Effects of dietary crude protein on antioxidant activity, immunocompetence and the structural properties of the rumen in Tibetan sheep (Ovis aries), as determined by transcriptomic analysis

doi:10.21203/rs.3.rs-3966713/v1

Background: The rumen is a balanced ecosystem harboring a variety of microorganisms and plays an important role in the digestion and absorption of nutrients for ruminant. However, there are few studies on the effects of dietary crude protein on development and function in rumen of Tibetan sheep. The objective of this study was to evaluate the effects of dietary crude protein (CP) on the antioxidant activity, immunocompetence and the structural properties in the rumen of Tibetan sheep. Sixty two-month-old rams with an average weight of 15.40±0.81 Kg were randomly assigned to low-protein diet (10.20% of dry matter, L group) and high-protein diet (11.58% of dry matter, H group). The experiment was conducted over 97 d, including 7 d of adaption to the diets.

Results: Hematoxylin & eosin (H&E) results showed that high-protein diet increased papilla length, papilla width and muscular layer in rumen (P < 0.05). Compared with L group, supplementation with 11.58% crude protein increased the activities of T-AOC and SOD significantly (P < 0.05). A total of 612 significant differentially expressed genes (158 up-regulated and 456 down-regulated) were found in response to high-protein diet. Pathways and genes related to fatty acid biosynthesis, nutrition metabolism and muscle development were verified by real-time quantitative polymerase chain reaction.

Conclusions: In conclusion, 11.58% crude protein diet had superior papillary development and antioxidant activity of Tibetan sheep, likely through modulating the expression of functional genes.

Dietary crude protein

Tibetan sheep

Rumen

Histomorphological

Antioxidant capacity

immune levels

RNA-seq

The Tibetan sheep (Ovis aries) is predominantly distributed in the Qinghai-Tibet Plateau with an altitude of over 3000 m, which provision various aspects such as the meat, hides and milk [1]. Additionally, the Tibetan sheep plays a crucial role in maintaining the balance of the natural ecosystems in the Qinghai-Tibetan plateau [2]. In recent years, with the orderly advancement of grassland eco-pastoralism, the feeding mode of Tibetan sheep has gradually changed from traditional natural grazing to drylot feeding [3]. Because of influence of low temperature and hypoxia, the energy requirement of Tibetan sheep is more important than that of protein in normal metabolism. Therefore, the current research mainly focuses on the effect of dietary energy level on Tibetan sheep, and there are few studies on the effect of dietary protein level on Tibetan sheep.

The rumen is a balanced ecosystem harboring a variety of microorganisms (i.e., bacteria, fungi, and protozoa), bacteria being the most abundant [4]. Due to the microbial interactions, the crude fibers were converted into volatile fatty acids by fermentation process and then met 70% of the energy needs of the host [5]. Notably, both rumen microbe and rumen phenotype were directly affected by the dietary nutrient level [6]. Therefore, understanding the nutritional requirement, especially dietary protein levels forms a basis for improving productivity in Tibetan sheep.

It is hypothesized that diets with different protein levels could change functional gene expression, thereafter affecting the response of immunity and development of epithelial papillae in rumen. Therefore, this study aimed to explore the impact of dietary protein levels on the antioxidant activity, immunocompetence and the structural properties of the rumen in Tibetan sheep. In addition, the impact of different protein level diets on the mRNA expression of rumen was evaluated.

Histological analysis of the rumen

Results of H.E. staining of rumen tissue sections were presented in Figure 1A. The papillae length in the L group was significantly shorter than that in the H group (P < 0.05). Both papillae width and muscular layer of H group was significantly wider than in the L group (P < 0.05). No obvious difference was observed in the stratum corneum between the groups (Figure 1B, P > 0.05).

Antioxidant and immune response index assays

As shown in Figure 2A, the activities of T-AOC and SOD in rumen of H group were significantly higher than those of L group (P < 0.05). There was no significant difference in CAT, GSH-Px activity and MDA content in rumen fluid between the two groups (P > 0.05). Although the difference was not significant, there was an increased tendency of IgA, IgM, IgG, IL-6, and IL-1β in the H group than the L group (Figure 2B, P > 0.05).

RNA-Seq and Mapping

Table 1 showed the basic statistics for RNA-seq reads generated from the rumen of Tibetan sheep. A total of 390.63 million raw reads were obtained through sequencing in ten samples. After quality control, an average of 65.01 million clean reads was generated (64.04 million for H group and 65.97 million for L group). For all samples, the percent of clean reads was 99.85%. The Q30 values were between 94.01% and 96.75%. Average of 96.71% of the clean reads was mapped to the Ovis aries reference genome (H group with 97.52%, L group with 95.91%).

Analysis of Gene Expression

Venn diagram showed that there were 1706 and 893 differentially expressed genes (DEGs) belonging to this item in treatment and control DEG datasets, respectively, and they shared 14,458 DEGs as the intersection (Figure 3A). According to the gene expression values (FPKM), the principal component analysis (PCA) exhibited that H group and L group was separated into two different groups (Figure 3B). Hierarchical clustering analysis of the DEGs showed that the same group of DEGs was clustered together, indicating the accuracy and reliability of samples (Figure 3C). A total of 612 significant DEGs (based on |(fold change)| > 1.2, P-value < 0.05, and false discovery rate (FDR) < 0.05) were found in response to dietary CP level. Of these genes, 20 were up-regulated and 426 were down-regulated in the H group (Figure 3D).

Enrichment Analysis of DEGs

Gene ontology (GO) enrichment analyses were carried out using the database for annotation, visualization and integrated discovery software (https://david.ncifcrf.gov) (Figure 4A). A total of 573 significantly enriched GO terms were annotated within three major function groups: biological process (BP, 354), cellular component (CC, 95), and molecular function (MF, 124). For the down-regulated terms, the most significant GO categories observed were perikaryon, long-chain fatty-acyl-CoA catabolic process, and endoplasmic reticulum polarization. For the up-regulated terms, the most significant GO categories observed were potassium ion homeostasis, muscle contraction, and cell periphery.

Based on Kyoto Encyciopedia of Genes and Genome (KEGG) pathway enrichment analysis, 33 KEGG pathways were significantly (P < 0.05) enriched in the rumen tissue (Figure 4B). The top five pathways with the most representation of DEGs were retinol metabolism (13 DEGs), metabolic pathways (56 DEGs), ECM-receptor interaction (8 DEGs), focal adhesion (12 DEGs), and protein digestion and absorption (8 DEGs). Compared to the L group, 13 differentially nutrition metabolism-related signaling pathway (i. e., protein digestion and absorption, nicotinate and nicotinamide metabolism, glutathione metabolism, carbon metabolism, and fatty acid biosynthesis) were identified in H group.

Nine significant DEGs, identified from the RNA-seq data, were randomly selected for RT-qPCR validation, including two fatty acid biosynthesis-related genes (ACACB and ACSF3), seven nutrition metabolism-related genes (ATP1B1, IDH2, NT5E and NNT) and three muscle development-related genes (MYH11, KCNMA1 and MYL9). The RT-qPCR confirmed that the DEGs exhibited the same pattern of expression as observed with the RNA-seq (Figure 5), indicating our transcriptomic analysis was highly credible.

Correlation analysis

Correlation analysis (Figure 6) showed that T-AOC was positively correlated (P < 0.01) with ACACB (0.93), ACSF3 (0.94), ATP1B1 (0.94), and MYH11 (0.92), and IgG was positively correlated (P < 0.01) with ACSF3 (0.93). T-AOC was positively correlated (P < 0.01) with IDH2 (0.91), NT5E (0.86), KCNMA1 (0.91), MYL9 (0.87) were positively correlated (P < 0.05). IgG was positively correlated with ACACB (0.90), ATP1B1 (0.88), IDH2 (0.87), ATP1A2 (0.90), MYH11 (0.92), KCNMA1 (0.86) (P < 0.01). IL-6 was positively correlated (P < 0.05) with ACACB (0.83), ATP1B1 (0.87), IDH2 (0.84), ATP1A2 (0.83), MYH11 (0.95), KCNMA1 (0.85), MYL9 (0.84).

Effects of dietary protein level on rumen tissue morphology

The interior of rumen was lined with stratified epithelium consisting of papillae which provide surface area for short chain fatty acid (SCFA) absorption [7]. Both number and size of papillae were influenced by nutrition intake and the concentrations of SCFA to which they were exposed [8]. Simultaneously, the muscular layer participated in peristalsis of rumen, contributing to the nutrient utilization of ruminant [9]. Previously, Xia et al (2018) reported that with the increase of the dietary crude protein, the acetate, propionate, butyrate and total volatile fatty acid (VFA) concentrations increased in rumen of Holstein bulls [10]. Cristina et al (2022) found that sheep fed a high-protein (173g CP/kg dry matter) diet showed higher acetate, butyrate and VFA concentration compared with those fed a low-protein (134g CP/kg dry matter) diet [11]. A similar result was observed by Lv et al., who observed that the ruminal concentration of butyrate and isobutyrate increased with the high crude protein diet in weaned lambs [12]. According to previous studies, the fermentation parameters in the rumen, especially butyrate concentration promoted rumen papillae growth in ruminants [13]. In the present study, the papillae length, papillae width and muscular layer of the rumen were significantly affected by the dietary protein levels. With the increase of the dietary protein level, the development of rumen papillae increased. It speculated that the apparent digestibility of nutrient in high protein diet was higher than for low protein diet [14], and then altered rumen fermentation, which contributing to development of rumen papillae.

Effects of dietary protein level on antioxidant properties of rumen tissue

The equilibrium between oxidation and antioxidation is crucial for maintaining balanced redox homeostasis [15]. For ruminant, the ruminal epithelium predispose to oxidative stress due to oxidants produced endogenously by active oxidative metabolism [16]. The excess free radicals in ruminal epithelium are removed by antioxidant enzymes (e.g., GSH-Px, T-AOC, SOD, and CAT) [17]. The T-AOC reflects the compensatory ability of the antioxidant enzyme system to external stimulation and the metabolism state of free radicals [18]. In the present study, the T-AOC activity has increased significantly as the dietary crude protein level increased from 10.20% to 11.58%. Our result was consistent with Chen et al. (2023) findings, in which a higher T-AOC activity was observed when increased dietary crude protein level from 44% to 49% [19]. Via transforming the superoxide radicals into H₂O₂, the SOD is the first line of defense against excessive oxidative radicals [20]. Our results showed that the SOD activity was significantly increased with increase in the proportion of dietary crude protein. A similar result was observed by Liu et al., (2023) who found that diets supplemented with the 20% crude protein level increased the plasma CAT activity compared with the 12% crude protein levels [21]. It was a reasonable explanation that the inadequate protein nutrition can decrease the production of antioxidant enzymes, while lambs are sensitive to a diet with low crude protein levels.

Effects of dietary protein level on immune response of rumen tissue

Although no significant difference was observed in the immune response of rumen tissue, dietary crude protein increase tended to promoted the activities of IgA, IgG and IgM, while opposite trend was seen for the activity of TNF-α. In the plasma parameters of yak, the activities of IgA, IgG and IgM was lesser when fed the high-protein diet, whereas the activity of TNF-α was greater than that of the low-protein diet in yak [22]. The family of Igs including IgA, IgG, and IgM, were important non-specific immune factors, which participated in activation of the complement system and the enhancement of immunity [23]. Additionally, the TNF-α is one of the important potent inducer of pro-inflammatory cytokines that promoted cell proliferation and indirectly inhibits apoptosis through the induction of NF-кB [24]. In this study, the activity of TNF-α was reduced, and the activities of IgA, IgG and IgM were elevated, which indicated that the Tibetan sheep in the high-protein diet exhibited a stronger humoral and cellular immunity function.

Effects of dietary protein level on gene expression of rumen tissue

There were several metabolism-related pathways in the 33 significant KEGG pathways. Both protein digestion and absorption, and carbon metabolism pathways were essential for regulating the synthesis and transformation of protein and carbohydrate to maintain the balance of cell energy metabolism [25, 26]. The fatty acid biosynthesis metabolism pathway was the process by which the body converts acetyl-CoA and malonyl-CoA into fatty acids [27]. The nicotinate and nicotinamide were precursors of the coenzymes nicotinamide-adenine dinucleotide (NAD⁺) and were involved in tricarboxylic acid cycle [28]. Furthermore, the glutathione metabolism pathway was implicated closely in regulating the synthesis of proteins and nucleic acids, as well as maintenance of tissue antioxidant defenses [29]. Our results suggested that the different pathways mainly enriched in nutrition metabolism, which explained the reason of increasing dietary crude protein level improved the growth performance and nutrient utilization in ruminant [30].

Eleven of these DEGs, including two fatty acid biosynthesis-related genes (ACACB and ACSF3), seven nutrition metabolism-related genes (ATP1B1, SLC5A1, IDH2, ATP1A2, NT5E and NNT) and three muscle development-related genes (MYH11, KCNMA1 and MYL9). Correlation analysis revealed a positive correlation between ACACB and T-AOC, ACSF3 and T-AOC, IgG. For the fatty acid biosynthesis-related genes, the ACACB catalyzed the carboxylation of acetyl-CoA to malonyl-CoA, a major precursor of fatty acid synthesis [31]. The ACACB was involved in mitochondrial fatty acid (FA) oxidation and FA biosynthesis, which ubiquitously expressed in various developments of tissues, especially in heart, liver, and skeletal muscle [32]. ACACB-knockout mice exhibited insulin sensitivity, conferring protective effects against obesity and diabetes [33]. As an essential enzyme, the ACSF3 performed the first step of mitochondrial fatty acid synthesis II and activated fatty acids, served as the substrate for both de novo fatty acid synthesis and oxidation [34]. Deletion of SIRT3 causes altered expression of ACSF3, leading to govern the capacity of ACSF3 to mediate fatty acid metabolism disorder [35]. The up-regulated expression of their transcripts in respond to increasing dietary protein levels may reflect the increase for fatty acids synthesis in rumen.

Moreover, three genes identified as MYH11, KCNMA1 and MYL9 were associated with muscle development. MYH11, a member of the myosin family, encoded the smooth muscle myosin heavy chain (SMMHC) and modulated the maturely differentiated SMCs in visceral tissues [36]. Overexpression of Protein inhibitor of activated STAT restrained proliferation and migration in vascular smooth muscle via promoting the expression of MYH11 [37]. Down-regulation of the long noncoding RNAMBNL1-AS1 increased the expression of KCNMA1, thereby regulating the proliferation and apoptosis in skeletal muscle cells via activation of the cGMP-PKG signaling pathway [38]. MYL9 played important roles in cytoskeletal dynamics and experimental metastasis by regulating the actomyosin contractility and stress fiber assembly [39].

Annotated DEGs in the rumen tissues of Tibetan sheep with high and low crude protein levels were compared. A number of potential functional candidate genes were screened through transcriptomic sequencing analysis. However, further research is needed to define those mechanisms and the optimal crude protein required to promote rumen papillae development.

In conclusion, the present study demonstrated that the different crude protein levels diet induced changes in the antioxidant activity, immunocompetence and the structural properties of the rumen tissue. Our findings show that diet containing 11.58% crude protein contributed to development of rumen papillae, and improved the antioxidant properties when compared to dietary 10.20% crude protein. The functional analysis of DEGs suggested that genes related to fatty acid biosynthesis, nutrition metabolism and muscle development were associated with rumen development. The identified genes and signaling pathways play an essential role in affecting the rumen tissue, and further studies should be performed to elucidate the mechanisms.

Experimental Design

The experiment was conducted with 2-month-old weaned lamb (initial live weight of 15.40±0.81 Kg). A total of sixty lamb were divided into two treatments (n = 30). Diets were formulated to be isoenergetic and contained 2 levels of CP (10.20% and 11.58% of dry matter). The experiment adopted a single-factor randomized block design. This trail continued for 97 days, 7 of which were the adaptive period and 90 of which comprised the test feeding period. Throughout the experiment, lambs were fed a total mixed ration (TMR) of 70% concentration and 30% forage on a dry matter basis. Dietary dry matter (DM), CP, and ether extract (EE) were measured using the method described in a previous study. Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were examined as described by Van Soest et al. The value of net energy (DE) was calculated. The ingredients and nutrient compositions of diets were presented in Table 2. Diets and fresh drinking water were offered ad libitum.

Table 2 Ration composition and nutrient levels (dry matter basis) %

Items	Group¹
Items	Group L	Group H
Ingredient
Oat hay	15.00	15.00
Oat silage	15.00	15.00
Corn	37.10	32.20
Wheat	7.70	7.70
Soybean meal	0.70	1.40
Rapeseed meal	7.00	11.20
Cottonseed meal	0.70	0.70
Maize germ meal	0.70	0.70
Palm meal	11.20	11.20
NaCl	0.60	0.68
Limestone	0.70	0.70
Baking soda	0.07	0.07
Premix²	0.42	0.42
Lys	2.52	2.52
Met	0.48	0.39
Total	0.11	0.13
Nutrient levels³	100.00	100.00
DE/(MJ·kg^－¹)	9.66	9.56
Crude protein	10.20	11.58
Ether extract	3.40	3.36
Neutral detergent fiber	32.82	33.03
Acid detergent fiber	15.04	15.43
Ca	0.95	0.99
P	0.55	0.57

¹The protein level in the L group was 10.20%, while the protein level in the M group was 11.58%.²The premix provided the following per kg of diets: Cu 18 mg, Fe 66 mg, Zn 30 mg, Mn 48 mg, Se 0.36 mg, I 0.6 mg, Co 0.24 mg, VA 24 000 IU,VD 4 800 IU,VE 48 IU.³Digestible energy is calculated and the rest are measured.

Sample Collection

At the end of the experiment, five sheep from each group were selected randomly for slaughter at a commercial slaughterhouse. The rumen tissue of Tibetan sheep was collected immediately after slaughter. Approximately 1.5 cm² of rumen tissue were taken and placed immediately into liquid nitrogen for RNA extraction, while the remaining tissue were fixed in 4% paraformaldehyde for tissue sectioning.

HE staining

After fixation in 4% paraformaldehyde for 48 h, rumen tissue was dehydrated gradually by ethyl alcohol (Hailun, Changsha, China), and then successively embedded in paraffin wax (Sangon, Shanghai, China) and sliced into thick sections by the mounting onto poly-L-lysine-coated glass slides (Hailun, Changsha, China). Those slides were stained with with eosin for 3 min. The rumen papilla length, papilla width, muscular thickness, and stratum corneum thickness in images were measured using the Image-Pro Plus 5.1 software (Media Cybernetics Inc., Bethesda, MD, USA).

Enzyme linked immunosorbent assay (Elisa)

Both antioxidant and immunity of rumen were determined using the Elisa kit (Meibiao Biotechnology Co., Ltd, Jiangsu, China). In brief, 0.1 g of rumen tissue with 900 μL of phosphate buffered saline (Hailun, Changsha, China) were centrifuged at 3,000 × g at 4°C for 20 min. The supernatant fluid was collected and filtered through a 0.22-μm membrane.

Transcriptome analysis

Total RNA was extracted from rumen following the instructions of the RNA Extraction Kit (Invitrogen, Carlsbad, CA, USA). The integrity and purity of RNA were determined by Agilent Bioanalyzer 4150 (Agilent Technologies, CA, USA) and Nanodrop ND-2000 (Thermo Scientific, USA), respectively. After verifying and quantifying the cDNA, the libraries were sequenced on the Illumina HiSeq 4000 platform. The original sequencing data with low-quality reads were filtered using SOAPnuke (https://github.com/BGI-flexlab/SOAPnuke). The HISAT2 (V2.04) was applied to map the clean reads to the sheep reference genome of Ovis aries (Oar_v3.1).

Differential expression analyses were carried out to identify differentially expressed gene between different samples. EdgeR was used to normalize the data and extract DEGs with FDR < 0.05 and fold change >1.2. The analyses of GO and KEGG were used to classify the DEGs based on the specific biological functions using DESeq2 software package [40].

Real-Time Quantitative PCR (qRT-PCR) Analysis

Five genes were randomly selected for qRT-PCR to verify the accuracy of the transcriptome sequencing data. The total RNA isolated from rumen tissue was reverse transcribed into cDNA using a PrimeScript^TM RT reagent kit (TaKaRa, China). The qRT-PCR was performed with the SYBR^® Premix Ex TaqTM kit (TaKaRa, China) in triplicate. The relative gene expression of the mRNAs was calculated using the 2^−ΔΔCt method. Primers sequences were designed at Online primer design (Bioengineering Co. LTD, Shanghai, China) (Table 3).

Table 3 Primer sequences of the differentially expressed genes (DEGs) for real-time polymerase chain reaction (PCR) analysis.

Name	Primer sequence (5’-3’)	Tm （℃）	Product length
MYL9	F:TGTGATCCGCAACGCCTTCG R:TGTGATCCGCAACGCCTTCG	60.0	127bp
KCNMA1	F:CAGGCGGATGGCACTCTCAAG R:CCCAGTCTTTCACGGAGGTCATC	60.0	88bp
MYH11	F:AGCCAGAGACGAGAGGACCTTC R:AAGCCGTTGGAGAGGAATGTGTAG	60.0	120bp
NNT	F:TACGGATGCGGCAGCCAATC R:TAGGCAACCAAAGACCCACTGAAG	60.0	90bp
NT5E	F:CCATTCTTCTCAACAGCAGCATCC R:GAGCGGTGCCATCCAGATAGAC	60.0	132bp
IDH2	F:GGAGATGGACGGCGATGAGATG R:TCATTGGTCTGGTCACGGTTCG	60.0	129bp
ATP1B1	F:TACGGCTACAAAGAGGGCAAACC R:TGAACAGGCAGGACATACGGATTG	60.0	137bp
ACSF3	F:ACCACACGTACAAGGACCTCTATTC R:AAGGAGACATCGTTGGAGCACAG	60.0	130bp
ACACB	F:GAGACAAGATCGCCTCCACCATC R:CACTCCACCGTCAGACCACTTC	60.0	85bp

Ethics statement

The experimental animals used in this study were Tibetan sheep of the Qinghai Plateau type in China, located at the Tibetan sheep experimental base in Haiyan County, Haibei Prefecture, Qinghai Province. Animal care and experimental protocols were approved (QUA-2020-0710) by the Institutional Animal Care and Use Committee of the Qinghai University, China.

Statistical Analysis

The difference of the histomorphology, Elisa data and mRNA expression between two groups was evaluated using two-tailed Student’s t-test with SPSS software (V19.0). Results are presented as the mean values ± standard error of the measurement (SEM). P value < 0.05 was considered statistically significant.

Authors' contributions

ZLW analyzed and explained the transcriptome data of the rumen of Tibetan sheep. ZLW and FSZ performed H.E. staining and measurement on the rumen. Perform Elisa testing on antioxidant and immune levels in rumen tissue using ZLW, FSZ, and QRJ. ZLW, QYAMS, KNZ, and YZ confirmed gene expression through RT-qPCR. ZLW, ZYW, and LSG are the main contributors to manuscript writing. LSG and SZH provided financial support for this experiment. All authors have read and agreed to the published version of the manuscript.

Funding

The current work was funded by Construction of Standardized Production System for Improving quality and efficiency of Tibetan sheep industry (2022-NK-169).

Data availability statement

Sequence data that support the findings of this study have been deposited in the European Nucleotide Archive with the primary accession code PRJNA1077743.

Conflicts of interest

No conflict of interest existed in the submission of this manuscript, and manuscript was approved by all authors for publication.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Competing interest reported. No conflict of interest existed in the submission of this manuscript, and manuscript was approved by all authors for publication.

Effects of dietary crude protein on antioxidant activity, immunocompetence and the structural properties of the rumen in Tibetan sheep (Ovis aries), as determined by transcriptomic analysis

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