Abstract

   Neonatal calves’ diarrhea, which can be severe enough to cause death,
   has a significant impact on the global cattle industry. In this study,
   alfalfa polysaccharides and seaweed polysaccharides were found to
   significantly improve the diarrhea condition in neonatal calves. To
   explore the underlying mechanisms, further microbiomic and metabolomic
   analyses were conducted. This study investigated the impact of alfalfa
   polysaccharides and seaweed polysaccharides on growth performance,
   serum metabolites, gut microbiota, and metabolomics in neonatal
   Holstein calves. A total of 24 newborn calves were randomly assigned to
   three groups, with 8 calves per treatment group. The control (CON)
   group was fed a basal diet, the alfalfa polysaccharide (AP) group
   received a basal diet supplemented with alfalfa polysaccharides (4
   g/calf/day), and the seaweed polysaccharide group (SP) received a basal
   diet supplemented with seaweed polysaccharides (4 g/calf/day). These
   polysaccharides were plant extracts. Compared to the CON group, the
   results indicated that SP significantly enhanced the body weight,
   height, chest circumference, and average daily gain of Holstein calves
   (p < 0.05), while also reducing the diarrhea rate and improving manure
   scoring (p < 0.05). Compared to the CON, AP also reduced the diarrhea
   rate (p < 0.05). In terms of serum biochemistry, supplementation with
   AP and SP increased serum alkaline phosphatase (ALP) and insulin-like
   growth factor 1 (IGF-1) levels compared to the CON group (p < 0.05).
   Both AP and SP elevated serum catalase (CAT) and Total Antioxidant
   Capacity (T-AOC) levels, indicating enhanced antioxidant status (p <
   0.05). Regarding immune responses, supplementation with AP and SP
   significantly increased serum complement component 3 (C3) and
   immunoglobulin M (IgM) levels, while significantly reducing
   pro-inflammatory cytokines interleukin-18 (IL-18), tumor necrosis
   factor alpha (TNF-α), and interferon-gamma (IFN-γ) compared to the CON
   group (p < 0.05). Microbiota analysis revealed that AP modulated the
   abundance of Firmicutes, while SP influenced the abundance of
   Prevotella and Succiniclasticum. AP and SP differentially influenced
   intestinal metabolites compared to the CON group, leading to enrichment
   in pathways related to immunity, antibacterial, and anti-inflammatory
   functions. These pathways included the biosynthesis of alkaloids from
   ornithine, lysine, and nicotinic acid, glucocorticoid and
   mineralocorticoid receptor canothersis/antagonists, secondary
   metabolite biosynthesis, and alkaloid biosynthesis from histidine and
   purine, thus alleviating intestinal inflammation. Therefore, by
   supplementing with AP and SP, the diarrhea rate in calves was reduced,
   and the immune function of Holstein calves was enhanced, while
   simultaneously promoting a higher relative abundance of beneficial gut
   bacteria and suppressing the relative abundance of pathogenic bacteria.
   Additionally, gut pathways associated with immune response and
   inflammation were modulated by AP and SP. This study provided valuable
   insights and theoretical underpinnings for the use of AP and SP in
   preventing diarrhea in neonatal calves.

1. Introduction

   Neonatal calves’ diarrhea is prevalent on farms worldwide, adversely
   affecting animal health, welfare, and farm profitability [[46]1].
   Factors such as intestinal inflammation, tumor infiltration, and gut
   microbiota dysbiosis contribute to neonatal calf diarrhea [[47]2].
   These factors collectively disrupt gut function, leading to impaired
   nutrient absorption and the invasion of harmful microorganisms and
   toxins, such as Escherichia coli [[48]3], heat-stable toxins, and
   endotoxins [[49]4], resulting in diarrhea. Intestinal inflammation
   could damage the gut mucosa, blood vessels, and lymphatic system,
   increasing gut permeability, which causes protein and blood leakage,
   exacerbating malnutrition and reducing immunity [[50]5]. Dysbiosis of
   the gut microbiota leads to a reduction in beneficial bacteria and an
   increase in pathogenic bacteria, disrupting the balance of the gut
   microbiota and affecting the normal absorptive and secretory functions
   of the gut [[51]6]. For example, beneficial bacteria, such as
   Lactobacillus, Bifidobacterium, and Streptococcus thermophilus,
   decrease in levels [[52]7]. On the other hand, pathogenic bacteria,
   including Escherichia coli and Salmonella spp., increase in levels,
   causing severe gastrointestinal issues [[53]8].

   Maternal sources are the primary origin of beneficial gut
   microorganisms in newborn calves, as calves acquire these bacteria
   during birth and through contact with their mother’s skin, milk, and
   feces [[54]9]. Environmental exposure and dietary intake (feed and
   milk) also contribute beneficial bacteria [[55]9]. Prenatal
   colonization through translocation from maternal sources may start in
   the womb and continue via exposure to beneficial microbes during birth
   and ingesting colostrum and milk postpartum [[56]10]. The initial
   microbiota is both innate and acquired, rapidly diversifying as calves
   interact with their surroundings and diet. Seaweed polysaccharides are
   utilized by Bifidobacterium spp. as prebiotics [[57]11], while alfalfa
   polysaccharides are utilized by Lactobacillus spp. [[58]12].

   The use of antibiotics to prevent diarrhea in newborn calves has led to
   issues such as antibiotic resistance, as well as disruption in the
   establishment of early-life microbiota [[59]13]. Therefore, natural
   bioactive substances have been investigated by scientists as
   alternatives. Natural polysaccharides such as alfalfa polysaccharides
   (APs) and seaweed polysaccharides (SPs) have garnered widespread
   attention due to their rich bioactive components and potential health
   benefits [[60]14,[61]15]. AP, which is extracted from alfalfa, has been
   found to exhibit various bioactivities including anti-inflammatory,
   antioxidant, and immunomodulatory effects [[62]14]. Research has shown
   that AP activates splenic B cells through TLR4, primarily exerting its
   immune functions via the MAPK and NF-κB signaling pathways [[63]16].
   SP, which is extracted from various seaweeds, possesses multiple
   functions including antibacterial, antiviral, antioxidant, antitumor,
   and immunomodulatory effects [[64]17]. Studies have indicated that
   seaweeds and their derived metabolites hold potential in aquaculture
   feed, particularly in enhancing fish immune responses and gut health
   [[65]18].

   The mechanisms of complex diseases have been revealed through
   multiomics analysis, which also enhanced the understanding of the
   interactions among various substances in the body and their biological
   significance [[66]19]. Few studies have used multiomics techniques to
   investigate the effects of AP and SP on diarrhea in neonatal Holstein
   calves. This study analyzed the growth performance, serum metabolites,
   gut microbiota, and gut metabolites of neonatal Holstein calves fed
   with AP and SP, exploring the processes and mechanisms by which AP and
   SP regulate diarrhea in neonatal Holstein calves. The findings aim to
   provide data references for the application of these polysaccharides in