Lactobacillus salivarius and Berberine Alleviated Yak Calves’ Diarrhea via Accommodating Oxidation Resistance, Inflammatory Factors, and Intestinal Microbiota

Simple Summary Yaks are economically important food animals in the plateau regions of China, but bacterial diarrheal diseases frequently occur, with limited effective treatments. In this study, we investigated the effects of Lactobacillus salivarius and berberine on diarrheal yak calves. Our results showed that yaks treated with Lactobacillus salivarius and berberine had higher weight growth rates and lower diarrhea scores. Serum analysis indicated that these treatments increased the levels of T-AOC, SOD, GSH-Px, and IL-10 while reducing MDA, TNF-α, IL-1β, and IL-6. Microbiota sequencing identified significant changes in two phyla and twenty-seven genera, including beneficial genera, such as Faecalibaculum and Parvibacter, and harmful genera, like Marvinbryantia and Lachnospiraceae UCG-001. These findings provide novel insights for developing new therapies to combat ruminant diarrhea. Abstract Yaks are important food animals in China; however, bacterial diarrheal diseases frequently occur on the plateau, with limited effective therapies. The objective of this research was to evaluate the effectiveness of Lactobacillus salivarius (LS) and berberine in alleviating diarrhea in yak calves. For this purpose, eighteen healthy yak calves were divided into control (JC), infected (JM), and treatment (JT) groups. Yaks in the JT group were treated with 2 × 1010 CFU/calf L. salivarius and 20 mg/kg berberine, and yaks in the JM and JT groups were induced with multi-drug-resistant Escherichia coli. The results showed that the weight growth rate in the JM group was significantly lower than that in the JC and JT groups. The diarrhea score in the JM group was significantly higher than that in both the JC and JT groups. Additionally, the contents of T-AOC, SOD, GSH-Px, and IL-10 were significantly lower in the JM group than those in the JC and JT groups, while MDA, TNF-α, IL-1β, and IL-6 were significantly higher in the JM group. Microbiota sequencing identified two phyla and twenty-seven genera as significant among the yak groups. Notably, probiotic genera such as Faecalibaculum and Parvibacter were observed, alongside harmful genera, including Marvinbryantia and Lachnospiraceae UCG-001. Our findings indicate that treatment with L. salivarius and berberine significantly reduced diarrhea incidence, improved growth performance, and positively modulated intestinal microbiota, which could provide novel insights for developing new therapies for ruminant diarrhea.


Introduction
The third pole, the Qinghai-Xizang plateau, is in internal Asia [1] and is characterized by a frigid, anoxic climate with strong ultraviolet radiation [2,3].In this rugged environment, numerous "boat of the plateau" ruminants-yaks-have been living at altitudes of 3000-5000 m for thousands of years.There are 14 million yaks on the Chinese plateau, which represents 90% of the total yak population globally [4,5].In the Chinese plateau regions, yaks are economically and religiously important ruminants, providing nourishing meat and milk products, furs, medicinal horns, and dung fuel for native herdsmen [6].Hence, cattle disease, an especially infectious disease, can negatively affect the yak industry and the local economy on the plateau [4].
Cattle diarrhea caused by intestinal microbes is usually associated with higher morbidity and mortality, especially in calves [7,8].Infectious diarrhea caused by bacteria, viruses, and parasites is considered a major cause of economic loss in the cattle industry [9].Besides the mortality caused by serious diarrhea, growth retardation and treatment expenses also reduce the economic benefits of cattle farming, and antibiotic resistance is a growing health concern due to antibiotic overuse [10,11].Among these microbial pathogens, the opportunistic Gram-negative Escherichia coli is a common inhabitant of the gut in warm-blooded hosts and is often detected in many secondary infections.The predominant pathotypes of E. coli responsible for diarrhea in yak calves are enterotoxigenic E. coli (ETEC), with serotypes O91 and O145 among the most prevalent serotypes [12].E. coli infection in cattle not only causes severe diarrhea but also impedes meat production and trade [13].Nowadays, antimicrobial resistance is a serious issue for human and animal health [14,15], and many studies have reported antimicrobial resistance in E. coli strains on cattle farms [16,17].Therefore, developing a novel therapeutic method for E. coli infection is urgent and important.
Probiotics were first defined as live microbes that promote health when ingested in appropriate amounts by the World Health Organization [18].Nowadays, probiotics are popularly utilized in food supplements, cosmetics, and for gastrointestinal diseases [19,20].Lactobacillus salivarius is a beneficial bacterium that can mediate gut microbiota and the inflammatory response and maintain the intestinal barrier [21,22].A previous study confirmed that a strain of L. salivarius could alleviate intestinal damage in mice caused by E. coli [23].Berberine is a well-known isoquinoline alkaloid found in plants like Coptis chinensis, Phellodendron chinense, and Berberis aristate, etc. [24,25].In traditional Chinese medicine, herbs like Coptis chinensis are used to treat digestive system diseases like diarrhea and jaundice [26].Previous studies have verified that berberine has anti-bacterial and anti-diarrhea effects [27] and can alleviate colitis and inflammatory bowel disease [28,29].These studies suggest that L. salivarius and berberine could be employed to treat bacterial diarrhea in animals.
The gut microflora is composed of an enormous number of microbes, including bacteria, fungi, protozoa, and viruses [30], which play considerable roles in various physiological processes, such as digestive absorption, nutrition metabolism, and immunity [31].The balance of microbial immunity is positively related to intestinal homeostasis [32], while microbiome changes are associated with inflammatory bowel disease [33] and irritable bowel syndrome [34].Previously, microbial dysbiosis has also been reported in diarrheic cattle and yaks [35][36][37].Until now, little awareness has been mastered regarding the treatment effect of Lactobacillus salivarius (LS) and berberine on diarrheic yaks on the plateau.The objective of this research was to evaluate the effectiveness of Lactobacillus salivarius and berberine in alleviating E. coli (enterotoxigenic E. coli (ETEC))-induced diarrhea in yak calves.This was achieved by analyzing their impact on antioxidant capacity, inflammatory factors, and intestinal microbiota.

Experiment Design
Eighteen healthy four-month-old male Qinghai-Tibet Plateau-origin yak calves were selected from a yak industrial farm in Linzhou country, China.The calves were housed in individual pens with concrete flooring covered by straw bedding.The housing area was maintained at a temperature of 20 ± 3.5 • C with adequate ventilation, and all calves were given three days to acclimate.Each pen was equipped with a feeding trough and fed on hay and concentrates with pelleted starter feed (265.9 g/kg CP, DM basis) with water supplied ad libitum.The pens were refreshed every 3 days, and manure was removed daily to keep the bottoms of the pens dry and clean.For the experimental study, yak calves were randomly grouped into three groups: control (JC), infected (JM), and treatment (JT).From the 4th to the 10th day, yaks in the JT group were treated orally with previously isolated L. salivarius (PP859184) at 2 × 10 10 CFU/calf and 20 mg/kg berberine (Meryer, Shanghai, China.Yaks in the JC and JM groups received an equal volume of distilled water.On the 11th day, ruminants in groups JM and JT were induced with multi-drug-resistant E. coli (PP859186) isolated from diarrheal yaks.By contrast, the JC group was induced with an equal volume of Luria-Bertani medium (Solarbio, Beijing-China).On the 12th day, blood and rectal content samples were obtained from all yak calves (Figure 1).Fecal samples were collected directly from the rectum using sterile gloves and stored at −80 • C until analysis.Body weight was measured, and the diarrhea scores of the animals were evaluated.The diarrhea score was assessed on the basis of diarrhea severity as reported in a previous study: normal (0), slightly moist (1), temperately moist (2), loose (3), and watery stools (4) [38].
ivarius and berberine in alleviating E. coli (enterotoxigenic E. coli (ETEC))-induced diarrhea in yak calves.This was achieved by analyzing their impact on antioxidant capacity, inflammatory factors, and intestinal microbiota.

Experiment Design
Eighteen healthy four-month-old male Qinghai-Tibet Plateau-origin yak calves were selected from a yak industrial farm in Linzhou country, China.The calves were housed in individual pens with concrete flooring covered by straw bedding.The housing area was maintained at a temperature of 20 °C ± 3.5 °C with adequate ventilation, and all calves were given three days to acclimate.Each pen was equipped with a feeding trough and fed on hay and concentrates with pelleted starter feed (265.9 g/kg CP, DM basis) with water supplied ad libitum.The pens were refreshed every 3 days, and manure was removed daily to keep the bottoms of the pens dry and clean.For the experimental study, yak calves were randomly grouped into three groups: control (JC), infected (JM), and treatment (JT).From the 4th to the 10th day, yaks in the JT group were treated orally with previously isolated L. salivarius (PP859184) at 2 × 10 10 CFU/calf and 20 mg/kg berberine (Meryer, Shanghai, China.Yaks in the JC and JM groups received an equal volume of distilled water.On the 11th day, ruminants in groups JM and JT were induced with multidrug-resistant E. coli (PP859186) isolated from diarrheal yaks.By contrast, the JC group was induced with an equal volume of Luria-Bertani medium (Solarbio, Beijing-China).On the 12th day, blood and rectal content samples were obtained from all yak calves (Figure 1).Fecal samples were collected directly from the rectum using sterile gloves and stored at −80 °C until analysis.Body weight was measured, and the diarrhea scores of the animals were evaluated.The diarrhea score was assessed on the basis of diarrhea severity as reported in a previous study: normal (0), slightly moist (1), temperately moist (2), loose (3), and watery stools (4) [38].

Intestinal Microbiota Sequencing of Yak Calves
Eighteen rectal samples from yak calves in the JC, JM, and JT groups were selected for microbial DNA extraction via QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.The quality and concentration of these extracts were examined using 1% agarose gel electrophoresis and Qubit 3.0 fluorimeter (Thermo Scientific, Waltham, MA, USA), as described in previous studies [39,40].The V3-V4 zone of the 16S rRNA gene of all yak microbiomes was amplified using the primer pairs 341F/785R [41].
The PCR amplification products were examined to confirm their quality via electrophoresis.Finally, these products were used for library construction using TIANSeq DirectFast Library Kit (Illumina) (Tiangen, Beijing, China) and sent for amplicon sequencing via the Illumina MiSeq platform (Bioyi Biotechnology Co., Ltd., Shanghai, China).

Sequencing Data Analyzing of Yak Calves
The raw data from calf ruminants were processed with cutadapt and QIIME2 (QIIME 2 2020.6.)software to filter sequences and generate an amplicon sequence variant (ASVs) abundance table [42,43].All yak ASVs were annotated by blasting against the SILVA database (V138).Alpha diversity was analyzed to explore the richness and diversity of microflora within the yak samples by calculating the Shannon, Simpson, ACE, Coverage, Chao1, Observed species, and PD_whole_tree indexes [44].Beta diversity was assessed to compare the intestinal microbiota composition of different yak groups through principal component analysis [45], principal coordinates analysis [46], and non-metric multidimensional scaling [47].Significantly different species between calf groups (JC, JM, and JT) were identified using LEfSe and multiple t-tests [48,49].

Statistical Analysis
Valid difference analysis among yak groups was performed using SPSS version 36.0.The results are presented as means ± standard deviation (SD), and statistical significance was determined at p < 0.05.Data normality was assessed using the Shapiro-Wilk test.For normally distributed data, one-way ANOVA was conducted, followed by Tukey's post hoc test.For non-normally distributed data, log transformation was applied before analysis.Statistical significance was consistently set at p < 0.05.

Lactobacillus salivarius and Berberine Reduced Diarrhea in Yak Calves
There were no marked differences in the initial (Day 4) and final (Day 12) body weight between the JC, JT, and JM groups (Figure 2a).However, the weight growth rate in the JM group was notably lower (p < 0.01) than that in the JC group, while yaks supplemented with LsB showed a higher growth rate (p < 0.05) compared with those in the JM group (Figure 2b).The diarrhea score in the JM group was significantly higher than in the JC (p < 0.0001) and JT (p < 0.01) groups (Figure 2c).

Discussion
Yaks are pivotal livestock in the Chinese plateau region, crucial for producing highquality meat and dairy products that have gained popularity in recent decades.However, cattle, especially calves, are susceptible to diarrhea caused by E. coli, which seriously threatens their health and productivity [50,51].
As observed in previous studies, E. coli induces significant infection in yaks [52], resulting in the higher diarrhea scores and impaired weight gain observed particularly in the JM group.Interestingly, supplementation with LsB in yak calves led to higher growth rates and mitigated weight impairment.To elucidate the underlying mechanisms of LsB on diarrheal animals, we investigated serum antioxidant capacity and inflammation in calves.Markers such as T-AOC, SOD, GSH-Px, and MDA are well-established indicators of antioxidant enzyme activity, reflecting the oxidative stress status of the host [53].In our study, E. coli infection induced oxidative damage characterized by decreased T-AOC, SOD, and GSH-Px and increased MDA levels in the JM group, consistent with findings in bacteria-infected animals [54,55].Conversely, treatment with LsB reversed these trends in the JT group, indicating that LsB enhances the antioxidant capacity of ruminants.TNF-α, IL-1β, and IL-6 are critically inflammatory factors [56], while IL-10 plays a crucial role in limiting proinflammatory responses and inflammatory injury [57,58].In this study, E. coli infection resulted in elevated levels of TNF-α, IL-1β, and IL-6, accompanied by decreased IL-10 levels in the JM group, consistent with findings in animals induced with bacterial and Lipopolysaccharide challenges [59,60].Interestingly, LsB administration reduced the levels of these inflammatory factors and increased IL-10 levels, suggesting that LsB alleviates intestinal damage by modulating inflammatory responses in yaks.
Subsequently, we conducted micro-community sequencing and obtained 1,879,179 raw and 1,809,878 filtered sequences, resulting in alignment to 9648 ASVs, with 434 ASVs shared across yak calves.There was no remarkable difference observed in the alpha diversity index, which partially aligns with findings in E. coli-challenged animals [61,62] but contrasts with a study by Wang et al., 2024 [63].Analysis at different taxonomic levels revealed that E. coli infection altered the microbiome of yaks, while LsB partially restored the microbial structure.For instance, at the phylum level, Firmicutes (JC = 53.01%,JM = 54.13%,JT = 44.54%),Bacteroidota (JC = 29.00%,JM = 22.77%, JT = 25.18%), and Actinobacteriota (JC = 13.29%,JM = 17.86%,JT = 26.81%)were dominant across all yak groups, albeit with varying relative abundances.The Firmicutes/Bacteroidota ratio was notably higher in the JM group (2.48) than in the JC (1.83) and JT (1.77) groups; this is a commonly used indicator of dysbiosis [64].Our findings suggest that LsB mitigates intestinal damage by modulating the gut microbiota.
We further detected marked differences in bacterial composition among yak groups, identifying two phyla and twenty-seven genera that showed significant variations.Among these, Marvinbryantia, known for its higher abundance in depressive mice [65] and animals with intestinal damage induced by high-fat feeds [66], as well as Lachnospiraceae UCG-001, which was observed at higher levels in ulcerative colitis mice [67], were found to be less abundant in JT yaks, suggesting that LsB could potentially reduce these pathogenic bacteria.Similarly, UBA1819 and Solobacterium, genera associated with various diseases [68,69], and Sharpea, which was found at higher levels of abundance in Cryptosporidium-infected pigs [70], were also less abundant in JT yaks, indicating a potential role of LsB limiting the colonization of these pathogenic bacteria.Previous research has linked a lower abundance of Lachnospiraceae UCG-010 with chronic kidney disease in pigs [71], while Christensenella is recognized as an important genus in healthy hosts [72].Faecalibaculum, a probiotic genus negatively associated with inflammation [68], and Parvibacter, which produces short-chain fatty acids [34], were found at higher abundances in JT calves, suggesting that LsB may alleviate intestinal injury by promoting the proliferation of these genera in plateau ruminants.Our results indicate that LsB restores the balance of the intestinal microbiota in yaks.

Conclusions
In conclusion, this study investigated the role of Lactobacillus salivarius and berberine in the prevention and treatment of yak calves' diarrhea.The results were consistent with our expectation that Lactobacillus salivarius and berberine can mitigate bacteria diarrhea in yaks by regulating oxidation resistance, inflammatory factors, and microbiota.Moreover, these interventions increase the abundance of probiotic genera (Faecalibaculum and Parvibacter) and decrease harmful genera (Marvinbryantia and Lachnospiraceae UCG-001).Our results provide novel insights for developing new therapies for ruminant diarrhea.

Figure 2 .
Figure 2. Effects of Lactobacillus salivarius and berberine on weight (kg), growth rate (%), and diarrhea score in yak calves.(a): Weight gain (kg) in yak calves over the experimental period.(b): Growth rates among the different treatment groups.(c): Diarrhea severity scores assessed using a standardized scoring system.Significance levels are indicated as follows: **** p < 0.0001, ** p < 0.01, and * p < 0.05.Data are presented as the mean ± SEM (n = 6 per group).These results demonstrate the beneficial effects of Lactobacillus salivarius and berberine on reducing diarrhea and promoting weight gain in yak calves.

Figure 2 .
Figure 2. Effects of Lactobacillus salivarius and berberine on weight (kg), growth rate (%), and diarrhea score in yak calves.(a): Weight gain (kg) in yak calves over the experimental period.(b): Growth rates among the different treatment groups.(c): Diarrhea severity scores assessed using a standardized scoring system.Significance levels are indicated as follows: **** p < 0.0001, ** p < 0.01, and * p < 0.05.Data are presented as the mean ± SEM (n = 6 per group).These results demonstrate the beneficial effects of Lactobacillus salivarius and berberine on reducing diarrhea and promoting weight gain in yak calves.
rhea score in yak calves.(a): Weight gain (kg) in yak calves over the experimental period.(b): Growth rates among the different treatment groups.(c): Diarrhea severity scores assessed using a standardized scoring system.Significance levels are indicated as follows: **** p < 0.0001, ** p < 0.01, and * p < 0.05.Data are presented as the mean ± SEM (n = 6 per group).These results demonstrate the beneficial effects of Lactobacillus salivarius and berberine on reducing diarrhea and promoting weight gain in yak calves.

Figure 4 .
Figure 4. Effects of Lactobacillus salivarius and berberine on the microbial community structure and diversity in calves.The panels illustrate different aspects of microbiota analysis.(a): Amplicon Sequence Variants (ASVs) Venn diagram showing the overlap and unique ASVs across treatment groups.(b): Rarefaction curve depicting the relationship between the number of observed ASVs and sequencing depth, indicating the completeness of sampling.(c): Rank abundance plot illustrating the distribution of ASV abundances across different treatment groups.(d): Alpha diversity index analysis representing the microbial diversity within each treatment group.Data are shown as the mean ± standard error of the mean (SEM), with n = 6 for each group.

Figure 4 .
Figure 4. Effects of Lactobacillus salivarius and berberine on the microbial community structure and diversity in calves.The panels illustrate different aspects of microbiota analysis.(a): Amplicon Sequence Variants (ASVs) Venn diagram showing the overlap and unique ASVs across treatment groups.(b): Rarefaction curve depicting the relationship between the number of observed ASVs and sequencing depth, indicating the completeness of sampling.(c): Rank abundance plot illustrating the distribution of ASV abundances across different treatment groups.(d): Alpha diversity index analysis representing the microbial diversity within each treatment group.Data are shown as the mean ± standard error of the mean (SEM), with n = 6 for each group.

Figure 5 .
Figure 5. Impact of Lactobacillus salivarius and berberine on the microbial community structure in calves at different taxonomic levels.(a): Phylum: Distribution of microbial communities at the phylum level across different treatment groups.(b): Class: Variation in microbial communities at the class level between treatment groups.(c): Order: Representation of microbial communities at the order level in different treatment groups.(d): Family: Microbial community structure at the family level across treatment groups.(e): Genera: Distribution of microbial communities at the genus level for each treatment group.

Figure 5 .
Figure 5. Impact of Lactobacillus salivarius and berberine on the microbial community structure in calves at different taxonomic levels.(a): Phylum: Distribution of microbial communities at the phylum level across different treatment groups.(b): Class: Variation in microbial communities at the class level between treatment groups.(c): Order: Representation of microbial communities at the order level in different treatment groups.(d): Family: Microbial community structure at the family level across treatment groups.(e): Genera: Distribution of microbial communities at the genus level for each treatment group.

Figure 6 .
Figure 6.Heat map analysis of the effect of Lactobacillus salivarius and berberine on calf microbiota in all groups.(a): Phylum.(b): Genera.Color intensity represents the relative abundance of each taxon, with warmer colors indicating higher abundance.

Figure 6 .
Figure 6.Heat map analysis of the effect of Lactobacillus salivarius and berberine on calf microbiota in all groups.(a): Phylum.(b): Genera.Color intensity represents the relative abundance of each taxon, with warmer colors indicating higher abundance.

Figure 9 .
Figure 9. Identification of marker species among different yak groups using multiple t-tests.Identification of significant marker species among different yak groups at various taxonomic levels using

Figure 9 .
Figure 9. Identification of marker species among different yak groups using multiple t-tests.Identification of significant marker species among different yak groups at various taxonomic levels using multiple t-tests: (a) Phylum: Marker species identified at the phylum level across different yak groups.(b) Genera: Marker species identified at the genus level among the yak groups.The statistical analysis highlights species with significant differences among the JC, JM, and JT groups, providing insight into the distinct microbial profiles of each group.Statistical significance is indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001.