An Investigation into Rumen Fungal and Protozoal Diversity in Three Rumen Fractions, during High-Fiber or Grain-Induced Sub-Acute Ruminal Acidosis Conditions, with or without Active Dry Yeast Supplementation

“, 2013) which had been de novo aligned in-house using MUSCLE (Edgar, 2004).”

“The Internal Transcribed Spacer 1 region (ITS1) of fungi was amplified using the primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAG G-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) (White et al., 1990).”

“1.38 (Schloss et al., 2009).”

“Linear discriminant analysis (Segata et al., 2011) was used to determine discriminatory OTUs by treatment group, with significance at P 0.05 usingWilcoxon rank test.”

“Multivariate analyses showed diet type was very significant in driving diversity, followed by ADY treatment, for AMOVA, ANOSIM, and weighted UniFrac (Table 2).”

“…diversity could be attributed to the change in pH or the change in substrate as grain and concentrate diets often reduce microbial diversity (Wu et al., 2011; Belanche et al., 2012a; Li et al., Frontiers in Microbiology | www.frontiersin.org 10 October 2017 | Volume 8 | Article 1943 2013;…”

“Nucleic acids were extracted and prepared for Illumina MiSeq (Illumina, SanDiego, CA) at the University of Guelph sequencing facility as previously described (AlZahal et al., 2014, 2017) using the repeated bead-beating method (Yu and Morrison, 2004).”

“seqs in mothur to parse by primer, and then sequences were culled if they contained ambiguous bases, were shorter than 90 (Zimmerman and Vitousek, 2012) or longer than 487 bases, or which did not align or classify to the Findley fungal ITS database (Findley et al., 2013) which had been de novo aligned in-house using M…”

“Additionally, acidosis can lead to other systemic health problems, such as liver abscesses or inflammation (including laminitis) (reviewed in Plaizier et al., 2008).”

“…also appear to directly compete with fungi through enzymatic destruction and predation (Hsu et al., 1991; Gruninger et al., 2014), although a meta-analysis has suggested that rumen defaunation more often causes a reduction in cellulolytic microorganisms, including fungi (Newbold et al., 2015).”

“For statistical analysis, protozoa were subsampled (normalized) to 5,000 sequences/sample, and fungi were subsampled to 500 sequences/sample due to low reads/sample (Supplemental Table 3; Smith et al., 2014).”

“…in feed intake and downstream gastrointestinal dysfunction (i.e., diarrhea) of SARA cause subsequent reductions in rumen function, microbial diversity, host performance, and host immune function (Khafipour et al., 2009; Hook et al., 2011; Petri et al., 2013; McCann et al., 2016; Sato, 2016).”

“Yet almost nothing is known about its effect on ruminal protozoal and fungal diversity, despite their roles in fiber degradation (Williams and Withers, 1993; Lee et al., 2000; Krause et al., 2003; Sun et al., 2006; Belanche et al., 2012a,b).”

“intracellular pH in the context of an acidic extracellular pH can cause some acidic volatile fatty acids to disperse into cells and accumulate to toxic levels as intracellular anions (Russell and Diez-Gonzalez, 1998).”

“The effects of yeast supplementation on preventing or treating SARA, as well as on bacterial diversity, have been previously characterized (Khafipour et al., 2009; Petri et al., 2013; AlZahal et al., 2014, 2017; Uyeno et al., 2015; McCann et al., 2016).”

“An inhouse ruby script was used to truncate sequences at the reverse primer or at homopolymers after 8 bases (Luo et al., 2012; Ishaq et al., 2017).”

“A meta-analysis of S. cerevisiae supplementation indicated that it would increase protozoal growth (Desnoyers et al., 2009).”

“Fungi (Warner, 1966; Orpin, 1975; Gruninger et al., 2014) and protozoa (Hook et al., 2012; Williams and Coleman, 2012) are known to associate with rumen epithelial cells until chemotaxis draws them into the liquid and solid fractions.”

“…16S sequences using trim.seqs in mothur to parse by primer, and then sequences were culled if they contained ambiguous bases, were shorter than 90 (Zimmerman and Vitousek, 2012) or longer than 487 bases, or which did not align or classify to the Findley fungal ITS database (Findley et al., 2013)…”

“Both datasets were clustered using the nearest neighbor method, protozoal at a 4% species-level cutoff (Ishaq and Wright, 2014) and fungi at a 3% species-level cutoff (Blaalid et al., 2013).”

“…amplified using previous protocols (Ishaq and Wright, 2014; Ishaq et al., 2015) that utilized the primers P-SSU-316F (5′-GCTTTCGWTGGTAGTGTATT-3′) (Sylvester et al., 2004) and GIC758R (5′-CAACTGTCTCTATKAAYCG3′) (Ishaq and Wright, 2014) which target the V3–V4 region of the 18S rRNA gene and…”

“Protozoa also appear to directly compete with fungi through enzymatic destruction and predation (Hsu et al., 1991; Gruninger et al., 2014), although a meta-analysis has suggested that rumen defaunation more often causes a reduction in cellulolytic microorganisms, including fungi (Newbold et al.”

“Dietary management is the most widely-used technique for preventing the onset of SARA in cattle (Stone, 2004).”

“Yet almost nothing is known about its effect on ruminal protozoal and fungal diversity, despite their roles in fiber degradation (Williams and Withers, 1993; Lee et al., 2000; Krause et al., 2003; Sun et al., 2006; Belanche et al., 2012a,b).”

“Many species of rumen protozoa and methanogenic archaea are known to interact symbiotically (Vogels et al., 1980; Sharp, 1998; OheneAdjei et al., 2007), but there exists an antagonism between fungi and protozoa.”

“…in feed intake and downstream gastrointestinal dysfunction (i.e., diarrhea) of SARA cause subsequent reductions in rumen function, microbial diversity, host performance, and host immune function (Khafipour et al., 2009; Hook et al., 2011; Petri et al., 2013; McCann et al., 2016; Sato, 2016).”

“…11 October 2017 | Volume 8 | Article 1943 Thrune et al., 2009), but alters the redox potential of rumen fluid, and with its ability to survive in the rumen allows for a more continuous control of rumen pH, it has an advantage over chemical pH buffering with sodium bicarbonate (Marden et al., 2008).”

“Yet almost nothing is known about its effect on ruminal protozoal and fungal diversity, despite their roles in fiber degradation (Williams and Withers, 1993; Lee et al., 2000; Krause et al., 2003; Sun et al., 2006; Belanche et al., 2012a,b).”

“Error is presented as standard deviation. study and have been previously identified in grain (Abe et al., 2015; Lee et al., 2015).”

“…in feed intake and downstream gastrointestinal dysfunction (i.e., diarrhea) of SARA cause subsequent reductions in rumen function, microbial diversity, host performance, and host immune function (Khafipour et al., 2009; Hook et al., 2011; Petri et al., 2013; McCann et al., 2016; Sato, 2016).”

“In vivo studies under SARA conditions showed treatment with S. cerevisiae active-dry yeast (ADY) improved rumen pH (Bach et al., 2007; Thrune et al., 2009; AlZahal et al., 2014), as well as adherent bacteria (ex.”

“Many species of rumen protozoa and methanogenic archaea are known to interact symbiotically (Vogels et al., 1980; Sharp, 1998; OheneAdjei et al., 2007), but there exists an antagonism between fungi and protozoa.”

“While fungi are negatively affected by a decrease in pH, they may be positively affected by the reduction in bacteria with which they are often in competition for nutrients (Møller et al., 1999).”

“…in feed intake and downstream gastrointestinal dysfunction (i.e., diarrhea) of SARA cause subsequent reductions in rumen function, microbial diversity, host performance, and host immune function (Khafipour et al., 2009; Hook et al., 2011; Petri et al., 2013; McCann et al., 2016; Sato, 2016).”

“In vitro investigation using fermentation chambers showed pH was a larger driver of fermentative ability than substrate; low pH reduced microbial fiber digestion, nitrogen circulation, and volatile fatty acid production, especially acetate and butyrate (Calsamiglia et al., 2007).”

“…findings which showed no effect (Hristov et al., 2012; Boots et al., 2013), a reduction in total abundance (Belanche et al., 2012a), a reduction of diversity within three common genera (Denman et al., 2008), and a reduced diversity in sequenced libraries (Kumar et al., 2015; Tapio et al., 2017).”

“…change in pH or the change in substrate as grain and concentrate diets often reduce microbial diversity (Wu et al., 2011; Belanche et al., 2012a; Li et al., Frontiers in Microbiology | www.frontiersin.org 10 October 2017 | Volume 8 | Article 1943 2013; Fernandes et al., 2014; Kumar et al., 2015).”

“Once rumen pH is below 6.0, the extent and duration of the lowered pH will differentially affect the ability of cellulolytic bacteria to attach to fiber particles (Roger et al., 1990; Mouriño et al., 2001; Sung et al., 2007).”

“Fungal abundance and cellulolytic potential were found to increase in the presence of hydrogen-utilizing species, such as methanogenic archaea (Joblin et al., 1990; Marvin-Sikkema et al., 1990), presumably due to the pH-modulating effect.”

“Diet-induced SARA can cause damage to the rumen epithelium (Steele et al., 2011) and increase the expression of host genes responsible for rumen epithelial barrier function (McCann et al.”

“However, bacterial-fungal interactions can be rather positive, and can even include cross-domain production of growthpromoters (reviewed in Tarkka et al., 2009).”

“In vivo studies under SARA conditions showed treatment with S. cerevisiae active-dry yeast (ADY) improved rumen pH (Bach et al., 2007; Thrune et al., 2009; AlZahal et al., 2014), as well as adherent bacteria (ex.”

“Likewise, relative abundances of Ophryoscolex caudatus, Ostracodinium trivesiculatum, Epidinium ecaudatum, Eremoplastron rostratum, Eudiplodinium rostratum, and Dasytricha ruminantium were significantly decreased in the HG diet.”

“…diets have been shown to have no effect (Hristov et al., 2012; Boots et al., 2013), to reduce total abundance (Belanche et al., 2012a), to reduce diversity within three common genera (Denman et al., 2008), and to reduce diversity in sequenced libraries (Kumar et al., 2015; Tapio et al., 2017).”

“Fungal abundance and cellulolytic potential were found to increase in the presence of hydrogen-utilizing species, such as methanogenic archaea (Joblin et al., 1990; Marvin-Sikkema et al., 1990), presumably due to the pH-modulating effect.”

“Type A will out-compete Type B when added to naïve Type B (Eadie, 1967; Coleman et al., 1972).”

“Diversity was calculated using themothur-integrated versions of CHAO (Chao and Shen, 2003), ACE (Chao and Shen, Frontiers in Microbiology | www.frontiersin.org 3 October 2017 | Volume 8 | Article 1943 2010), Good’s Coverage (Etsy, 1986), Inverse Simpson (Simpson, 1949), and Shannon Diversity (Shannon and Weaver, 1949)…”

“For example, many protozoa produce hydrogen during fiber digestion (Krumholz et al., 1983), there is competition for fiber substrates, some protozoal enzymes have been shown to degrade fungal cell walls (reviewed in Gruninger et al., 2014), while others consume fungal spores (Hsu et al., 1991;…”

“cerevisiae; AB Vista, Marlborough, UK; 8× 1010 cfu/head per day) which was applied as a top dressing, and which was prepared weekly by mixing 4 g of ADY (2 × 1010 cfu/g of DM) with 250 g of ground dry corn (AlZahal et al., 2014).”

“Fungi (Warner, 1966; Orpin, 1975; Gruninger et al., 2014) and protozoa (Hook et al., 2012; Williams and Coleman, 2012) are known to associate with rumen epithelial cells until chemotaxis draws them into the liquid and solid fractions.”

“Error is presented as standard deviation. study and have been previously identified in grain (Abe et al., 2015; Lee et al., 2015).”

“However, in the present study, diet-induced SARA increased fungal diversity, which is contrary to some previous findings which showed no effect (Hristov et al., 2012; Boots et al., 2013), a reduction in total abundance (Belanche et al.”

“Cellulase enzyme activity requires acid catalysis, and as such cellulase activity most often occurs extracellularly in the rumen, is sensitive to local pH, and works best in a slightly acidic environment (pH 6–7) (Weimer, 1993; Russell andWilson, 1996; Sung et al., 2007).”

“Likewise, entodiniomorphid protozoa consume lactate (Newbold et al., 1987), thus acidosis does not affect all species similarly.”

“Supplementation with ADY mitigated the reduction in protozoal diversity caused by diet or pH (objective 4), consistent with previous studies on bacteria (AlZahal et al., 2017).”

“Likewise, relative abundances of Ophryoscolex caudatus, Ostracodinium trivesiculatum, Epidinium ecaudatum, Eremoplastron rostratum, Eudiplodinium rostratum, and Dasytricha ruminantium were significantly decreased in the HG diet.”

“…in feed intake and downstream gastrointestinal dysfunction (i.e., diarrhea) of SARA cause subsequent reductions in rumen function, microbial diversity, host performance, and host immune function (Khafipour et al., 2009; Hook et al., 2011; Petri et al., 2013; McCann et al., 2016; Sato, 2016).”

“Fungi have the slowest life cycles of rumen microorganisms (24–32 h) (Theodorou et al., 1996; Hobson and Fonty, 1997), and association with the rumen epithelium may help cells avoid wash-out.”

“However, in the present study, diet-induced SARA increased fungal diversity, which is contrary to some previous findings which showed no effect (Hristov et al., 2012; Boots et al., 2013), a reduction in total abundance (Belanche et al.”

“…mixed A/B populations in ruminants (Towne et al., 1988a,b), indicating a potential for stasis in competition at the species’ level, as well as Type O (Coleman, 1979), consisting only of Entodinium, Isotricha, and Dasytricha which are more acid-tolerant (Lyle et al., 1981; Dennis et al., 1983).”

“Type A will out-compete Type B when added to naïve Type B (Eadie, 1967; Coleman et al., 1972).”

“E. furca monolobum and Neocallimastix are cellulolytic and have been associated with methanogens seeking hydrogen (Regensbogenova et al., 2004; Wei et al., 2016), potentially they may be competing for fiber substrate or hydrogentrophs in the rumen.”

“An inhouse ruby script was used to truncate sequences at the reverse primer or at homopolymers after 8 bases (Luo et al., 2012; Ishaq et al., 2017).”

“, 1988a,b), indicating a potential for stasis in competition at the species’ level, as well as Type O (Coleman, 1979), consisting only of Entodinium, Isotricha, and Dasytricha which are more acid-tolerant (Lyle et al., 1981; Dennis et al., 1983).”

“Isotrichids have been shown to associate with plant particles due to chemotaxis toward a variety of sugars (Orpin and Letcher, 1978; Diaz et al., 2014a,b), thus their increase in the present study with ADY treatment may result from the associated improvement in fiber digestion and availability of…”

“Protozoal sequences were identified in epimural fractions, contrary to a previous study which used a different variable region of the 18S rRNA gene (Shin et al., 2004).”

“Yet almost nothing is known about its effect on ruminal protozoal and fungal diversity, despite their roles in fiber degradation (Williams and Withers, 1993; Lee et al., 2000; Krause et al., 2003; Sun et al., 2006; Belanche et al., 2012a,b).”

“For example, Neocallimastix frontalis, a cellulolytic fungus, was inhibited by the cellulolytic bacterium Ruminococcus flavefaciens (Bernalier et al., 1993).”

“In vitro studies found Saccharomyces cerevisiae yeast reduced bacterial protease activity (Chaucheyras-Durand et al., 2005), and could clear Escherichia coli from rumen fluid (Chaucheyras-Durand et al.”

“…diets have been shown to have no effect (Hristov et al., 2012; Boots et al., 2013), to reduce total abundance (Belanche et al., 2012a), to reduce diversity within three common genera (Denman et al., 2008), and to reduce diversity in sequenced libraries (Kumar et al., 2015; Tapio et al., 2017).”

“High-starch diets, on the other hand, favor the protozoa Entodinium (Dehority and Odenyo, 2003), although it should be noted that the Dehority and Odenyo results were likely differential by species, as only some Entodinium (i.”

“Yet almost nothing is known about its effect on ruminal protozoal and fungal diversity, despite their roles in fiber degradation (Williams and Withers, 1993; Lee et al., 2000; Krause et al., 2003; Sun et al., 2006; Belanche et al., 2012a,b).”

“Isotrichids have been shown to associate with plant particles due to chemotaxis toward a variety of sugars (Orpin and Letcher, 1978; Diaz et al., 2014a,b), thus their increase in the present study with ADY treatment may result from the associated improvement in fiber digestion and availability of…”

“…protozoa produce hydrogen during fiber digestion (Krumholz et al., 1983), there is competition for fiber substrates, some protozoal enzymes have been shown to degrade fungal cell walls (reviewed in Gruninger et al., 2014), while others consume fungal spores (Hsu et al., 1991; Morgavi et al., 1994).”

“Protozoa also appear to directly compete with fungi through enzymatic destruction and predation (Hsu et al., 1991; Gruninger et al., 2014), although a meta-analysis has suggested that rumen defaunation more often causes a reduction in cellulolytic microorganisms, including fungi (Newbold et al.”

“, 2005), and could clear Escherichia coli from rumen fluid (Chaucheyras-Durand et al., 2010).”

“Polyplastron, along with Ophyroscolex and Metadinium, dominate Type A rumen populations which are common in wild ruminants (Towne et al., 1988a,b), and may Frontiers in Microbiology | www.frontiersin.org 12 October 2017 | Volume 8 | Article 1943 represent a “wild-type community.””

“Pichia and Candida both contain species which utilize lactic-acid (Mendes de Almeida et al., 2012; Sirisan et al., 2013), and both of which were increased on a high-grain diet in the current study.”

“Pichia and Candida both contain species which utilize lactic-acid (Mendes de Almeida et al., 2012; Sirisan et al., 2013), and both of which were increased on a high-grain diet in the current study.”

“Protozoa were amplified using previous protocols (Ishaq and Wright, 2014; Ishaq et al., 2015) that utilized the primers P-SSU-316F (5-GCTTTCGWTGGTAGTGTATT-3) (Sylvester et al.”

“, 2014) and protozoa (Hook et al., 2012; Williams and Coleman, 2012) are known to associate with rumen epithelial cells until chemotaxis draws them into the liquid and solid fractions.”

“Isotrichids have been shown to associate with plant particles due to chemotaxis toward a variety of sugars (Orpin and Letcher, 1978; Diaz et al., 2014a,b), thus their increase in the present study with ADY treatment may result from the associated improvement in fiber digestion and availability of…”

“PCR product was cleaned and normalized with a SequalPrep Normalization Kit (Invitrogen, ThermoFisher Scientific, US) (AlZahal et al., 2016, 2017), and pooled at equimolar concentrations.”

“Sheep have also been shown to change from Type O to other types following diet changes (Kittelmann et al., 2016).”

“Fungi have the slowest life cycles of rumen microorganisms (24–32 h) (Theodorou et al., 1996; Hobson and Fonty, 1997), and association with the rumen epithelium may help cells avoid wash-out.”

“Differentiating between the effects of the availability of different feed substrates and the acid-production potential of feeds (Kim et al., 2012) on rumen microbial diversity is challenging, especially as different feed substrates or formulations can cause varied amount of saliva production, which…”

“Ishaq et al. (2017) then fed active dry yeast to the dairy cows with induced SARA and noted an increase in pH and rumen protozoal abundance.”

“The improvement in classification of fungi in the rumen thus enabled the recent assessment of their composition and dynamics across different animals and diets (Ishaq et al., 2017; Tapio et al., 2017a).”

“…and efficient analysis of the rumen protozoal populations in the rumen microbial community, including variations caused by different diets (Ishaq et al., 2017; Zhang et al., 2017; Bainbridge et al., 2018), breed and lactation stage in dairy cattle (Cersosimo et al., 2016), and…”

“As found for bacteria [34], also the AF richness and diversity in the rumen declines in response to high-grain feeding of dairy cows [36,38,39], which was also observed in the rumen of goats switched from low to high-grain [40].”

“These effects include the deactivation of specific biomass-degrading enzymes from some of the most efficient fiber degraders of the rumen microbiome but also system-level responses, such as the reduction of feed conversion within the rumen [7,8].”

“It is also possible that different types of anaerobic rumen fungi would be present when animals have access to different diets [13].”

“However, another study on dairy cows showed opposite trends [87].”

“Although several studies have been reported on the diversity and relative abundance of epimural microbial communities [41,42], further studies are needed to determine the establishment process of epimural microbial communities.”

“In this regard, abundant DFM Saccharomyces cerevisiae was found in epithelial or solid-associated fractions in the rumen (Ishaq et al., 2017) near the top where oxygen content is higher.”

“Members of the genus Thelebolus have been found in ruminant samples before [50].”

“[28] fed active dry yeast to the dairy cows and recorded an increase in pH and rumen protozoal abundance.”

“cerevisiae may not serve a functional purpose [9].”

“The greater relative abundance of fungi in the SOL phase of DCP-fed fermenters compared to EM ones is in accordance with the greater NDF disappearance and increased pH values observed in the DCP-fed fermenters, as fungi actively contribute to fiber degradation in the rumen [58] and they are negatively affected by a de…”

“Animals submitted to a low-forage diet, for example, showed a higher abundance of Entodinium and Prevotella species (Carberry et al. 2012) whereas animals with induced sub-acute ruminal acidosis (SARA) presented an increase of rare fungal taxa (Ishaq et al. 2017).”

“Contrary to the findings of Ishaq [30], a change in ph did have an impact on the variety and composition of the fungi community.”

“Based on our results, it appears that the time of starch feeding is more closely associated with increasing protozoal abundance, which agrees with previous research suggesting that starch is a more potent stimulus of protozoal growth than fiber (Ishaq et al., 2017).”

“Prior research indicated that supplementation with active dry yeast failed to restore the original microbiota composition that existed before the commencement of SARA in dairy calves, implying the restricted functional effectiveness of this commercial yeast strain [17].”

“Some researchers noted a marked reduction in the relative abundance of anaerobic fungi, including Neotrichobena and Pyristobena , in dairy cows transitioning from coarse to high-concentrate diets, which precipitated subclinical acidosis [48].”

“1 3 important role in carbohydrate degradation, and its diversity increased with the increased fiber content (Ishaq et al. 2017).”

“…animals, sustaining in the rumen 1013 to 1014 microbes, the food chain is thus reachable by UI, NI, DI (Cruchaga et al., 2011; Doran et al., 2018; Ishaq et al., 2017; Legay et al., 2016; Pronk et al., 2017; Raul et al., 2016; Rodrigues et al., 2018; Scheurer et al., 2016; Soliveres et al., 2016;…”

“31-33 For example, despite intensified husbandry and stringent biosecurity in modern intensive production systems, the incidence of certain digestive and metabolic disorders remains persistently high, suggesting that excessively sanitized, controlled environments may displace ancestral indige -…”

“…in multiple culture-independent diversity surveys (Tuckwell et al. 2005; Griffith et al. 2009; Liggenstoffer et al. 2010; Nicholson et al. 2010; Kittelmann et al. 2012, 2013; Ishaq et al. 2017; Paul et al. 2018; Mura et al. 2019; Joshi et al. 2022; Young et al. 2022; Meili et al. 2023, 2024).”

“…the CSP group; however, its functional role in ruminal fiber fermentation remains poorly defined, and its 10.3389/fmicb.2026.1905439 Frontiers in Microbiology 13 frontiersin.org enrichment patterns are inconsistent across diverse hosts and dietary conditions (Ishaq et al., 2017; Wei et al., 2024).”