One of the most prosperous subspecies of terrestrial mammals is the ruminant family. They live in a wide range of diverse environments around the world and have a big impact on various ecosystems, industries including agriculture, leisure activities, and cultures [16]. The ability of this group of animals to survive and procreate on low-quality, low-protein, and high-fiber plant resources is a major factor in their success. Most of the various stakeholders believe that animal health is crucial to the production of livestock; however, there is a disagreement among consumers, farmers, and veterinarians over what constitutes an acceptable health state.

The term “animal health’’ lacks a precise scientific definition (ranging from the absence of disease to a broad definition of health as a state of unrestricted physical, physiological, and psychological well-being), as well as clear standards from which the condition of animals and the quality of their feed could be properly evaluated. One of the main goals of farmers is to assure the health and performance of their livestock through the adequate provision of suitable feed. By enhancing forage quality and creating rations with more balanced ratios of forage and grain, organic farming should attempt to boost the energy content of cattle diets to improve the efficiency of protein utilization and, as a result, reduce nitrogen loss to the environment [16]. Farm animals’ cell differentiation, biological development, and physiology are all significantly influenced by miRNAs. These processes include controlling muscle growth and hypertrophy, adipose tissue expansion, oocyte maturation, and early embryonic development [17]. Recent research has demonstrated the critical roles of miRNAs in sheep [18], goats [19], and bovine [20,21] rumen development, as well as the preservation of intestinal homeostasis. As a result, the miRNA expression profiles in the rumen, small intestine (duodenum and jejunum [22]), and large intestine (cecum and colon) of sheep and cattle have been identified. The study also revealed that some miRNAs are exclusively expressed in specific intestine segments, indicating that their roles may be constrained to the local microenvironment. In addition, taxonomic differences in how miRNAs regulate gene expression typically occur during the expression and processing stages [23]. In ruminants, the colon is a vital component of the hind gut’s digestive system. Cell wall polysaccharides, cellulose, and hemicellulose are fermented and used in large part by the colon in ruminants. According to Yan and colleagues’ study [23], from a total of 1572 miRNAs discovered in the colon tissues during the analysis of colon miRNA transcriptomes in preweaning and postweaning goats, 39 differentially expressed miRNAs and 88 highly expressed miRNAs were screened, and various functions of dynamic miRNAs in the regulatory system governing colon growth in goats were discovered [23]. The study provided evidence that miRNAs have a direct role in the host’s responses to nutrition by identifying potential miRNA control mechanisms of gene expression during the switch from FD to HG feeding and phytogenic supplements [24]. The study hypothesized a potential role as a mechanism of interaction between the host and the ruminal microbiota, and suggested that this communication is bidirectional, with the microbiota influencing the host’s miRNA expression pattern and the host potentially helping to shape the gut bacterial profile through the production of specific miRNAs [25].

The ruminant mammary gland (MG) is a crucial organ responsible for producing milk for human infant and adult nutrition [26]. Not only nutrition, genetics, breed, disease pathogens, and other environmental factors, but also post transcriptional regulation of gene expression affects MG productivity. Lactation, one of the amazing outcomes of evolution, is a very dynamic and complicated process [27]. The growth of the MG and the production and release of milk are all parts of lactation. Cattle, buffaloes, goats, sheep, and camels provide almost all the world’s milk. Yaks, horses, reindeer, and donkeys are additional less frequent milk-producing animals. Each species’ prevalence and significance vary greatly between different geographical areas and nations. Feed, water, and climate are the three main factors that influence the dairy species retained. Market demand, dietary customs, and the socioeconomic makeup of each household are other variables that could impact the existence of a dairy species (e.g., poorer families tend to rely more on small ruminants). Since milk from dairy ruminants such as cows, goats, and sheep has been shown to have positive benefits on humans, extensive work has been carried out to increase milk production and improve its nutritional value [28]. Most infant formulas are based on cow milk proteins, and both cow and goat milk are commonly used as dairy products. Results show that adults absorb significant levels of milk-derived miRNAs from commercial pasteurized milk. Additionally, investigations have shown that milk exosomes can be incorporated into kidney, intestinal, intestinal cancer, and peripheral cells, as well as into macrophages and colon cancer cells [29]. According to Golan-Gerstl and colleagues, 95 percent of the miRNAs expressed in human milk are likewise expressed in goat and bovine milk, and pasteurization of bovine milk does not appear to eliminate miRNAs [30]. Additionally, 24 validated sites that were engaged in immunomodulatory actions were shared by conserved miRNAs [31]. Unsaturated fatty acid-rich feed added to the diet can be a useful strategy to boost milk’s health-promoting qualities; albeit, the impacts on the genes and pathways involved in these processes have not yet been fully and accurately described [32]. There are a number of measures, including nutrition, seasonal feed changes, and genomic variation, that can be utilized to improve the beneficial components in milk in ruminants because the process for the synthesis of milk fat is complex and subject to multifactorial regulation [33].

Bovine, caprine, and ovine species’ genetic variations of miRNAs expressed in the mammary gland or found in milk and localized in dairy quantitative trait loci (QTLs) were examined to find variations that might be the causes of dairy features. Using whole genome data to find miRNA genetic variants expressed in the mammary gland and localized in dairy QTLs, the study identifies miRNA genetic variations of interest in the context of dairy production [34].

A. Cattle

Proteins, lipids, and amino acids, as well as other bioactive substances such as hormones and cytokines, are all readily available in cow milk, which is also a good source of many other vital nutrients. Cow milk has been commercialized and regularly used by people for growth and health benefits due to its nutritional relevance. Li and colleagues describe the miRNA expression spectra of three milk fractions (fat, whey, and cells), contrast the milk fraction miRNome profiles with those of mammary gland tissue, and determine which milk fraction miRNome profile might be a better indicator of the miRNome profile of mammary gland tissue. Their findings demonstrated that the miRNAome of mammary gland tissue was accurately represented by miRNAs from milk fat. Top expressed miRNAs in milk were functionally annotated, and this revealed their crucial regulatory roles in mammary gland functions and perhaps to milk recipients [35]. Udder diseases, particularly mastitis brought on by bacterial infections, are significant issues for the dairy industry globally. Mastitis continues to be a major issue for the dairy industry globally, resulting in significant losses every year from reduced milk production (both quantity and quality), expensive treatments, and early animal culling, as well as having a significant impact on the development of antimicrobial resistance in cattle due to the widespread use of dry cow antibiotics [36]. Improved tools that can accurately detect early mammary inflammation in cattle are urgently needed given the relevance of early disease detection for minimizing the considerable financial and animal welfare implications of mastitis globally. Monitoring mammary gland health and spotting early inflammation require different methods. The most used method is somatic cell counting (SCC), which can be carried out in large quantities of milk or as individual milk samples, directly or indirectly through colorimetric quantification, frequently with the California Mastitis Test (CMT) [36]. Mammary epithelial cells release milk fat globules through a budding mechanism that encloses a crescent of the mammary epithelial cells cytoplasm in the plasma membrane [35]. Whether the miRNAs found in milk are specific to the mammary gland or come from the blood is the key question in determining the involvement of miRNAs in lactation. Tzelos and colleagues investigated the relationship between CMT scores (0 to 3), which were derived from many individual quarter milk samples (n = 236) taken from dairy cows at various lactations, and the levels of four inflammation-associated miRNAs (bta-miR-26a, bta-miR-142-5p, bta-miR-146a, and bta-miR-223). They confirmed that higher miRNA levels during lactation 1 than later lactations were responsible for the significant lactation number effect (P 0.01) for bta-miR-26a, bta-miR-142-5p, and bta-miR-146a. They also showed that bta-miR-223 and bta-miR-142-5p levels could accurately (100% sensitivity, >81% specificity) identify early inflammation. They stated that their findings offer further evidence of the usefulness of miRNAs as early diagnostic indicators of cow mastitis [36]. Using microarray and quantitative PCR analysis, Izumi and colleagues identified variations between colostrum (100 miRNAs) and mature milk (53 known miRNAs). They confirmed that some mRNA was present in cow’s milk, but that naturally occurring miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. Synthesized miRNA spiked in the raw milk whey were degraded [37]. Wang’s research team used transcriptome studies of mammary gland tissues from dairy cows during the high-protein/high-fat, low-protein/low-fat, or dry periods to investigate the molecular mechanisms governing milk secretion and quality in dairy cows. They discovered 25 core differentially expressed miRNAs (DE miRNAs) that were important for mammary gland growth and epithelial cell terminal differentiation during lactation, as compared to non-lactation. Their findings suggested that during mammary gland development, DE miRNAs might function as regulators of milk quality and milk secretion [38]. Xia and colleagues used miRNA and transcriptome data from the mammary epithelial cells of dairy cattle with high (H, 4.85%) and low milk fat percentages (L, 3.41%) during mid-lactation to screen and identify differentially expressed miRNAs, candidate genes, and co-regulatory pathways related to the metabolism of milk fat. In the co-expression networks of the dairy cattle mammary system, they discovered functional miRNAs and regulatory candidate genes involved in lipid metabolism (Table 1). This information advances the understanding of potential regulatory mechanisms of genetic elements and gene signaling networks involved in milk fat metabolism [39]. Since small non-coding RNAs have been linked to various phenotypes in bovine sperm, Werry and colleagues hypothesized that some differences in bull fertility may be reflected in the levels of various miRNAs in sperm. However, efforts to identify sperm-borne molecular biomarkers of male fertility have so far failed to identify a robust profile of expressed miRNAs related to fertility. The most abundant miRNAs in both populations were identified (miRs -34b-3p, -100-5p, -191-5p, -30d-4p, and -21-5p), and variations in both the total levels and particular patterns of isomiR expression were assessed. The findings offer a thorough description of the bovine sperm miRNAome and point to numerous potential roles in fertility [40]. To support the use of milk fat globules as a source of small non-coding RNAs to diagnose mastitis, their abundance from five cows before and after lipopolysaccharide (LPS) challenge was compared. Six miRNAs that are known to be regulated in the mammary gland during inflammation were also examined. The results showed that milk fat globules might be an easily accessible source of miRNAs that are possible biomarkers to detect early mastitis, and enable the application of a quick and efficient treatment. Four miRNAs (miR-494-3p, -148a-3p, -99a-5p, and -125b-5p) were variably abundant depending on the inflammatory status [41]. The Cui study team found 497 known miRNAs and 49 new ones using mRNA sequencing in the mammary glands of milking dairy cows. One of them, miRNA-71, was expressed differently in cows whose milk contained high and low levels of protein and fat [42]. A total of 21 differentially expressed genes can be referred to as targets for some of the 71 DE miRNAs based on their prior RNA sequencing data, suggesting that they may be crucial regulators of the milk protein and fat characteristics in dairy calves [43]. The miRNAomes of five essential metabolic tissues (rumen, duodenum, jejunum, liver, and mammary gland tissues) involved in protein synthesis and metabolism from 18 dairy cows fed high- and low-quality diets were studied to better understand the molecular regulatory mechanisms of milk protein production in dairy cows. There were 340, 338, 337, 330, and 328 miRNAs expressed in the rumen, duodenum, jejunum, liver, and mammary gland tissues, respectively. The findings indicated that miRNAs expressed in these tissues may play a part in controlling the transfer of amino acid for milk production downstream, which is a critical mechanism that may be related to low milk protein under poor forage feed [44]. When the mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, with different mammogenic potentials that are related to differences in dairy performance, were compared, 754 distinct miRNAs were found in the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) midlactating cows. The outcome demonstrates variations in the mammary gland miRNomes of two dairy cattle breeds, and suggests that miRNAs may have a role in the flexibility of the mammary gland and the synthesis of milk components, both of which are connected to the quantity and composition of milk [45].

With Illumina RNA-sequencing, it was discovered that miRNAs have a regulatory role in the early development of the gastrointestinal tract (GIT). As a result of the findings, which included temporal and regional variations in miRNA expression as well as a connection between miRNA expression and the microbial population in the GIT during early life, there is now additional support for the theory that host–microbial interactions regulate gut development through a different mechanism [20]. Based on the variations in meat quality attributes and 90 differently expressed mRNAs, an integrated study of miRNA and mRNA expression profiling was carried out between bulls and steers, and 18 DE miRNAs were discovered. The findings offered compelling proof of the molecular genetic regulation and gene interactions in cattle [46].

B. Goats

Goat milk is typically made into cheese in Mediterranean nations and Latin America; in Africa and South Asia, it is typically drunk raw or acidified. One of the most significant livestock animals is the goat (Caprahircus) [47]. Numerous studies on goats have looked at the impact of various feeding methods on milk fat content; nevertheless, the physiological underpinnings of this reaction are still not well understood.

A study that examined the relationship between differentially expressed miRNAs in goat mammary tissue and the fatty acid composition of goat milk found that levels of miR-183, targeting MST1 (Macrophage Stimulating 1), were positively associated with the milk’s fatty acid content [48]. The MST1 gene is targeted by miR-183 in the cytoplasm of goat mammary epithelial cells, which results in an inhibition of milk fat metabolism. One of the most crucial aspects of the nutritional quality of goat milk is its lipid composition. For instance, the findings of the study by Ollier and colleagues revealed that whole intact rapeseeds or sunflower oil in high-forage or high-concentrate diets affected milk yield and composition, but not the mammary mRNA expression of the important genes involved in lactose (for example, α-lactalbumin), protein (for example, β-casein), and lipid metabolism (e.g., lipoprotein, lipase) [49]. The response to dietary interventions did not appear to be mediated by changes in the mRNA abundance of genes encoding essential lipogenic enzymes and the associated transcription factors, according to [50] on lactating goats fed a supplement of sunflower seed oil. Thirty highly expressed miRNAs, including miR-103, whose expression correlates with lactation, were found by high-throughput sequencing in the mammary gland of lactating goats. This study’s conclusions provided new insight about the roles of miR-103 and the molecular processes that control milk fat synthesis [51]. Diets containing extruded linseed alone or in conjunction with fish oil in lactating goats exhibited effects on mRNA connected to protein metabolism and transport pathways rather than lipid metabolism, as well as a significant alteration in the FA composition of milk [52]. MiR-25 mimics in goat mammary epithelial cells lowered the expression of genes involved in lipid metabolism, which was inversely correlated with milk production at various phases of lactation. The study’s findings revealed the role of the miR-25/PGC-1beta regulatory axis during lactation and suggested that miR-25 may be involved in lipid metabolism [53] (Table 1). A total of 1487 unique miRNAs, including 45 novel miRNA candidates and 1442 known and conserved miRNAs with 378 differentially expressed and 758 co-expressed miRNAs, were found between early and late lactation. The study’s findings suggested that miRNAs may be crucial to early and late lactation throughout the development of the dairy goat mammary gland, which will help researchers better understand how genes regulate mammary gland development and lactation [54]. The goat genome was sequenced, and 487 miRNAs were identified. The greatest miRNA clusters were discovered on chromosome 21 [55]. Overall, 131 novel and 300 conserved miRNAs were identified after analyzing goat MG tissues during early lactation using the Illumina-Solexa high-throughput sequencing method [56]. Additionally, 346 conserved and 95 novel miRNAs were discovered in goat MG tissues from dry off, and peak lactation does use the same technique (Illumina-Solexa sequencing).

C. Sheep

Most sheep in the world live in developing nations. In colder regions they are even more common than goats. Sheep farming includes a variety of products that can be produced, including milk, meat, skin, fiber, and dung, although most small-scale farmers in developing nations grow sheep for meat or for sale as livestock in local markets. Milk production and lactation duration have not significantly increased because of genetic selection in dairy sheep. Awassi, East Friesian, and Lacaune are sheep breeds used for dairy products [47].

In a study with different fat-tailed sheep breeds, 155 DE miRNAs, including 78 up-regulated and 77 down-regulated miRNAs, were found between the tail fat tissue of Hu sheep (short-fat-tailed) and Tibetan sheep (short-thin-tailed) using miRNA-Seq. The findings might offer a useful theoretical framework for research into the molecular processes behind sheep tail adipogenesis [57]. Using RNA sequencing and cell-level validation (an error-based approach to design and optimization) is crucial when dealing with a complicated process such as NGS. The degree of validation and quality control required for specific process steps can be determined by carefully considering the likelihood of potential failure spots. It also helps in troubleshooting any errors and validating changes made to various test system components. The role of miRNA in the deposition of intramuscular fat (IMF) was investigated, and 59 DE-miRNAs were discovered between 2-month-old (Mth-2) and 12 month-old (Mth-12) Aohan fine-wool sheep (AFWS). In an effort to enhance the quality of sheep meat, the study identified lists of miRNAs linked to intramuscular lipid deposition in sheep and their prospective targets [58]. A study compared the microstructures and the miRNA expression profile of mammary gland (MG) tissues at peak lactation in small-tailed Han and Gansu Alpine Merino sheep, with various milk production attributes. Eighteen of the one hundred and twenty-four mature miRNAs produced were differentially expressed between the two breeds. The findings indicated that the functions of miRNAs in the growth and lactation of MG in sheep can be improved. The results also indicated that the targeted genes of differentially expressed miRNAs were mainly involved in metabolic pathways and signaling pathways related to MG development, milk protein, and fat synthesis [59] (Table 1).