Abstract

As a result of the excessive use of synthetic antibiotics as animal growth promoters and production stimulants, new strains of pathogenic microbes have been emerged. The growing awareness about the necessity of the animal feed safety, as well as consumers' concern about their food safety, has led to a steady increase in the use of direct-fed microbial (DFM) as alternative to antibiotics in the dairy farm animals. The direct fed microbial (DFM) are definedas live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance. Lactic acid producing bacteria (LAB), lactic acid utilizing bacteria (LUB) and yeast (Saccharomyces cerevisiae) represent the most components of the DFM strains. The DFM beneficial affects included: 1) Stimulating the growth of beneficial microbes in the gastrointestinal tract (GIT) by producing of growth factors likes certain amino acids and vitamins to enhance GIT microbial balance and modulate its microbial population. 2) Compete with pathogens in the intestinal mucosa for the site of adherence, colonization and nutrients sources. 3) Produce antimicrobial agents (e.g. bacteriocins, H₂O₂ and organic acids) that can inhibit pathogens in the GIT. 4) Regulate gene expression of the host immune cells (Host immunomodulation) and stimulate immune system function through production of killer cells and cytokines. 5) Increasing the efficiency of feed digestion and flow of nutrients through the GIT by stimulating the digestive enzymes secretion. 6) Detoxification of toxic materials through activates the host metabolism process. 7) Alleviating the harmful effects of the stress by enhancing host immune response. The improvement of newborn animal health, decrease diarrhea occurrence and mortality rates, significant increase in milk production and improvement of its composition by the adult animals was associated with DFM diet supplementation. In this review, the importance of the DFM in the diet of dairy animals, their mode of action, and their various effects on feed intake, nutrients digestion, blood parameters, milk production, and its composition will be discussed.

Keywords

Direct fed microbial, Dairy animals, Immunity, Deed, Milk yield.

Introduction

The animal production sector represents one of the most important sources of food for all countries of the world. Therefore, continuous development of this sector is required through research to finding ways to increase the number of animals that produce milk and meat in parallel with increasing the productivity of the animal unit to the maximum extent. Increasing production of milk and meat has become necessary nowadays to fill the food gap in some countries and to keep pace with the large increase in the world population, which is expected to reach 9.7 billion people by the year 2050 ¹. To achieve this desired goal, several strategies have been adopted in the past, including developing efficient breeding systems and selection for the high-producing animals and raising them intensively¹. Also, the use of genetic engineering techniques to obtain the maximum animal production is one of these strategies². In addition, some non-feed additives have been used (e.g.; hormones and antibiotics) to maximize the animal's ability to benefit from its feeds and makes it resistant to diseases, thus supporting the animal's health and doubling its production³.

As a result of the excessive use of synthetic antibiotics as animal growth promoters and production stimulants, new strains of pathogenic microbes have been emerged. These pathogenic microbes have the ability to resist action of the most known antibiotics, which represents an imminent danger to the health of animals and humans too⁴. Due to this danger, most of the countries have taken strict measures to use antibiotics in animal feeding. The European Union countries banned the use of antibiotics as growth and production stimulants in 2003 and turned their attention to looking for natural alternatives to the antibiotics⁵. In this regard, the use of beneficial microbes as natural microbial feed supplements to combat pathogenic microbes and improve animal health and productivity was one of the most prominent options⁶. The first concrete observation of the positive role played by the beneficial bacteria was made by the Russian scientist Metchnikoff⁷, who postulated the possibility of modifying the microbial population in the intestine by replacing harmful microbes with beneficial “friendly” ones. Lilly and Still well⁸ gave the name “probiotics” to microorganisms that produce substances which promoted the growth of other microorganisms. Fuller⁹ defined the "probiotics" as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance". In 2003, the FDA's Office of Regulatory Affairs, in cooperation with the Association of American Feed Control Officials (AAFCO), recommended use the term "Direct-Fed Microbial" (DFM) instead of "Probiotics" to describe feed products that contains a source of beneficial live microbes.

The growing awareness about the necessity of the animal feed safety, as well as consumers' concern about their food safety, has led to a steady increase in the use of direct-fed microbial (DFM) in feeding of farm animals. According to the probiotics in animal feed market report¹⁰, the global probiotics market in animal feed was valued at 5.4 billion US$ in 2025, and is expected to reach approximately 8.27 billion US$ by 2030. The demand for animal DFM preparations expanded at a compound annual growth rate (CAGR) of 8.9% which makes the DFM industry an attractive field for investment. In this review, the importance of the DFM in the diet of dairy animals, their mode of action, and their various effects on feed intake, nutrients digestion, blood parameters, milk production, and its composition will be discussed.

Direct-Fed Microorganisms and their Modes of Action

Impact of Direct-Fed Microbial on Animals Incidence of Diseases

The added DFM to the diets of livestock animals has a vital role in maintaining animal health through enhancing GIT beneficial microbial population and minimizing the abundances of the harmful and pathogenic microbes⁴⁴. It has been reported that DFM had effective role in combating harmful and pathogenic microbes such as Escherichia coli, Clostridium perfringens and Salmonella which are responsible for infection of animals with diarrhea, necrotic enteritis, anorexia, gut dysbiosis, chronic inflammation and dysregulation of the immune system^45-46. In this regard, positive impact of lactic acid bacteria as DFM against Salmonella in the gut of young calves fed milk replacer has been observed⁴⁷. Also inclusion of Lactobacillus rhamnosus (Lcr35) as DFM significantly reduced adhesion ability of enteropathogenic Escherichia coli and enterotoxigenic Klebsiella pneumoniae to intestinal mucosa⁴⁸. Most of direct fed bacterial species, including lactic acid bacteria, Bifidobacteria and Bacillus spp. are capable of producing many types of thermostable bacteriocins⁴⁹, with a wide spectrum range of antimicrobial activity against pathogenic bacteria in the gut of the farm animals including Listeria, Bacillus cereus, Enterococcus, Staphylococcus, and different species of Salmonella ⁵⁰. It is worth mentioning that bacteriocins of lactic acid bacteria antagonize the action of the pathogenic bacteria within the host animal GIT by interfering with DNA replication of target pathogenic bacteria⁵¹.

It is known that the beneficial bacteria that colonize the intestines of dairy animal’s newborn come from their parents or through the rest of the adult animals in the farm. Thus, newborn animals become protected against intestinal pathogens by the parent’s beneficial bacterial effect⁵². However, the intensive production of animals in dairy farms has reduced the normal microbial colonization of the gut, making newborn animals more vulnerable to the challenges of pathogenic bacteria in the gastrointestinal tract ⁵³. In newborn animals,probiotics can mimic the action of the natural colonization in protecting the intestinal mucosa from pathogens colonization⁵². The presence of hydrophobic surface layer proteins in most probiotic strains, especially Bifidobacterium & Lactobacillus spp., enables them to adhere to the surface of host intestinal cells. These adherent probiotic strains bind to specific receptor sites on the intestinal epithelium and thus prevent pathogens from reaching and colonizing intestinal epithelial cell surfaces⁵⁴.

Some strains of DFM can cause a disturbance in the cell to cell communication in the pathogenic bacteria, which occurs through the secretion of specific chemical signals⁵⁵. On the other hand, it has been found that probiotical bacteria can limit the pathogenic effect of the pathogenic bacteria as well as viruses by influence the (quorum sensing) communication between bacteria and their hosts⁵⁶. in this regard, it has been reported that the fermentation products of Lactobacillus acidophilus La-5 caused inhibition for secretion of extracellular autoinducer-2 of the enterohaemorrhagic E. coli serotype O157:H7, which suppressed the expression of the locus of enterocyte effacement virulence gene in E. coli serotype O157:H7. Consequently, quorum sensing can become disrupted so that colonization and pathogenesis by E. coli serotype O157:H7 in the GIT is prevented⁵⁷. The expression of Listeria monocytogenes virulence gene in the intestinal villi of rats has been decreased after the treatment with Lactobacillus salivarius. Also, the Listeria count in the spleen was decreased after the treatment with both L. salivarius and Lactococcus lactis⁵⁸. In addition, Dong et al⁵⁹ reported  that Lactobacillus plantarum CICC6257 treatment led to repression in the expression of three virulence genes (InlA, InlB and prfA) of the L. monocytogenes.

Maintaining dairy animal health requires maintaining the safety of its digestive system from pathogens, especially in the lower part of the intestine (Table 3). The damage caused by enteropathogens in the intestine induces inflammation, which in turn may lead to a thicker intestinal wall.⁶⁰

Probiotic organism	Inhibitory effect by	Antagonistic effect against
Enterococcus faecium, Pediococcus pentosaceus, and Bacillus subtilis	Bacteriocins	C. perfringens and Listeria monocytogenes
Lactobacillus salivarius	Bacteriocins	Campylobacter jejuni
Bacillus subtilis	Competitive exclusion	E. coli
Clostridium butyricum	Increased populations of Lactobacillus and Bifidobacterium	Salmonella spp. and C. perfringens
E. faecium, P. acidilactici, L. salivarius, and L. reuteri	Competitive exclusion	Campylobacter jejuni
Lactobacillus crispatus	Competitive exclusion	E. coli O157:H7 and S. typhimurium
Lactobacillus spp.	Competitive exclusion	Campylobacter jejuni

Elam et al.⁶¹ stated that lumina propria (loose connective tissue in the intestine supports the delicate mucosal, allows the epithelium to move freely with respect to deeper structures, and provides for immune defense) at ileal region was thinner in the treated steers with live cultures of Lactobacillus acidophilus and Propionibacterium freudenreichii than control steers, suggesting that dietary DFMs supplementation may provide the treated animals with more energy for growth has well as reduced the danger of the intestine inflammation. It has been reported that the dietary inclusion of Lactobacillus rhamnosus improves the growth of the intestinal epidermal cells and thereby leading to reduction of the intestinal epithelial apoptosis as well as improve the ability of the GIT to fight against diseases⁶². Moreover, the DFM act for enhancement of the epithelial barrier function through making compositional changes in the tight junction of the epithelia with blocking of the intestinal epithelial surface receptors⁶³ or stimulation of mucins synthesis to form a physical barrier along the epithelial monolayer⁶⁴. Finally, to give a clear understanding for the mode of action of the direct fed microbial in decreasing incidence of the disease, all methodologies must be considered on a case-by-case basis. Also, additional studies on host - direct fed microbial interaction should be done.

Impact of Direct-Fed Microbial on Animal’s Immunity

The immune system is the primary defense mechanism that protects animals against invading pathogens and other foreign substances present in body tissues and fluids⁶⁵. It consists of two complementary components: the innate (non-specific) immune system and the adaptive(specific) immune system, which work in concert to prevent infection and maintain host health. The innate immune system provides the first line of defense by rapidly recognizing and eliminating invading microorganisms through physical barriers, such as the skin and mucosal surfaces, immune cells including phagocytes and natural killer cells, and antimicrobial proteins and enzymes⁶⁶. Although this response is rapid, it lacks antigen specificity. In contrast, the adaptive immune system mounts a highly specific immune response after pathogen recognition and is primarily mediated by T and B lymphocytes together with antibodies circulating in the blood and other body fluids⁶⁶. T lymphocytes play several essential roles in adaptive immunity. Helper T (Th) cells coordinate immune responses by secreting cytokines that activate other immune cells, whereas cytotoxic T cells eliminate virus-infected and abnormal cells. Following pathogen clearance, a subset of T cells differentiates into memory T cells, enabling a rapid and effective response upon subsequent exposureto the same pathogen. Activated helperT cells also stimulate B lymphocytes to proliferate and differentiate into plasma cells, which produce large quantities of antigen-specific antibodies. In addition, some activated B cells become memory B cells that contribute to long-term immunological memory. Communication among immune cells is mediated through direct cell-to-cell interactions and solublesignaling molecules, particularly cytokines. Antibodies specifically recognize and neutralize antigens while facilitating pathogen elimination by other immune cells⁶⁵.

Direct-fed microbials (DFM) have emerged as promising nutritional tools for enhancing both innate and adaptive immune responses in dairy animals⁵³. Their immunomodulatory activity begins with their ability to colonize or transiently adhere to the intestinal mucosa, thereby forming a competitive barrier that limits pathogen attachment and invasion. This interaction also stimulates the production of γ-interferon, immunoglobulins, macrophages, and lymphocytes, leading to improved mucosal and systemic immunity⁵². In addition to competitive exclusion, DFM modulate immune function through the production of biologically active microbial components, including yeast-derived mannan-oligosaccharides (MOS), β-glucans, and bacterial peptidoglycans⁶⁷. These molecules function as microbial-associated molecular patterns (MAMPs) that interact with pattern-recognition receptors expressed on intestinal epithelial and immune cells, thereby initiating intracellular signaling pathways involved in immune regulation⁶⁸. One of the principal mechanisms involves modulation of Toll-like receptor (TLR) expression in intestinal epithelial cells, which contributes to the maintenance of mucosal immune homeostasis and immune tolerance⁶⁹. Furthermore, DFM activate dendritic cells through TLR2-dependent signaling and increased expression of transforming growth factor-β (TGF-β), resulting in enhanced secretion of secretory immunoglobulin A (IgA) in the small intestine⁷⁰. They also preserve intestinal epithelial integrity by preventing cytokine-induced apoptosis, thereby strengthening the intestinal barrier against pathogen invasion¹. Moreover, DFM regulate macrophage function and cytokine secretion by influencing intracellular signaling pathways that control the balance between pro-inflammatory and anti-inflammatory cytokines, particularly the IL-12/IL-10 axis⁶⁹.

Numerous studies have demonstrated the immunostimulatory effectsof specific DFM strains in dairy animals. Dietary supplementation with Bacillus subtilis natto increased serum concentrations of immunoglobulin G (IgG) and γ-interferon in calves⁷¹. Likewise, Saccharomyces cerevisiae and its metabolites upregulated the expression of genes associated with pathogen recognition and T-cell differentiation⁷². Similarly, Lactobacillus casei Shirota and Lactobacillus rhamnosus Lr23 enhanced macrophage activation and stimulated tumor necrosis factor-α (TNF-α) secretion, thereby promoting innate immune responses in ruminants⁷³. Despite these encouraging findings, the immunomodulatory effects of DFM cannot be generalized across all dairy animals because they are highly strain-dependent and are influenced by numerous factors, including animal species, age, physiological status, diet composition, environmental conditions, and the resident gut microbiota. Consequently, further well-designed mechanistic and in vivo studies are needed to elucidate the interactions between specific DFM strains and the immune system and to identify the most effective microbial candidates for improving immune competence and disease resistance in dairy animals.

Impact of DFM on Young Dairy Animals’ Performance

The rearing of young dairy animals, including calves, lambs, and goat kids, is a fundamental component of dairy production systems, as these animals represent the future productive herd and the primary source of replacement heifers and dairy cows. It is widely recognized that management practices during the early stages of life have profound and long-lasting effects on animal health, growth performance, and future productivity. Consequently, the following section reviews the effects of direct-fed microbials (DFM) on the performance, health, and development of young dairy animals throughout the different stages of their early life.

Impact of Direct-Fed Microbial on Pre-Weaning or Weaning Animals’ Performance

The application of direct-fed microbials (DFM) in the nutrition of young dairy animals has attracted considerable attention because the digestive system of newborn and pre-weaned animals is not yet fully developed. During this early stage of life, young animals are highly susceptible to a variety of stressors, including nutritional, environmental, and management-related challenges. Moreover, their risk of infection by pathogenic microorganisms, particularly those colonizing the gastrointestinal tract, is elevated due to the immaturity of their immune system and the incomplete establishment of a stable and beneficial gut microbiota⁷⁴. Because the rumen is functionally immature during the neonatal and pre-weaning periods, young dairy animals rely primarily on the lower gastrointestinal tract for nutrient digestion. Consequently, supplementation with direct-fed microbials (DFM) is expected to facilitate the establishment and maintenance of a beneficial intestinal microbiota, thereby promoting gastrointestinal development and improving gut function³⁶. Accordingly, numerous studies (Table 4) have investigated the effects of DFM supplementation, using various microbial strains and dosage levels, on the health, growth, and overall development of young dairy animals under dairy production systems.

Diarrhea is the mean reason for high mortality and morbidity for the newborn and young animals in the dairy farms. It has been reported that lactic acid bacteria, especially Lactobacillus, can suppress diarrhea and improve growth of newborn or stressed young dairy animals³⁶. In this regard, Casper et al⁷⁵ reported that feeding newborn dairy calves on milk replacer supplemented with L. plantarum (GB LP-1) at 4 or 8 (g/d) improved animal'sbody weight gain (BWG) and feed efficiency as well as improving fecal scores with the highest dosage of the L. plantarum supplementation. Similarly, Frizzo et al⁴⁷ found that supplementation of milk replacer with combination of Lactobacillus and Pediococcus lead to higher average daily gain (ADG) for the pre-weaning calves, higher starter (spray-dried whey powder) intake and lower fecal consistency index. Also, Agazzi et al⁷⁶ stated that feeding dairy calves during their first month of life on Lactobacillus and Bacillus combination led to higher growth rate and higher animal’s body weight gain, increase intake of the concentrates and reduction incidence of diarrhea. It has been reported that inclusion of the lactic acid bacteria and spores of Bacillus sp. in the diet of the young dairy animal generally target small intestine, where they act mainly by limiting E. coli and Salmonella colonization through competitive exclusion and improving immunity, thereby improving feed efficiency, animal growth rate and supporting animal health through preventing diarrhea and faecal shedding of coliform bacteria¹. In this respect, Novak et al⁷⁷ observed immunity enhancement in the calves supplemented orally with electrolyte containing Bacillus subtilis than those calves supplemented with electrolyte alone or non- supplemented calves.

Yeast-based direct fed preparations act to early establishment of the ruminal cellulolytic bacteria and ciliate protozoa³⁹, which supporting digestion of the complex forage carbohydrates and guarantees rapid development of the rumen function as well as improvement in feeding and growth rates⁷⁸. In this regard, a previous study on neonatal Holstein calves showed that S. cerevisiae boulardii (0.5 g/d) promoted starter intake and animal's growth rate with no effect on alleviating diarrhea⁷⁹. Moreover, Villot et al⁸⁰ found that S. cerevisiae boulardii CNCM I-1079 maintained average daily gain (ADG) in scouring pre-weaning calves compared with non-diarrheic calves. Also, the inclusion of S. cerevisiae boulardii CNCM I-1079 and L. acidophilus BT1386 in the dairy calves’ diet during weaning led to decrease count of the pathogenic Streptococcus and Tyzzerella_4 in the gut with increase count of Fibrobacter beneficial bacteria⁸¹. In addition, Broadway et al⁸² reported that B. subtilis inclusion in the diet of weaned calves with Salmonella challenge led to decrease Salmonella concentrations in the gut digesta and increase animal’s feed intake. It is noteworthy here that the DFM supplementation is not-effective in feeding calves that have healthy conditions during pre-weaning to weaning transition^83-84.

Impact of Direct-Fed Microbial on Post-Weaning Animals Performance

The post- weaning ruminant faces many sources of stressors (e.g. transport, regrouping, overcrowding, castration, fasting, transition to high-grain diets and respiratory diseases) which negatively impact the growth rate and increased rates of morbidity and mortality³⁶. Many studies have been done to improve health and growth performance e of the post-weaning animals in the growing stage by using direct fed microbial as feed supplements ^85-88. In this respect, Saleem et al⁸⁹ found that supplementation of lambs’ diet with Pediococcus acidilactici and Pediococcus pentosaceus during the post-weaning period significantly improved their average daily weight gain, final body weight and feed conversion ratio (FCR). Hillal et al⁹⁰ observed significant increase in the average daily weight gain of the growing lambs fed diet supplemented with combination of fungal and bacterial DFM including Saccharomyces cerevisiae and Lactobacillus, Aspergillus and Streptococcus genres. Moreover, inclusion of L. acidophilus in the diet of buffalo calves increased animal's average daily weight gain by 31.4% ⁹¹. In addition, Hassan et al⁹² observed that growing lambs received R. flavefaciens showed higher feed digestibility and growth rate. Moreover, Finck et al⁹³ reported that the newly weaned steers that received S. cerevisiae in their diet showed a cumulative increase in dry matter intake. Also, Tripathi and Karim⁹⁴ reported that yeast DFM showed positive impact on feed intake, body weight gain and feed efficiency by the growing lambs and they attributed that to improvement of feed digestion and microbial protein synthesis. The utilization of yeast DFM in growing lambs feeding indicate that their impact may be associated with animal diets, yeast strains and level of the inclusion³⁶. It has been observed that the multi-strain DFM preparations of M. elsdenii + Lactobacilli + S. cerevisiae showed advantage over other consisted of Lactobacilli+ yeast in improving growing lambs average daily weight gain and feed efficiency as a results to higher dry matter digestibility, nitrogen intake, absorption and retention^95-96. Nevertheless, some recent studies stated that no effect was observed on the growth and performance of ruminants supplemented with direct fed microbial^86-87. The difference in the DFM strains, dose, animal breed, farm management, feeding practice and other environmental factors may account for their contrary results^31, 34.

Impact of Direct-Fed Microbial on Adult Dairy Animal’s Performance

Numerous studies have investigated the effects of direct-fed microbials (DFM) on the health status and productive performance of dairy animals during the transition and lactation periods (Table 5). Dairy producers pay particular attention to animals during these critical stages because they are exposed to multiple physiological and metabolic stresses associated with calving, dietary and feed ingredient changes, the onset of lactation, negative energy balance, and an increased risk of inflammatory and metabolic disorders, including acidosis and ketosis³⁶.

Impact of Direct-Fed Microbial on Feed Intake

The effect of the added direct fed microbial to the diets of dairy animals on dry matter intake (DMI) was not consistent. Some studies have proven the positive effect of the DFM on the DMI⁹⁷, while in others, no significant effect was detected⁹⁸, and in some cases, DMI was reduced as a result to DFM addition⁹⁹.

DFM	Dosage	Delivery method	Animal	Health	Performance
S. cerevisiaeE. faecium	5 × 10⁹ cfu/d2 × 10⁹ cfu/d	Mixed with 0.5 kg ground corn and top-dressed	Transition dairy cows	–	No effect on DMI, milk yield, BW, plasma BHBA, NEFA, glucose, haptoglobin
S. cerevisiaeE. faecium	5 × 10⁹ cfu/d2 × 10⁹ cfu/d	Mixed with 0.5 kg ground corn and top-dressed	Lactating dairy cows (60–70 d in milk)	–	No effect on DMI, milk yield, milk and blood parameters; lower fecal starch content, greater apparent total-tract digestibility of starch
S. cerevisiae	2 × 10¹⁰ cfu/d	In ration	Primiparous lactating dairy cows challenged with SARA	Tended to alleviate SARA symptoms	No effect on DMI and milk yield
S. cerevisiae	8 × 10¹⁰ cfu/d	Mixed with ground corn	Multiparous lactating dairy cows	Improved ruminal pH	Increased DMI, milk yield, total VFA production, higher propionate
S. cerevisiae CNCM I-1077	1 × 10¹⁰ cfu/d	In ration	Lactating dairy cows	–	No effect on DMI, eating time, milk yield, production efficiency; tended to improve rumination, rumen temperature and milk fat production
S. cerevisiae CNCM I-4407	5 × 10¹⁰ cfu/d	Top-dressed on ration	Lactating dairy cows	Lower ruminal lactate, serum NEFA and BHBA, liver enzyme activities	Increased milk yield, rumen pH 4 h after morning feeding, total VFA and acetate concentration; No impact on propionate or butyrate concentrations; higher glucose at peak lactation
S. cerevisiae	4 × 10⁹ cfu/d	Incorporated into a grape by-product and mixed with basal diet	Early lactating dairy goats	Similar plasma metabolites and liver enzymes; reduced fecal E. coli and increased Lactobacilli (greater stability of intestinal ecosystem)	Greater DMI, milk production
S. cerevisiae	2 × 10¹⁰ cfu/d	In ration	Primi- and multiparous transition and early lactating dairy sheep	Suppressed pro-inflammatory gene expression during peripartum period	Increased milk yield; tended to increase milk fat; enhanced energy utilization
Propionibacterium P63L. plantarum 115L. rhamnosus 32	P63 or P63 + Lp or P63 + Lr (10¹⁰ cfu/d of each strain)	In high-starch or low-starch diet	Lactating dairy cows	–	Rumen pH increased; no effect on ruminal VFA; P63 + Lr tended to reduce CH₄ emission with low-starch diet
M. elsdenii	4.8 × 10¹² cfu/d	Inoculation through ruminal cannula for 2 d	Lactating dairy cows challenged with SARA	Increased protozoa count, decreased S. bovis count	Increased total VFA concentration in the corn-based group; decreased VFA concentration in the wheat-based group
P. bryantii 25A	2 × 10¹¹ cells/dose	In ration	Dairy cows in mid-lactation challenged with SARA	No effect on SARA symptoms	No effect on rumen pH
L. casei ZhangL. plantarum P-8	1:1: 6.5 × 10¹⁰ cfu/d	In ration for 30 d	Primiparous lactating dairy cows	No effect on fecal bacteria richness and diversity; enhanced rumen fermentative and beneficial bacteria; suppressed potential pathogens	No effect on milk fat, protein and lactose contents; increased milk production, milk immunoglobulin G, lactoferrin, lysozyme, lactoperoxidase; decreased somatic cell counts
S. cerevisiaeLactococcus	L: 1.6 × 10¹⁰ cfu/d;SC + L: yeast 8 × 10¹⁸ cfu/d and L 8 × 10⁹	In ration	Healthy or mastitis lactating dairy cows	Alleviated mastitis by relieving mammary gland inflammation, reducing milk somatic cell counts, decreasing abundance of mastitis-causing pathogens	–

Higher DMI is usually attributed to the development of growth and activity of fiber-degrading microbes in the rumen, especially cellulose-degrading bacteria and rumen ecology homeostasis^100-101. In the same context, it was found that Lactobacillus plantarum work to separate the carbohydrates into simpler units of the monosaccharides, and there by improve animal's feed intake and supporting the rumen ecology homeostasis³³. In this concern, Daivis¹⁰² stated that supplemented dairy heifers with 5 g per head of Propionibacteria P169 and Saccharomyces cerevisiae mixture showed increase in the daily feed intake from 9.32 kg to 10.09 (kg/day). Positive impact of Propionibacteria freudenreichii (P169) supplementation on DMI by dairy cows under summer heat and humidity conditions was reported¹⁰³. Moreover, So et al¹⁰⁴ reported positive impact of Lactobacillus casei TH14 on intake of sugarcane bagasse by mid-lactating Holstein Friesian cows. In contrast, Francisco et al¹⁰⁵ observed that dairy cows supplemented with Propionibacteria showed lower daily DMI at the first week of lactation. Also, Weiss et al⁹⁹ reported that supplemented dairy cows with high level (6×10¹¹CFU/day) of Propionibacterium P169 had lower feed intake. In addition, Boyd¹⁰⁶ observed lower dry matter intake in mid lactating Holstein cows that supplemented with a combination of Lactobacillus acidophilus NP51 and Propionibacterium freudenreichii NP24 during the hot weather. However, no effect on DMI has been detected when dairy cattle supplemented with combination of L. acidophilus and P. freudenreichii ¹⁰⁷. Also, no change in the DMI was detected in Holstein cows fed diet supplemented with Lactobacillus acidophilus or Propionibacterium freudenreichii⁹⁸. In addition, West and Bernard¹⁰⁸ observed that lactating Holstein cows diet supplementation with P. freudenreichii, L. acidophilus NP51 and L. acidophilus NP45 had no impact on the dry matter intake. Furthermore, Azzaz et al¹⁰⁹ reported that no effect of E. faecium supplementation on DMI by early lactating Holstein dairy cows. Also, Kholif et al¹¹⁰ found that probiotics multi-species supplementation did not affect the dry matter intake by lactating Farafra ewes. On the other hand, active yeast or yeast culture play an important role in raising of the ruminal pH as well as O₂ scavenging in the rumen, and thereby stimulate growth and activity of cellulolytic microbes, which in turn improves feed intake and feed digestion¹¹¹. Compared to the control animals, supplemented diet of the dairy cows with live yeast increased DM and organic matter (OM) intake by 3.2% and 4%, respectively¹¹². In this regard, Gaafar et al¹¹³ observed that feeding buffalo with baker’s yeast increased DM and OM intake by 2.7% and 3.2% as compared to the control. In another study, Cai et al⁹⁷ reported that feeding of crossbred goats with yeast and Clostridium butyricum increased DM intake.

Impact of Direct-Fed Microbial on Nutrients Digestibility

The efficient digestion of the feed nutrients is the key to improvement of the health and production in the dairy animals. The DFM play effective role in development of nutrients digestibility by supporting growth of cellulolytic bacteria in the rumen as well as enhancement of the GIT enzymes activity⁹⁷. Moreover, lactic acid bacteria help to prevent acidosis occurrence by controlling the volatile fatty acid ratios in the rumen³³. Also, active dry yeast has effective role in stabilizing ruminal pH by stimulating specific ciliate protozoan populations, which quickly engulfs starch, and compete effectively with the amylolytic lactate-producing bacteria¹¹⁴. Regarding the role of yeast in improving digestion parameters in dairy animals, Bitencourt et al¹¹² reported that supplementation of the dairy cows with yeast improved DM, OM and neutral detergent fiber (NDF) digestibility by 2.7%, 2.3%, and 10%, respectively. Moreover, Azzaz et al¹¹⁵ reported that lactating Egyptian buffaloes fed diet supplemented with yeast culture showed significant increase in all nutrients digestibility coefficients than those of control.

Regarding the effect of direct fed bacteria on the digestive parameters in dairy animals, Qiao et al¹¹⁶ observed that Chinese Holstein cows’ diet supplementation with Bacillus licheniformis cultures improved OM, crude protein (CP), NDF and acid detergent fiber (ADF) digestibility coefficients. In addition, Boyd et al¹¹⁷ reported that lactating Holstein cows diet supplementation with Lactobacillus acidophilus NP51 and Propionibacterium freudenreichii NP24 showed higher NDF and CP digestibility. Furthermore, Azzaz et al¹¹⁸ observed that lactating goats fed diet supplemented with L. acidophilus showed significant increase in DM, OM, CP, crude fiber (CF) and nitrogen-free extract (NFE) digestion coefficients than goats of control. Also, So et al¹⁰⁴ reported positive impact of Lactobacillus casei TH14 supplementation on nutrient digestibility of diet containing sugarcane bagasse by mid-lactating Holstein cows. Similarly, Azzaz et al¹⁰⁹ reported that locally isolated E. faecium fed to Holstein cows improved the digestibility of the dietary DM, OM, CP, NDF and ADF digestibility compared to the cows of control. In another study on lactating ewes, Azzaz et al¹¹⁹ observed that E. faecium NRC-3 and L. rhamnosus as dietary supplementation improved DM, OM, CP, NDF, ADF and non structural carbohydrates (NSC) digestion coefficients. Ultimately, the observed positive impact of the direct fed bacteria may be attributed to its positive effect on rumen and intestine ecology³³.

Impact of Direct-Fed Microbial on Blood Metabolites

There is a conflict in the obtained results about the effect of direct fed microbial on dairy animals’ blood parameters and metabolites. Although some studies showed no impact of DFM on blood glucose concentration in the treated dairy animals^{99, 106,108,115}, Bruno et al¹²⁰ reported significant decreases in the blood glucose concentrations. Conversely, many of studies showed positive significant impact of the DFM supplementation on the dairy animals’ blood glucose concentration. In this regard, Nocek and Kautz¹²¹ reported that postpartum cows supplemented with Enterococcus faecium showed higher blood glucose and insulin concentrations. Similarly, Aleman et al¹²² observed that primiparous Holstein cows supplemented with Propionibacteria showed greater plasma glucose concentration than control cows. In addition, Abu et al¹²³ noted higher blood glucose concentration in Holstein dairy heifers fed rice straw treated with DFM mixture containing L. rhamnosus. In recent studies, Azzaz et al¹⁰⁹ found that Holstein dairy cows fed diet supplemented with two different strains of Enterococcus faecium showed higher blood glucose concentration. Similarly, Azzaz et al¹¹⁹ observed that lactating ewes received diet supplemented with Enterococcus faecium or L. rhamnosus showed higher blood glucose concentration. In the majority of dairy animal studies, the higher blood glucose concentration may attributed to significant improvement in ruminal digestion of feed nutrients especially ON, CP, and NSC which resulted in a higher propionate concentration in the rumen¹²⁴ and subsequently in the blood which activate liver enzymes to convert propionate to glucose through gluconeogenesis process¹²⁵.

In general, adding DFM to dairy animal diets had no significant effect on the blood levels of protein, albumin, globulin, urea, triglycerides, cholesterol and total lipids, suggesting safety of DFM use on animal health⁵². But there are some exceptions, as Peng et al¹²⁶ found that dairy cows received diet supplemented with Bacillus subtilis natto showed low level of blood non-esterifies fatty acids (NEFA) which indicates efficiency of energy utilization. Other studies have reported a decrease in the blood urea concentration in dairy animals’ received DFM supplemented diets^{115, 121}. This may be attributed to improve utilization of nitrogen by ruminal microbial population, which may lead to metabolic process improvement as a direct response to DFM supplementation⁵². In addition, Azzaz et al¹⁰⁹ revealed that lactating Holstein cows that received E. faecium showed lower serum cholesterol and triglycerides than cows of control. This may reflect the ability of E. faecium to deconjugate of the bile salts by a specific hydrolysis process causing a reduction in cholesterol and triglycerides absorption at the intestinal level¹²⁷.

Impact of Direct-Fed Microbial on Milk Production and Milk Composition

The inclusion of the DFM preparations in the feeding of dairy animals may not only improve nutritional and health status of the animals, but also developed their ability to produce more milk with high quality⁵². Several studies showed that dairy animals that received diets supplemented with different DFM preparations had higher milk production with a higher presence of functional components (e.g. fat, unsaturated fatty acids and protein) with a lower somatic cells count^{109,119,128,129,130}. It has been reported that inclusion of Saccharomyces cerevisiae in the early lactating dairy cows’ diets increased milk production by 1.18 kg/day, milk fat yield by 0.06 kg/day and protein yield by 0.03 kg/day¹²⁹. Also, Ayad et al¹³¹ found that during 42 days in milk, S. cerevisiae as DFM supplement increased lactating cow’s milk yield by 23%. In another study, Maamouri et al¹³² observed that mid-lactating Holstein Friesian cows that received diet supplemented with S. cerevisiae produce higher milk yield and milk fat and protein yields than those of the control. Moreover, Azzaz et al¹¹⁵ found that Egyptian buffaloes fed diet supplemented with a mixture of yeast culture and Propionibacterium (P169) showed higher milk yield and yields of fat corrected milk (FCM), protein, fat, lactose, total solids (TS) and solid not fat (SNF) than buffaloes of the control, with no impact of the yeast culture+ Propionibacterium (P169) on milk fatty acid profile. Furthermore, Bakr et al¹³³ observed that early lactating Egyptian Holstein cows fed diet supplemented with S. cerevisiae had higher milk yield and milk fat percentage, but lower milk protein percentage and somatic cell count than cows of control. Xu et al¹³⁰ reported that tropical dairy cow’s diet supplementation with Lactobacillus plantarum P-8 and Lactobacillus casei, increased cows' milk yield by 37%. Kafilzadeh et al¹³⁴ reported that Sanjabi ewes supplemented with Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium thermophilum and Enterococcus faecium mixture during the last third of the lactation period showed significant increase in milk yield and its components yield. Elaref et al¹³⁵ found that Sohagi ewes fed diet supplemented with dry yeast produced 20.3% more milk than ewes of control. In recent study, Azzaz et al¹⁰⁹ observed that supplemented diet of Holstein Friesian cows in early lactation with newly isolated E. faecium lead to increase cows' daily milk production by 17.1 and FCM by 20.9%, respectively. Compared to the control, the E. faecium not affect the concentrations of milk components, but significantly increases the proportion of C18:1 trans-9, C18:2 cis-9-12 and C18:2 trans-10 cis-12 of milk fatty acids. In the latest study, Azzaz et al¹¹⁹ reported that lactating ewes supplemented diet with E. faecium NRC-3 or L. rhamnosus increased milk yield by 21.7% and 20.6%, and fat corrected milk yield by 23.3 and 21.9% compared to control. Compared to the control, E. faecium NRC-3 or L. rhamnosus supplementation had no significant effect on the milk fatty acid profile. Different mechanisms have been reported to explain milk production and milk composition improvement in response to direct-fed microbial supplementation (Figure 4).

Some studies attributed the improvement of milk production to the ability of the direct fed microbial strains to reduce inflammation of the udder as well as somatic cell count in the milk¹³⁰. while the others studies have suggested that milk production improvement is subject to direct effect of the DFM on the rumen ecology through stimulating growth and activity of rumen microbes especially fiber-degrading microorganisms⁵².

Conclusion

Direct-fed microbials (DFM) are an effective and sustainable alternative to antibiotic growth promoters in dairy animals. By improving gastrointestinal microbial balance, enhancing nutrient utilization, strengthening immune function, and inhibiting pathogenic microorganisms, DFM contribute to better animal health, feed efficiency, milk yield, and milk quality while reducing the risk of antimicrobial resistance. However, their effectiveness depends on factors such as microbial strain, dosage, diet, and animal management. Future research should focus on identifying more effective microbial strains and optimizing DFM application strategies to maximize their benefits and support sustainable dairy production.

Acknowledgements

None

Funding

None

Conflict of Interest

The author declares no potential conflicts of interest with respect to the research, authorship, and/or publication of this article

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