MicroRNAs (miRNAs), defined as short non-coding RNA (ncRNA) molecules of about 22 nucleotides in length, regulate a variety of biological processes through the post-transcriptional regulation of gene expression. miRNAs or microRNAs are essential for controlling gene expression in a variety of organisms, including livestock. By attaching to messenger RNA (mRNA) and either destroying it or preventing its translation, they play a role in post-transcriptional control. The function of miRNAs in a variety of biological processes, such as immune response, growth, development and reproduction, has been investigated in livestock.
Recent data has shown that various miRNAs go through different processing routes. Tumorigenesis and miRNA processing dysregulation are tightly connected (Jiang and Yan, 2016). More recently, the function of miRNA in a variety of inflammatory and immune illnesses, including allergic inflammation, has also been identified (Rebane and Akdis, 2014). miRNA regulates gene expression post-transcriptionally, either by targeting the 3’untranslated region (UTR) or coding region of mRNA. One cell type can have multiple genes targeted by a single miRNA and vice versa; the impact of a single miRNA on a target gene may be modest (Lu and Rothenberg, 2013). Consequently, the relationship between miRNAs and gene expression becomes extremely complex. A number of miRNAs exhibit tumor-suppressive or oncogenic properties and are crucial for the initiation, growth and spread of cancer (Romano et al., 2017). MicroRNA-140-5 p suppresses hypoxia induced nephrotoxicity and renal fibrogenesis in in vitro conditions (Wu et al., 2022). High stability in the biofluids (serum, plasma, milk) of livestock, active secretion from cells, cell-to-cell communication and ease of extraction confer the potential for circulating miRNA to act as a novel biomarker for the identification and tracking of disease progression in animals (Correia et al., 2017).
MicroRNAs (miRNAs) play a crucial role in regulating gene expression and their involvement in stress responses in livestock has been a subject of research. Stress unleashes negative impacts on cattle productivity and culminates in significant financial losses for livestock firms (Gupta et al., 2018). Producing safe and high-quality food requires keeping farm animals in perfect physical condition, free from illness or misery. Unpredictable and uncontrollable circumstances include metabolic stress, immunological stress, stress brought on by regrouping/splitting of livestock’s during their productive life, stress related to weaning, stress resulting from dietary changes, transport stress, environmental stress and rough handling (Muroya et al., 2016). MiRNAs are part of the molecular mechanisms that help animals adapt to stress.
Expression profiling in domestic livestock has shown that miRNA exhibits unique temporal (at cellular and tissue level) and geographic expression patterns (Kuosmanen et al., 2017). The dysregulated expression of miRNA would result in improper regulation of several cellular processes and genetic networks. Research suggested that circulating miRNAs are significantly involved in metabolic disorders (Gupta et al., 2018), foot and mouth diseases (Basagoudanavar et al., 2018) and in paratuberculosis in livestock (Deiuliis et al., 2016). This article provides a comprehensive overview of the regulatory importance of miRNA in livestock stress.
miRNAs associated with immune system and diseases of livestock
Animal illnesses are a major source of financial losses for the livestock sector and serious problems for animal welfare (Bishop and Woolliams, 2014). Furthermore, a variety of diseases that affect livestock can spread to human, potentially leading to serious health problems or even death. For the livestock industry, effectively managing diseases is a global concern that calls for several layers of control and intervention. For the treatment of diseases in livestock, miRNA can be employed as biomarkers with diagnostic, prognostic, or therapeutic capabilities. Acute and chronic events exhibit distinct effects on the metabolic pathways of bovines, which can alter animal’s quality of life and production. MicroRNAs influence immune function, mammary gland health, milk composition and quality, as well as other metabolic factors.
Small ruminant species have been found to contain hundreds of microRNAs; many of these microRNAs’ functions in the various regulatory systems are still unknown. Economic factors like milk, meat and wool production have frequently been the focus of studies on sheep and goats (Zhai et al., 2019), but only a small number of studies have examined how their health status relates to their overall well-being.
Immunity
In large ruminants
Diseases, illnesses and certain environmental alterations related stresses can decrease the immune response through stimulating the Hypothalamic-Pituitary-Adrenal (HPA) axis and the SAM (Sympathetic-Adrenal-Medullary) system (Minton 1994). Recently studies have suggested that miRNAs regulate many immune cell activities and are essential for bovine immunity (Dong et al., 2017).
According to a recent experimentation, miRNAs are significant controllers of the host’s gene expression and immunological responses and also performs as potential biomarkers to identify mycobacterial infection susceptibility (Wright et al., 2020). During mycobacterial infections in cattle, numerous miRNAs have been shown to be modified (Gupta et al., 2018; Iannaccone et al., 2018). When infected with M. avium subspecies paratuberculosis and M. bovis, the c-miRNAs miR-19b, miR-196b and miR-146 undergo changes associated to the immune system and inflammation (Iannaccone et al., 2018).
Studies sought to identify possible genes and miRNAs beneficial for developing mastitis prevention, diagnosis and treatment methods as well as to confirm the importance of miRNAs during innate immune responses (Chen et al., 2019; Srikok et al., 2020) and concluded that bovine serum of mammary tissue infected with Streptococcus uberis faces downregulation of miR-181a, 16 and miR-31 as compared to healthy tissue, whereas miR-223 was highly upregulated (Naeem et al., 2012). miRNAome and transcriptome of milk along with blood CD14+ monocytes were described using S. uberis to infect Holstein Friesians cows through next-generation sequencing done by Lawless et al., (2014). Interestingly, the majority of miRNAs found to be downregulated in milk monocytes recovered from mastitis infected animals and were anticipated to exclusively target genes associated in innate immunity, such as those implicated in the TLR, NOD and RIG-I-like receptor signaling pathways. MiR-26a was anticipated to target a bridging molecule (FGA) important in host defensive mechanisms against S. aureus (Ju et al., 2018). Due to the fact that miR-144 and miR-451 were markedly upregulated in mammary glands infected with E. coli and significantly downregulated in those infected with S. aureus, researchers determined that these two miRNAs could be useful for differentiating between the two pathogens (Luoreng et al., 2018). When compared to healthy animals, the serum of calves with mastitis caused by S. aureus has been shown to have a number of miRNAs that are differentially expressed (DE). Among these, miR-144 and miR-125 were discovered to be upregulated. DE-miRNAs play a significant role in innate immunity and inflammatory responses, as confirmed through KEGG pathway analysis as noted by Luoreng et al., (2018). Further research must be conducted on these DE-miRNAs as possible biomarkers for mastitis brought on by E. coli. Numerous studies have suggested that miRNAs may be used as MAP prognostic and diagnostic tools. In the blood of MAP-positive animals compared to unexposed animals, Malvisi et al., (2016) found two downregulated (miR-6517 and miR-7857) and seven upregulated (miR-19b, miR-19b-2, miR-1271, miR-100, miR-301a, miR-32 and one novel miRNA) miRNAs. Four miRNAs (miR-1976, miR-873-3p, miR-520f-3p and miR-126-3p) were combined by Gupta et al., (2018) to create a prediction model that could distinguish between animals with moderate to severe JD infection and healthy animals. Wang et al., (2019) reported the participation of a different group of miRNAs in the regulation of MAP response in the ileum and ileal lymph as well as the possible engagement of miR-100, miR-330 and miR-2447 in the Th17 cell differentiation pathway during MAP infection in the ileal lymph node. Additionally, as certain other prominent infectious diseases of cattle progress, such as tuberculosis, foot and mouth disease (FMD) and BVD, miRNA expression is altered (Iannaccone et al., 2018; Taxis et al., 2017).
In small ruminants
Globally, sheep and goats are among the small ruminants that provide a significant amount of meat, milk and wool. Mammary gland development (Galio et al., 2013), muscle (Lie et al., 2014), embryo/ovary (Chang et al., 2018), milk-related phenotypes (yield and composition) (Lin et al., 2013) and hair/skin related phenotypes (Wu et al., 2014) have all been the focus of miRNA studies in small ruminants. Stress-activated immune responses and the HPA axis have been connected and C-miRNAs have been suggested as potential biomarkers in this connection (Naylor et al., 2020). MiR-145, miR-233 and miR-1246 expression levels rise in correlation with the inflammatory response, as determined by the assessment of immunological and stress markers (Naylor et al., 2020). Additionally, it has been demonstrated that miRNAs influence the immunological response of small ruminants to infectious illnesses. Peste des petits ruminants virus (PPRV) is a Morbilli virus that is extremely infectious and lethal to domestic and wild small ruminants (Kumar et al., 2014). However, miR-21-5p, which inhibits interferons (IFNs) antiviral activity and miR-30b, which limits initial macrophages ability to process and present antigen, were shown to be downregulated in PPRV infection (Khanduri et al., 2018). PPRV affected the levels of miRNA expression in the lungs and spleen of sick sheep and goats (Pandey et al., 2017). Comparing the two species, the spleen and lungs exhibited 20 and 11 DE-miRNAs, respectively. Du et al., (2017) identified 25 known and 240 novel DE miRNA candidates in primary sheep testicular cells infected with bluetongue virus as well as the significant enrichment of target genes in MAPK, PI3K-Akt, endocytosis, Hippo, NF-κB viral carcinogenesis, FoxO and JAK-STAT signaling pathways in an effort to investigate the mechanisms of bluetongue virus infection.
miRNAs as biomarker found in milk
In large ruminants
At birth, calves are devoid of circulating antibodies. Newborns’ potential to resist infections in the early days of life is entirely dependent on the passive immunity conferred by ingested colostrums (Chase et al., 2008). Colostrum is rich in nutrients, bioactive compounds and microRNAs than milk. Recently, the potential consequences of colostrum ingestion on calves were evaluated (Van et al., 2020). Using a systematic sequencing-based investigation of milk and colostrum at different stages of lactation, miR-181a, miR-155 and miR-223, were considerably more abundant in colostrum than in milk (Chen et al., 2010). miRNA related to immune system were also discovered in microvesicles derived from milk. Several microRNAs (miRNAs) were discovered to be DE among the two breeds after sequencing and secondary confirmation by RT-qPCR (Özdemir et al., 2020). These included miR-21-5p, miR-142-3p, miR-222, miR-340, miR-1248, miR-29c, miR-101 and let-7a-5p. Functional enrichment assessment found that the overall amount of microRNAs in the colostrum of DAK cows was significantly closely associated with immune pathways than in milk (Özdemir et al., 2020). The concentration and distribution of microRNAs in milk and colostrum may also be affected by management-related parameters, such as nutrition (Jacometo et al., 2018) and subclinical diseases (Cai et al., 2018). Milk exosomal microRNAs found to balance nutrition between buffalo breeds with low and high milk production (Pang et al., 2022).
In small ruminants
The development of the mammary gland throughout various stages of pregnancy, lactogenesis and colostrogenesis in small ruminant species has been linked to the presence of numerous miRNAs (Galio et al., 2013). The enhanced expression of both microRNAs towards the end of lactation and pregnancy had a significant influence in mammary gland development (Galio et al., 2013). For dairy goats, profiles of miRNA expression throughout milk lactation and during the dry periods have been observed. During breastfeeding, miR-7, miR-423- 5p and miR-378 have been recognized as serving crucial regulatory roles in milk component transport and ingredient synthesis (Dong et al., 2013). MiR-574 may be connected to the development of the mammary gland and milk production in goats through oxytocin, estrogen, adipocytokine, endocrine and MAPK signaling pathways (Hou et al., 2017). The mammary gland of a dairy goat and the raw milk it produced were both discovered to contain high concentrations of 13 microRNAs, proving that the miRNAs in the milk came from the mammary gland’s cells (Li et al., 2012).
miRNAs and genetic background
Cattle’s genetic make-up has an effect on microRNAs that are related to stressor stimuli. Ioannidis et al., (2018) studied the potential use of c-miRNAs as genetic performance indicators in age, fertility and welfare traits in dairy cattle. Certain miRNAs, such as miR-127 or 140, were related to immunological responses, inflammation and health attributes (mastitis, fertility, lameness). Perinatal mortality and dystocia are quantifiable features that profoundly influence animal welfare and productivity. Using genome-wide association studies and quantitative trait loci mapping, researchers discovered many significant genomic sites in Holstein dairy cows that are associated with perinatal mortality and dystocia. MiRNA expression in livestock genetic selection is in its infancy; further research is needed to assess their functional significance and potential use in selective breeding programs.
MicroRNAs as biomarkers in livestock stress
A biomarker is a measurable indicator of a certain biological state, such as health and disease. Growing body of research is supporting the important role of miRNAs in stress reactions as well as in several diseases where they can be used as potential biomarkers for both illnesses and stressors, indicating their significance in diagnostic and monitoring applications. The identification of particular miRNAs in cow blood is an extremely useful approach for determining how the animals react to thermal alterations. Animal health and productivity might suffer from high ambient temperatures. Cow blood samples were obtained in experimentation by Lee et al., (2020), to examine the mRNA expression under various environmental situations. Different types of miRNAs as signature biomarkers with functions addressing various livestock diseases are listed in Table 1.
miRNAs and management induced stress
In large ruminants
Diverse factors associated to housing and management can induce stress in cattle and severely alter physiological and productive metrics. Stress alters miRNA synthesis, mRNA target expression and the functionalities of miRNA-protein complexes
(Olejniczak et al., 2018). As a stress model,
Colitti et al., (2019) investigated the reformation of the hierarchy in social group in lactating cows by concentrating on composition of milk exosome and discovered that a number of miRNAs were differently expressed. Genes associated with glucocorticoid receptor signaling and neurotrophic factor-mediated TRK receptor signaling was discovered by functional pathway investigation of potential target genes of DE microRNAs
(Jeanneteau et al., 2012).
In small ruminants
Inability to adapt to a different diet causes inexperienced stress in small ruminants, which is characterized by growth stagnation
(Magistrelli et al., 2013). Weaning stress is able to adversely affect the growth in the animals by reducing cell proliferation, the researchers suggested that drastically changed miRNAs may be effective biomarkers to assess the degree of weaning stress in goat production
(Liao et al., 2019). Yang et al., (2018) evaluated the levels of estrus rate, body state score and hypothalamus–ovary–nutrition-related microRNAs in sheep fed with or without concentrate. A substantial number of DE miRNAs were discovered, in which 113 were released in the ovary and 148 in the hypothalamus, respectively. MiR-200b, miR-200c and miR-200a were considerably DE-miRNAs that were expressed in both ovary and hypothalamus. The scientists discovered that food deprivation had the greatest impact on the production of miR-204-5p and miR-223-3p. Since the welfare of livestock is a pressing concern, researchers have been attempting for many years to determine the optimal housing system for small ruminants considering their geographical climate in order to maximize productivity and welfare. The researchers concluded that housing systems for small ruminants needed to be economical, well-ventilated, hygienic and constructed from materials that could be found nearby. Materials for flooring and roofing should be chosen with consideration for the climate, animal preferences and parasite burden. Similar to this, when building a shed for small ruminants in the age of climate change and global warming, consideration must be given to behavioral and physiological changes, ventilation rate, wind speed and direction
(Wadhwani et al., 2016).
miRNAs and environmental stress
Heat stress
In large ruminants
One of the most influential environmental elements effecting cattle productivity, welfare and health is high temperature. In dairy and feedlot farms, cooling methods (such as shade, fans and sprinklers) are utilized to counteract the harmful effects of thermal stress. miRNAs perform a crucial contribution in the transcriptional control of genes encoding proteins implicated in heat stress response pathways
(Li et al., 2018). Heat stress has a detrimental impact on dairy cow homeostasis. Heat stress, in particular, appears to impact lipolysis and lipogenesis-related enzymes in bovine adipocytes (
Faylon et al., 2015).
A bovine mammary epithelial cell line exhibited significant variations in miRNA expression in response to thermal stressed conditions relative to thermal neutral conditions
(Salama et al., 2019). Mutations or SNPs (Single Nucleotide Polymorphisms) in the 3'-untranslated regions (UTRs) of genes might change expression of protein by introducing misleading miRNA binding sites
(Clop et al., 2006).
In small ruminants
Using miRNA-mRNA linkages in hypoxia-related pathways, such as the one linked with HIF-1 (Hypoxia Inducible Factor 1), a comparative study of miRNA transcriptome of two goat populations at differing elevations generated convincing evidence for acclimatization. The research concluded that miRNA-509-3p, miR-3069-1-3p, miR-208-3p and miR-409-5p, were downregulated, while miR-106a-5p was upregulated, in hypoxia-sensitive tissues such as the liver, heart, kidney, spleen and muscle encouraging that miRNAs play a central role in the regulation in mechanisms attributed to high adaptation
(Feng et al., 2020). These DE-miRNAs have genes engaged in a variety of processes as possible targets, including energy metabolism, erythropoiesis, DNA repair and apoptosis
(Feng et al., 2020). Furthermore, it was reported that miR-106a-5p targeted the VEGFR1 (or FLT1) gene, exerting an adverse regulatory impact on angiogenesis and paving the way for enhanced use of genetic resources in plateau areas.
High-altitude stress
Animals’ immune systems are impacted by high-altitude hypoxia, which reduces cytokine release and modifies immunoglobulin secretion
(Mishra et al., 2015). These occurrences are consistent with persistent stress having an impact on an animal’s physical health and making them more susceptible to diseases and infection
(Kong et al., 2019).
Regulation and control of livestock’s thermal stress responses by microRNA19a/b
miRNA-19a/b regulates innate immunity in response to temperature stress by directly targeting the TLR2 gene
(Zheng et al., 2014). Stress related to temperature is detrimental to animal health, since it suppresses the immune system and increases susceptibility to illness
(Abdelnour et al., 2019). TLRs play a crucial role in detection of pathogens and protection (
O’Neill, 2006) and through modulating anti-inflammatory responses to pristine tissue wounds, they serve as guards of tissue deterioration
(Ju et al., 2014). miRNA-19a/b is hypothesized to adversely influence cytokine secretion, notably interleukin-6 (IL-6)
(Ju et al., 2014). In both murine and human cells, miRNA-19b was discovered to influence the activities of nuclear factor-B (NF-B)
(Gantier et al., 2012). The ability to quickly respond to any unfavorable ecological factors and pro-inflammatory stimuli depends on the NF-B transcription dysfunction
(Gantier et al., 2012). However, additional research is needed to establish the relationship between heat stress effects and the protective function of NF-B
(Liu et al., 2018) postulated a distinct role for an NF-B signaling pathway comprising, reactive oxygen species (ROS), heat shock protein 27 (HSP27) and mitogen-activated protein kinases (MAPKs) in preventing heat stress-induced cell death.
