in sterile cryovials, labeled, and kept at −80 °C for further analysis. Digesta from the ileum and cecum was collected and placed in sterile cryovials, labeled, and stored at −80 °C. Additionally, ileal digesta samples were collected in sample bags and stored at −20 °C for the determination of amino acid content and exogenous marker chromium.

Chemical analysis

The following parameters were assessed: moisture content, DM, CP, ether extract (EE), CF, neutral detergent fiber (NDF), acid detergent fiber (ADF), ash content, energy (GE), free gossypol, chromium, and calcium and phosphorus content. The moisture content in the sample was determined in accordance with GB/T 6435-2014 (SAC, 2014), and the DM content of the sample was calculated. CP content was quantified following GB/T 6432-2018 (SAC, 2018a) using an automated Kjeldahl nitrogen analyzer. EE content was determined using GB/T 6433-2006 (SAC, 2006), with the assistance of a fat extraction apparatus. CF, ADF, and NDF contents were all determined using the filter bag method with an automatic fiber analyzer, following the Van Soest analytical system. The ash content was measured in accordance with GB/T 6438-2007 (SAC, 2007). Total Energy: The energy content was determined using a PARR-128 oxygen bomb calorimeter. Amino acid content was measured following the procedures specified in GB/T 18246-2019 and GB/T 15399-2018 (for sulfur-containing amino acids; SAC, 2018b, 2019). Free gossypol was analyzed using high-performance liquid chromatography. Amino acids were quantified using an L-8800 high-speed automatic amino acid analyzer. Chromium was measured using a colorimetric method as an external indicator. The calcium content was determined according to GB/T 6436-2018 (SAC, 2018c). The total phosphorus content was determined according to the GB/T 6437-2018 method (SAC, 2018d).

Scanning electron microscopy

The surface morphology of cottonseed meal and CPI was examined using scanning electron microscopy. The procedure is as follows: Conductive adhesive tape was applied to the specimen holder. Small amounts of CSM and CPI samples were sequentially placed on the adhesive tape. Excess, unadhered samples were gently removed by blowing. The samples were then subjected to observation under the electron microscope.

Serum biochemical parameters

The following serum biochemical parameters were measured: total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (UN), serum glucose (GLU), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG). Serum biochemical analyses were conducted using the Hitachi 3100 Automatic Biochemical Analyzer (Hitachi Diagnostic Products Shanghai Co., Ltd.) at the Animal Nutrition Research Institute, Sichuan Agricultural University.

Enzyme activity

Lactase, maltase, sucrose, α-amylase, protease, and TP content were measured using reagent kits from Nanjing Jiancheng Bioengineering Institute.

Intestinal morphology analysis

As previously described (Wang et al., 2023), duodenal, jejunal, and ileal sections were stained using the hematoxylin and eosin staining method. Imaging was performed using the DS-U3 system (Nikon, Japan). The Case Viewer software was used to measure the depth of more than ten complete villi and their corresponding crypts and to calculate the villus-to-crypt ratio.

Microbiota composition of digesta

Total bacterial counts, including those of Lactobacillus, Bacillus, and Bifidobacterium, in the cecal and colonic contents were determined using real-time quantitative PCR. The specific DNA primers and probe sequences employed in the experiment are detailed in Table S1.

Volatile fatty acids analysis

The concentrations of acetate, propionate, and butyrate were quantified using a gas chromatograph (VARIAN CP-3800, California, USA). For this analysis, cecal and colonic digesta samples (0.5 g) were first homogenized in 1.3 mL of ultrapure water. The homogenate was allowed to stand for 30 min and subsequently centrifuged at 10,000 × g at 4 °C for 15 min. To the resultant supernatant, 0.2 mL of a 25% (w/v) metaphosphoric acid solution and 23.3 mL of a 10 mmol/L crotonic acid solution were added. This mixture was then incubated at 4 °C for 30 min before being centrifuged at 10,000 × g at 4 °C for an additional 10 min. Following this, 0.9 mL of chromatographic methanol was introduced to 0.3 mL of the supernatant, and the mixture was centrifuged at 10,000 × g at 4 °C for 5 min. The resulting supernatant was filtered through a 0.22-µm filter membrane before analysis. A 1 µL aliquot of the prepared sample was then injected into the GC column for measurement.

Calculations

The apparent digestibility, nitrogen availability, and apparent ileal digestibility (AID) of the diets were calculated using the equations provided by Lewis (Lewis and Southern, 2001), with chromium serving as the indigestible marker.

$${\displaystyle \begin{array}{lll}{\displaystyle \mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=}& (\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\text{-}\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t})& /(\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e})\times 100\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{T}\mathrm{r}\mathrm{u}\mathrm{e}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=\frac{\begin{array}{c}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-(\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}-\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n})\end{array}}{\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}\times 100}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{U}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=\frac{\begin{array}{c}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}-\mathrm{U}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\end{array}}{\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}\times 100}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{T}\mathrm{r}\mathrm{u}\mathrm{e}\mathrm{U}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=\frac{\begin{array}{c}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-(\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}-\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n})-(\mathrm{U}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}-\mathrm{E}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{U}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n})\end{array}}{\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}\times 100}\end{array}}$$

The nutrient digestibility of CSM and CPI was calculated using the following method:

$${\displaystyle \begin{array}{llll}{\displaystyle \mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}}& \mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{S}\mathrm{p}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}(\mathrm{\%})=& \frac{\begin{array}{c}\mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}-\mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{N}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}{\begin{array}{c}\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{f}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{M}\mathrm{i}\mathrm{x}\mathrm{e}\mathrm{d}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}& +A\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{u}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}$$

The digestible and metabolizable energy of the experimental diets were directly measured using the total fecal collection method. The digestible energy values of the ingredients were determined through a substitution method.

$${\displaystyle \begin{array}{l}{\displaystyle \begin{array}{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}(\mathrm{M}\mathrm{J}/\mathrm{k}\mathrm{g})=\frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}}{\mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}\end{array}}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}(\mathrm{M}\mathrm{J}/\mathrm{k}\mathrm{g})=\frac{\begin{array}{c}\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}-\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{F}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}-\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{U}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\end{array}}{\mathrm{F}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}}}\end{array}}$$

$${\displaystyle \begin{array}{llll}{\displaystyle \mathrm{I}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}}& \mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}(\mathbf{M}\mathbf{J}/\mathbf{k}\mathbf{g})=& \frac{\begin{array}{c}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}{\begin{array}{c}\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}{\mathrm{I}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}}^{\prime }\mathrm{s}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{M}\mathrm{i}\mathrm{x}\mathrm{e}\mathrm{d}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}& +(\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t})\end{array}}$$

$${\displaystyle \begin{array}{llll}{\displaystyle \mathrm{I}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}}& \mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}(\mathrm{M}\mathrm{J}/\mathrm{K}\mathrm{g})& =\frac{\begin{array}{c}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{y}\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}-\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}{\begin{array}{c}\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}{\mathrm{I}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{t}}^{\prime }\mathrm{s}\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{M}\mathrm{i}\mathrm{x}\mathrm{e}\mathrm{d}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}& +(\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{E}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t})\end{array}}$$

The following equations were utilized to calculate the ileal digestibility of amino acids and their endogenous losses:

$${\displaystyle \begin{array}{lll}{\displaystyle \mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{I}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{l}}& \mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{s}(\mathrm{\%})=& (1-\frac{\begin{array}{c}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}\times \mathrm{C}\mathrm{h}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t};\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\end{array}}{\begin{array}{c}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\times \mathrm{C}\mathrm{h}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}\end{array}})\times 100\end{array}}$$

$${\displaystyle \begin{array}{lll}{\displaystyle \mathrm{E}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{u}\mathrm{s}}& \mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{L}\mathrm{o}\mathrm{s}\mathrm{s}(\mathrm{g}/\mathrm{k}\mathrm{g}\mathrm{D}\mathrm{M}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e})& =\frac{\begin{array}{c}\mathrm{C}\mathrm{h}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}\times \mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}\end{array}}{\mathrm{C}\mathrm{h}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}}\end{array}}$$

$${\displaystyle \begin{array}{llll}{\displaystyle \mathrm{S}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}}& \mathrm{I}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{s}(\mathrm{\%})& =\mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{I}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{l}\mathrm{D}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{s}& +\frac{\mathrm{E}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{L}\mathrm{o}\mathrm{s}\mathrm{s}}{\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{D}\mathrm{i}\mathrm{e}\mathrm{t}}\times 100\end{array}}$$

Statistical analysis

Data were processed using Excel 2019, and statistical analyses were conducted with SPSS 27. The homogeneity of variance across treatments was confirmed using Levene’s test. Variance analysis was performed via ANOVA, followed by multiple comparisons using Duncan’s method. The statistical model used for data analysis was Y_ij = μ + t_i + e_ij, where Y_ij represents the observed value for the j-th pig in the i-th treatment group; μ is the overall mean; t_i is the fixed effect of the i-th treatment (i = 1 for CSD, i = 2 for CSMD, and i = 3 for CPID); and e_ij is the random error term specific to the j-th pig in the i-th treatment. All experimental data are presented as means ± standard error. Statistical significance was set at P < 0.05, while values of 0.05 ≤ P < 0.10 were considered to indicate a trend toward significance.

Results

The content of free gossypol and nutritional value in CPI

The concentrations of free gossypol and nutritional value in CPI are presented in Table 2. CPI contains 69.50% CP, 2.21% CF, 21.64% NDF, 2.34% ADF, and 5.37% Ash. Compared to CSM, CPI exhibits a significantly higher CP content (P < 0.01) while showing a significant reduction in CF, NDF, ADF, and ash content (P < 0.01). Additionally, the concentrations of various amino acids in CPI are noticeably higher (Table S2). Notably, Lysine, Isoleucine, Histidine, Glycine, and Alanine increased by 138.77%, 102.96%, 100.00%, 102.09%, and 101.44%, respectively. The total amino acid content rose from 32.54% in CSM to 61.63% in CPI.

Table 2.

Comparison of nutrient levels between CSM and CPI (DM basis)

Item	CSM	CPI	Growth rate, %	SEM	P-value
FG, mg/kg	666.97a	38.26b	−94.26	140.842	<0. 01
CP, %	45.34b	69.50a	53.29	5.414	<0.01
EE, %	0.41	0.69	68.29	0.079	0.080
CF, %	12.50a	2.21b	−82.32	2.303	<0.01
NDF, %	42.71a	21.64b	−49.33	5.101	<0.01
ADF, %	17.20a	2.34b	−86.40	3.462	<0.01
Ash, %	6.27a	5.37b	−14.35	0.203	<0.01

ADF, acid detergent fiber;. CF, crude fiber; CP, crude protein; CSM, cottonseed meal; CPI, cottonseed protein isolate; DM, dry matter;. EE, ether extract; FG, free gossypol; NDF, neutral detergent fibber.

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

Surface morphology

Figure 1 presents scanning electron microscopy images of CSM before and after processing, magnified 1,000 times. Prior to treatment, the surface of CSM appeared smooth and tightly structured. In contrast, after extraction, CPI displayed a rougher, more porous surface with significantly increased voids. This structural change suggests that CPI may be more conducive to digestion and utilization by animals.

Figure 1.

Scanning electron microscopy of cottonseed meal and cotton meal protein isolate. Note: A/B: CSM (1,000×); C/D: CPI (1,000×).

Growth performance

As shown in Table 3, compared to the CSMD group, pigs in the CPID group exhibited a significant increase in G:F ratio (P = 0.012) and a trend towards increased ADG (P = 0.067). No significant differences were observed in G:F ratio and ADG between the CPID and CSD groups.

Table 3.

Effect of CPI on growth performance

Item	CSD	CSMD	CPID	SEM	P-value
IBW, kg	19.70	19.93	20.19	0.357	0.866
FBW, kg	22.69	22.08	23.64	0.447	0.357
ADG, kg/d	0.30	0.24	0.35	0.020	0.067
ADFI, kg/d	0.79	0.79	0.86	0.017	0.350
G:F	0.39b	0.27a	0.38b	0.024	0.012

ADG, average daily gain; ADFI, average daily feed intake; CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet; FBW, final body weight; G:F, gain-to-feed ratio; IBW, initial body weight.

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

Apparent nutrient digestibility

According to Table 4, there are no significant differences in the apparent digestibility of DM, GE, CF, and ash among the 3 diets. The apparent digestibility of CP in the CSD and CPID groups was significantly higher than that in the CSMD group (P < 0.01). Using the method of substitution, the apparent nutrient digestibility of CPI and CSM ingredients was determined. While there were no significant differences in the apparent digestibility of DM, GE, CF, and ash for the entire intestinal tract, the apparent digestibility of CP in CPI was significantly higher than in CSM (P < 0.01).

Table 4.

Comparison of apparent digestibility between CSM and CPI diets (fed basis, %)

Item	CSD	CSMD	CPID	SEM	P-value
Diet DM	86.42	86.05	88.25	0.598	0.291
Diet CP	82.40a	80.40b	82.18a	0.303	<0.01
Diet GE	85.76	87.62	88.40	0.630	0.224
Diet CF	54.52	46.97	53.34	2.299	0.459
Diet ash	54.05	54.37	54.37	1.592	0.996
Ingredient DM	—	70.10	77.33	5.719	0.421
Ingredient CP	—	77.57b	83.11a	1.096	<0.01
Ingredient GE	—	85.76	84.66	5.111	0.877
Ingredient CF	—	43.69	48.60	7.923	0.481
Ingredient Ash	—	68.10	75.02	8.345	0.756

CF, crude fiber; CP, crude protein; CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet; DM, dry matter; GE, gross energy.

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

Availability of energy

No significant differences in digestible energy were observed among the 3 diets, as indicated in Table 5. However, the ME of the CPID group was significantly higher than that of the CSMD group (P = 0.020). By employing the substitution method, the digestible energy values for CPI and CSM were evaluated, showing no significant differences between the 2. In contrast, the ME of CPI was significantly greater than that of CSM (P < 0.01).

Table 5.

Comparison of digestible energy between CSM and CPI diets (as fed basis, MJ/kg)

Item	CSD	CSMD	CPID	SEM	P-value
Diet DE	14.33	14.35	14.55	0.069	0.406
Diet ME	12.49ab	12.05b	12.77a	0.116	0.020
Ingredient DE	—	13.26	14.30	0.839	0.578
Ingredient ME	—	10.68b	13.98a	0.642	<0.01

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet; DE, digestible energy; ME, metabolizable energy;.

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

Nitrogen utilization efficiency

According to Table 6, nitrogen retention in the CPID group is significantly higher than that in the CSMD group (P < 0.01). There are no significant differences in nitrogen apparent digestibility, true digestibility, utilization, and true utilization between the CPID and CSD groups; both are significantly higher than those in the CSMD group (P < 0.05). Using the method of substitution to evaluate nitrogen utilization in CPI and CSM, CPI shows significantly higher nitrogen apparent digestibility, true digestibility, utilization, and true utilization compared to CSM (P < 0.05).

Table 6.

Nitrogen utilization efficiency of CSM and CPI diets (as fed basis)

Item	CSD	CSMD	CPID	SEM	P-value
N retention, g/d	14.21ab	11.52b	17.68a	0.911	<0.01
N apparent digestibility in diets, %	82.39a	80.09b	83.27a	0.408	<0.01
N true digestibility in diets, %	86.40a	84.02b	86.65a	0.397	<0.01
N availability in diets, %	69.97a	61.93b	70.61a	1.562	0.024
N true availability in diets, %	74.21a	67.61b	73.78a	1.313	0.048
N apparent digestibility in ingredient, %	—	75.62b	84.92a	1.687	<0.01
N true digestibility in Ingredient, %	—	79.41b	88.07a	1.734	<0.01
N availability in Ingredient, %	—	51.58b	71.81a	5.103	0.040
N true availability in ingredient, %	—	56.18b	75.59a	4.272	0.018

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet.

AID and SID of amino acids

As shown in Table 7, compared to the CSMD group, the CPID group exhibits varying degrees of improvement in both AID and SID. Specifically, CPID group shows significantly higher AID for Asp, Glu, Ser, and total amino acids compared to CSMD group (P < 0.05), with a trend towards increased ADI for Arg (P = 0.083). Additionally, CPID group demonstrates significantly higher SID for Arg, Asp, Glu, Ser, and total amino acids compared to CSMD group (P < 0.05).

Table 7.

Effects of CPI on AID and SID of amino acids (%)

Item	AID, %				SID, %
	CSMD	CPID	SEM	P-value	CSMD	CPID	SEM	P-value
EAA
Arg	60.56	67.34	1.963	0.083	64.50	73.10	2.079	0.033
His	50.88	59.82	3.054	0.154	65.45	74.65	3.062	0.143
Ile	45.76	53.27	3.071	0.234	61.29	67.24	3.018	0.341
Leu	47.68	53.08	4.378	0.563	60.21	64.78	7.698	0.623
Lys	53.57	61.27	3.407	0.273	73.34	79.24	3.520	0.397
Met	41.55	52.06	3.901	0.192	69.77	71.31	3.591	0.842
Phe	51.92	56.25	3.890	0.596	61.98	65.88	3.877	0.632
Thr	50.53	62.80	4.126	0.142	85.54	90.50	1.943	0.211
Val	40.13	43.36	6.632	0.705	59.21	61.50	3.986	0.789
NEAA
Ala	44.47	47.72	3.171	0.644	62.22	70.04	2.740	0.169
Asp	45.17	65.14	1.848	0.002	54.18	76.08	3.977	0.002
Cys	65.58	66.20	2.334	0.900	79.39	84.33	2.419	0.324
Glu	44.83	60.56	3.692	0.027	50.37	66.10	3.691	0.027
Gly	48.70	55.62	3.379	0.323	80.03	83.55	3.286	0.611
Pro	33.83	39.82	4.251	0.502	81.46	81.55	4.223	0.991
Ser	50.43	37.18	2.378	0.002	65.55	91.00	3.679	<0.01
Tyr	49.75	55.32	3.243	0.411	68.17	69.44	3.161	0.850
TAA	44.45	55.55	2.707	0.035	58.37	69.27	2.692	0.037

AID, apparent ileal digestibility; CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet; NEAA, non-essential amino acids; SID, standardized ileal digestibility; TAA, total amino acids; TEAA, essential amino acids.

Serum biochemical parameters

As shown in Table 8, there are no significant differences among the groups in serum levels of TP, albumin, UN, aspartate aminotransferase, alanine aminotransferase, TC, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total serum glucose.

Table 8.

Serum biochemical parameters of CSM and CPI diets

Item	CSD	CSMD	CPID	SEM	P-value
ALT, U/L	88.88	93.87	79.23	5.416	0.554
AST, U/L	46.52	51.01	45.33	2.814	0.705
TP, g/L	52.59	54.13	52.62	0.661	0.576
ALB, g/L	20.19	20.13	18.24	0.607	0.345
BUN, mmol/L	2.63	2.82	2.25	0.153	0.293
TC, mmol/L	2.26	2.23	2.38	0.048	0.393
LDL-C, mmol/L	1.11	1.15	1.20	0.028	0.532
HDL-C, mmol/L	0.55	0.57	0.61	0.009	0.250
TG, mmol/L	0.43	0.39	0.43	0.012	0.492
GLU, mmol/L	4.96	4.49	4.55	0.097	0.135

ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet; GLU, glucose; GLU, glucose; HDL-C, high-density lipoprotein cholesterol; TP, total protein; TC, total cholesterol; TG, triglycerides.

Intestinal morphology

Table 9 illustrates that, compared to the CSMD group, the CSD and CPID groups showed significant increases in villus height and villus-to-crypt ratio in the duodenum and jejunum (P < 0.05). In particular, the CPID group had significantly higher villus height and villus-to-crypt ratio in both the duodenum and jejunum compared to the CSMD group (P < 0.05).

Table 9.

Comparison of intestinal morphology between CSM and CPI diets

Item	CSD	CSMD	CPID	SEM	P-value
Duodenum
Villus height, μm	470.00a	385.13b	455.81a	9.531	<0.01
Crypt depth, μm	379.48	395.36	379.07	6.847	0.562
Villus-to-crypt ratio	1.25a	0.98b	1.22a	0.030	<0.01
Jejunum
Villus height, μm	490.83a	434.23b	486.13a	9.672	0.028
Crypt depth, μm	248.96	251.18	239.57	6.107	0.730
Villus-to-crypt ratio	1.99a	1.75b	2.04a	0.048	0.042
Ileum
Villus height, μm	438.54	385.28	432.69	11.919	0.134
Crypt depth, μm	218.17	202.73	203.88	5.025	0.395
Villus-to-crypt ratio	2.05	1.93	2.13	0.053	0.319

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet.

Mucosa enzyme activity

As shown in Table 10, the CSD and CPID groups show significantly higher activities of maltase and α-amylase in the duodenal mucosa, as well as sucrose activity in the jejunum, compared to the CSMD group (P < 0.05). The CPID group also exhibits significantly higher sucrose activity in the jejunum than the CSD group (P = 0.024). Additionally, there is a trend toward increased maltase activity in the jejunum of both the CPID and CSD groups compared to the CSMD group (P = 0.075).

Table 10.

Effects of CPI on digestive enzyme activities in different intestinal segments (U/mg protein)

Item	CSD	CSMD	CPID	SEM	P-value
Duodenum
Lactase	32.02	27.00	35.11	2.159	0.316
Sucrose	25.66	22.39	21.19	1.162	0.278
Maltase	27.76a	22.50b	27.10a	0.879	0.037
Lipase	17.52	18.55	24.10	1.417	0.126
α-Amylase	1.72a	0.51b	1.84a	0.218	0.018
Protease	376.70	348.30	315.77	18.061	0.405
Jejunum
Lactase	75.13	80.28	73.04	2.848	0.586
Sucrose	111.11b	108.04b	136.12a	4.429	0.024
Maltase	75.00	57.48	70.64	3.303	0.075
Lipase	20.62	24.62	28.10	1.560	0.146
α-Amylase	1.91	1.51	2.09	0.169	0.374
Protease	341.55	332.78	266.47	16.470	0.124
Ileum
Lactase	3.86	3.35	3.44	0.318	0.800
Sucrose	125.62	111.57	117.70	5.945	0.639
Maltase	164.44	122.28	161.03	15.714	0.489
Lipase	43.73	38.86	43.56	2.308	0.640
α-Amylase	0.94	0.94	0.87	0.091	0.933
Protease	461.91	459.56	447.74	24.370	0.971

a, b mean values within a row with unlike superscript letters were significantly different, P < 0.05.

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet.

Short-chain fatty acids

According to Table 11, the isovaleric acid content in the cecum of the CPID group is comparable to that of the CSD group, with both showing a trend of being higher than the CSMD group (P = 0.089). Additionally, the CPID group shows a trend of higher concentrations of valeric acid in the colon compared to the CSMD group (P = 0.087).

Table 11.

SCFA profiles in the cecum and colon of CSM and CPI diets (μmol/g)

Item	CSD	CSMD	CPID	SEM	P-value
Cecum
Acetic acid	4.16	3.60	3.56	0.208	0.460
Propionic acid	2.24	1.85	2.32	0.241	0.169
Butyric acid	1.91	1.45	1.60	0.099	0.194
Valeric acid	0.48	0.37	0.38	0.037	0.287
Isovaleric acid	0.08	0.05	0.07	0.019	0.089
Colon
Acetic acid	4.06	3.82	4.12	0.148	0.717
Propionic acid	1.86	1.80	2.42	0.129	0.101
Butyric acid	1.26	1.07	1.52	0.081	0.677
Valeric acid	0.48	0.37	0.52	0.034	0.087
Isovaleric acid	0.28	0.22	0.29	0.020	0.321

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet.

Gut microbiota composition

As shown in Table 12, there are no significant differences among the 3 dietary groups in the quantities of total bacteria, Lactobacillus, Bifidobacteria, and Bacillus in the cecal and colonic contents. However, there is a trend for higher levels of Lactobacillus (P = 0.059) in the colon of the CPID group compared to the CSD group.

Table 12.

Gut microbiota composition in the cecum and colon of CSM and CPI diets

Item	CSD	CSMD	CPID	SEM	P-value
Cecum
Total bacteria	11.43	11.37	11.44	0.079	0.834
Lactobacillus	7.18	7.07	7.66	0.171	0.327
Bifidobacterium	6.50	6.43	5.65	0.193	0.119
Bacillus	8.24	7.97	8.05	0.052	0.102
Colon
Total bacteria	11.45	11.88	11.81	0.138	0.427
Lactobacillus	7.29	7.43	8.16	0.161	0.059
Bifidobacterium	7.06	7.12	6.31	0.187	0.110
Bacillus	8.40	8.25	8.24	0.069	0.449

CSD, corn–soybean meal diet; CSMD, cottonseed meal diet; CPID, cottonseed protein isolate diet.

Discussion

The global cottonseed production for the 2023 to 2024 season is projected to reach 41.50 million metric tons (USDA, 2024). CSM is the byproduct of cottonseed after extraction of oil, resulting in a slightly reddish or yellow granular substance(Tan et al., 2022). After oil extraction, most residual oil is removed from the cottonseed, leaving behind CSM. Due to its high CP content (Li et al., 2011), CSM is commonly used as a partial replacement for SBM to reduce feed costs (Sun et al., 2015; Qin et al., 2022). However, in the past, the inclusion of CSM in livestock and poultry diets was limited due to its lower nutritional availability compared to SBM, as well as concerns about its toxicity (Tian et al., 2018). To address these limitations, growing interest has emerged in advanced processing techniques aimed at reducing CSM’s toxicity and enhancing nutrient utilization (Xu et al., 2022; Zhang et al., 2022). The function of the alkaline extraction and acid precipitation process is to solubilize proteins under alkaline conditions and precipitate them under acidic conditions, thereby facilitating protein extraction and purification (Zhang et al., 2020). This method has been shown to effectively reduce the content of anti-nutritional factors such as gossypol and decrease CF, thus enhancing both the extraction yield and purity of the protein (Jan et al., 2022; Xiong et al., 2022). As a result, the extracted material exhibits higher nutritional value, making it more suitable for use in feed production. In this study, compared to conventional CSM, the CP content of CSM treated by an alkaline extraction and acid precipitation process was significantly increased, while the levels of free gossypol, CF, NDF, and ADF were significantly reduced. CPI and CSM were used to replace 35% of the nitrogen source in corn–soybean meal diets, respectively. The results showed that CPI significantly improved the growth performance of pigs, increasing the G:F ratio, which was otherwise limited by CSM. It has been reported that free gossypol can reduce protein digestibility by inhibiting pepsin and trypsin activities in the gut and by binding to dietary iron (Zhu et al., 2017). In addition, pigs lack fiber-digesting enzymes, and as the cell wall content in feed increases, indigestible components such as lignin and non-starch polysaccharides also increase, reducing growth performance (Gutierrez et al., 2016; Jaworski and Stein, 2017). In this study, the alkaline extraction and acid precipitation process effectively reduced the free gossypol and dietary fiber content in CPI, resulting in no significant difference in G:F ratio between the CPID and CSD groups.

Energy levels and nutrient digestibility in feed are key factors affecting the production performance of growing pigs and are important indicators of the nutritional value of feed (Shurson et al., 2021; Park et al., 2024). Studies on digestibility and energy metabolism further reinforce the nutritional advantages of CPI over CSM. It has been well-documented that anti-nutritional factors, such as free gossypol and CF, impede nutrient digestion and absorption in animals (Fombad and Bryant, 2004). Compared to CSM, CPI had significantly higher CP (69.50% vs. 45.34%), a more flexible protein structure, a reduction in CF by 82.32%, and lower free gossypol content. The apparent digestibility of CP in the CPI group was significantly higher than that in the CSM group. In addition, the amino acid digestibility results showed that the SID and AID of Arg, Asp, Glu, Ser, and total amino acids in CPI were higher than those in the CSM group, indicating improved digestibility and availability of specific amino acids that are crucial for protein synthesis and growth performance. Furthermore, the observed improvements in nitrogen retention and true digestibility suggest that CPI enhances nitrogen utilization efficiency, potentially leading to reduced nitrogen excretion and a more sustainable swine production system. While no significant differences in digestible energy were observed between diets, the ME derived from CPI was significantly higher than that from CSM. This finding suggests that pigs fed CPI are more effective in converting consumed energy into metabolizable energy, likely due to its higher protein digestibility and reduced fiber content compared to CSM, thereby improving overall energy utilization efficiency. These data suggest that CPI is nutritionally superior to CSM, as evidenced by the higher CP digestibility, nitrogen deposition, nitrogen utilization, and metabolizable energy in the CPID group.

Serum biochemical indicators are used to evaluate the metabolic status of the organs in animals. In this study, there were no significant differences in serum biochemical markers among the dietary treatments, suggesting that CPI did not adversely affect liver or kidney function, nor did it affect lipid metabolism or glucose homeostasis in pigs. This indicates that CPI is a safe protein source. Although no significant differences were observed in serum biochemical indicators, CPI significantly improved the morphology of the small intestine. The small intestine is a critical site for nutrient digestion and absorption, and its morphology is essential for animal health and production performance (Wu et al., 2020; Bröer, 2023). Increased villus height indicates an expansion in the absorptive area, which enhances the efficiency of nutrient transport; the villus-crypt ratio reflects the metabolic status of epithelial cells, and a reduction in this ratio often suggests increased mucosal damage, leading to compromised digestive and absorptive capacities (Wang et al., 2022; Ribeiro et al., 2023). In this study, the CPI group exhibited significantly increased villus height, suggesting an expansion in absorptive area and improved nutrient transport efficiency; the increase in the villus-crypt ratio indicated reduced mucosal damage, thus improving digestive and absorptive capacity. Compared to the CSMD group, there was no significant difference in villus height or the villus-crypt ratio between the CPID and CSD groups, suggesting that the alkali-solution and acid-isolation process effectively alleviated the detrimental effects of CSM on intestinal structure. Damage to the intestinal mucosa can weaken its digestive and absorptive capacity, altering the activity of digestive enzymes, such as reducing the activities of maltase and amylase, ultimately affecting overall nutrient absorption efficiency and the health of growing pigs (Shuai et al., 2023). In this study, maltase and amylase activities in the duodenal mucosa and sucrase activity in the jejunal mucosa were significantly higher in the CSD and CPID groups than in the CSMD group. This suggests that replacing 35% of the nitrogen in CSD with CSM not only affects the integrity of the intestinal mucosa but also further affects the activity of digestive enzymes and overall gut health, leading to an increased feed-to-weight ratio and reduced nutrient absorption efficiency. Conversely, no significant differences were observed between the CPID and CSD groups, suggesting that the CPI processing method may have contributed to maintaining intestinal health in growing pigs. This suggests that CPI may promote better gut health and nutrient absorption, potentially due to i) the lower content of anti-nutritional factors (gossypol and CF) in CPI compared to CSD, and ii) the enhanced protein quality and amino acid profile of CPI, supporting intestinal integrity and function.

The gut microbiota plays a key role in nutrient absorption and metabolism, immune system development, and maintenance of the intestinal mucosal barrier (Mathew et al., 1998). Previous studies have shown that feeding fermented CSM to piglets significantly promotes the proliferation of beneficial bacteria, such as Lactobacillus and Bacillus (Yu et al., 2023). During fermentation, the surface of canola meal becomes loosened and porous, disrupting the fiber structure, which may promote the growth of specific microorganisms (Mathew et al., 1998). The findings of this study are consistent with these results, as CPI exhibited a rough and porous surface, with an increased abundance of Lactobacillus in the colon of the CPID group. Moreover, the degradability of dietary fiber influences the production of SCFAs (Tan et al., 2023). In this study, the levels of propionate and valerate in the hindgut of the CPID group were higher than those in the CSMD group. It has been reported that SCFAs produced by microbial fermentation not only promote the proliferation of beneficial microbes but also improve intestinal mucosal integrity and function (Grela et al., 2018, 2019). For example, valerate has been found to enhance intestinal barrier function in animal studies, possibly by promoting the activation of the AMPK signaling pathway in intestinal epithelial cells (Gao et al., 2022). The significant improvement in intestinal integrity in the CPID group further confirms the positive effects of SCFAs on intestinal integrity.

Conclusions

In conclusion, this study demonstrates that CPI, produced by an alkali-solution and acid-isolation process from CSM, provides significant nutritional advantages over CSM in growing pigs. These advantages include significantly reduced free gossypol content, improved effective energy values, higher CP content and overall intestinal apparent digestibility, and enhanced nitrogen apparent and true digestibility. Additionally, CPI improved intestinal villus morphology and increased digestive enzyme activities. These findings provide not only scientific support for the application of CPI as a high-quality protein source in the livestock industry but also novel insights into the exploitation of CSM.

Glossary

Abbreviation

ADF

, acid detergent fiber

ADFI

average daily feed intake

ADG

, average daily gain

AID

, apparent ileal digestibility

ALB

albumin

ALT

, alanine aminotransferase

AST

, aspartate aminotransferase

CP

, crude protein

CPI

, cottonseed protein isolate

CSM

, cottonseed meal

CF

, crude fiber

DE

, digestible energy

DM

, dry matter

EE

ether extract

G:F

, gain-to-feed ratio

GE

, gross energy

HDL-C

, high-density lipoprotein cholesterol

LDL-C

, low-density lipoprotein cholesterol

ME

, metabolizable energy

NDF

, neutral detergent fiber

SBM

, soybean meal

SID

, standardized ileal digestibility

TAA

, total amino acids

TC

, total cholesterol

TG

, triglycerides

TP

, total protein

UN

, urea nitrogen

V/C

, villus-to-crypt ratio

Conflict of interest statement

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the content of this paper.

Author contributions

Kang Wang and Kaizheng Ren: Formal analysis, Data curation, Writing-original draft, Conceptualization. Yuheng Luo and Ping Zheng: Formal analysis, Data curation, Conceptualization, Supervision. Xiangbing Mao: Supervision. Hui Yan: Validation. Quyuan Wang: Data curation. Jun He: Funding acquisition, Project administration, Methodology, Writing- Review & Editing.