Livestock production systems in semi-arid rangelands in the western United States are challenged by limited forage quantity and quality. Shorter growing seasons in northern latitudes and high-elevation areas require reliance on harvested forage to support livestock during the winter. Forage quality is particularly important when livestock face stressors like cold weather or have increased nutritional requirements such as during pregnancy and lactation. In addition, trace mineral deficiencies are common in ruminants during lactation (Zhao et al., 2022). To meet livestock nutrient requirements, producers supplement low quality (low crude protein) forage with protein and mineral supplements (DelCurto et al., 2000). Producers have access to a variety of mineral delivery systems to meet nutrient requirements but often these systems have limitations. Self-fed systems are often limited by extreme variation in intake, while single dose delivery systems have rapid serum and liver response rates, but a short-term effect requiring multiple treatments to meet the animal’s nutrient requirements (Arthington and Swensont, 2004; Jackson et al., 2020).

Previous work has evaluated the use of sustained release mineral boluses on cattle performance (Sprinkle et al., 2006; Jackson et al., 2020; Sprinkle et al., 2021). Results have shown that supplementation with a long-acting trace mineral bolus decreased calving interval and increased calf-weaning weights from cattle grazed in a rangeland environment (Sprinkle et al., 2021). There are few studies characterizing degradation rates of sustained release mineral boluses and factors that impact degradation rates (Carlisle et al., 2021) and little is known regarding the interaction of mineral boluses and forage quality on the rumen microbial community structure and function.

Dietary nutritional profile, forage quality, and mineral availability are crucial in shaping the structure and function of the rumen microbial community. The rumen contains a complex microbial consortium that colonizes and degrades ingested plant material. End products of the anaerobic degradation of complex cellulolytic and hemicellulolytic feedstocks are primarily volatile fatty acids (VFAs) consisting of predominantly acetate, propionate, and butyrate (Newbold and Ramos-Morales, 2020). Dietary changes in nutrient availability can alter the rumen microbial community composition and associated functions such as fiber degradation and fatty acid hydrogenation (Newbold and Ramos-Morales, 2020). Studies have also shown that cattle fed high-concentrate diets tend to have higher abundances of Prevotella ruminicola, P. brevis, P. bryantii and P. albensis while forage-fed cattle had greater abundances of Fibrobacter and greater microbial diversity based on Simpson’s alpha diversity and beta diversity metrics (Henderson et al., 2015; Tapio et al., 2017). Research comparing different forage sources with supplemental protein and minerals have shown that cattle fed an alfalfa hay-based diets had greater microbial alpha diversity than cattle fed a triticale straw-based diet (Kong et al., 2010; Petri et al., 2018). Mineral availability can impact fermentation. Selenium can alter the rumen microbiome composition and VFA content (Hendawy et al., 2021) and mineral supplementation has been linked to greater abundance of propionate producing bacteria (Liu et al., 2017). In contrast, other studies have shown either no effect or a negative effect on rumen fermentation in response to manganese and zinc supplementation although the response varied based on the mineral source (Liu et al., 2017; Vigh et al., 2023).

While numerous studies have evaluated the effects of diet quality on rumen microbial community structure and function, few have evaluated forage quality in conjunction with sustained release mineral boluses in beef cattle. Previously we found that diet quality affected degradation rates of sustained release mineral boluses (Carlisle et al., 2021). The objective of this present study was to characterize the effect of forage quality in conjunction with two different time release mineral supplement boluses on the structure and function of the rumen microbiome. We hypothesized that; 1) Rumen microbial diversity would be higher in cows fed high quality forage and 2) Mineral bolus prototype would alter rumen microbial community structure or function.

All protocols and procedures were approved by the Montana State University Agricultural Animal Care and Use Committee (#2019-AA12). All animals used in this study were provided by the Montana Agriculture Experiment Station. This study was conducted at the Bozeman Agriculture Research and Teaching farm at Montana State University in Bozeman, MT, USA.

Samples used in this study were obtained in a previous experiment (Carlisle et al., 2021). Briefly, a diet quality and mineral bolus study was established with 16 ruminally cannulated Angus crossbred cows (age 2 – 3 years, average weight 644 kg). The study was conducted as a completely randomized design with a 2 × 2 factorial arrangement of treatments. Treatments consisted of high- or low-quality forage diets and a 90-day or 180-day sustained release mineral bolus. The low-quality forage had > 600 g/kg neutral detergent fiber and < 80 g/kg crude protein, dry matter basis while high-quality forage had < 500 g/kg neutral detergent fiber and > 150 g/kg crude protein, dry matter basis (Carlisle et al., 2021). High quality forage consisted of alfalfa grass hay while low quality forage was mature wheatgrass hay. The low-quality treatment was protein supplemented with canola meal supplement containing 463 g/kg crude protein (CP) at a daily rate of 0.91 kg/hd to meet the maintenance requirements of mature, non-lactating, beef cows per NRC requirements. Average daily intake in the high-quality treatment was 14.24 kg/d (2.5 kg CP/d) while the cattle in the low quality group consumed an average of 13.86 kg/d (1.4 kg CP/d) (Carlisle et al., 2021). This resulted in an average of 78% greater CP intake by cattle on the higher quality diet. Nutrient analysis of the high quality and low-quality hay are shown in Table 1 and nutrient composition of the sustained release mineral boluses are shown in Table 2. Cattle were adapted to basal diets for 14 days prior to initiating the intake and digestion period. This was followed with a 15-d intake and digestion period to quantify the effects of forage quality on intake, digestion, and rumen fermentation characteristics of individual cows. The period selected for sampling was based on a previous evaluation of the kinetic of bolus degradation (Carlisle et al., 2021). Animals were randomly assigned to individual pens according to diet and bolus treatment (Carlisle et al., 2021). Rumen samples (100 ml) were collected with a suction strainer prior to feeding (0-hr) and 8-hr post feeding for microbial community analysis. Samples collected at 0-hr represented a baseline prior to feeding, and 8-hr post feeding was selected based on rumen content evaluation which showed maximum VFA concentrations were achieved by this timepoint (Carlisle et al., 2021). Samples were stored at -80°C prior to submitting for DNA sequencing.

In this study we investigated the effects of diets consisting of alfalfa grass hay or mature wheatgrass hay in conjunction with sustained release mineral bolus supplements on the rumen microbiome in beef cattle. Previously we found that forage quality affected degradation rates of sustained release mineral boluses (Carlisle et al., 2021). Although differences were observed, the neutral detergent fiber digestibility (NDFD) for the grass alfalfa mix hay was less than the NDFD of the wheatgrass hay and levels of non-fiber carbohydrates were similar which suggests hemicellulose content was greater in the low quality diet. The differences in ruminal response may relate to the low digestibility of stems in legumes compared to stems in grasses (Soest, 1994) and intake and total tract digestion for both diets may have been limited by fiber digestion rate and ruminal fill limitations (Galyean and Defoor, 2003). Despite the lower NDFD, ruminal volatile fatty acids and ammonia differed among the two diets suggesting differing ruminal environments which was hypothesized to be related to the rumen microbiome (Carlisle et al., 2021). The results of this present work showed that the rumen bacterial community structure and function was significantly altered by forage quality but were not affected by sustained release mineral bolus type. Rumen VFA and NH₃-N concentrations are important markers of rumen function (Wei et al., 2019). Redundancy analysis showed total VFAs were associated with high quality forage 8 hours post-feeding which suggests that high quality forage contributed to an increased fermentation rate in the rumen (Wei et al., 2019). VFAs are an important energy source contributing to the production performance of ruminants (Wang et al., 2020) thus, it is important to understand how forage quality and supplements effect microorganisms associated with VFA production.

Mineral bolus type was not associated with changes in the bacterial community composition or any rumen parameter. Our results are consistent with studies which have shown that mineral supplementation did not significantly alter bacterial abundances in dairy steers (Son et al., 2023) and yak (Bos grunniens) (Zhao et al., 2022). In contrast, other studies have shown a response to specific minerals. Copper (Cu), manganese (Mn), and zinc (Zn) have been linked to enhanced microbial enzyme activity (Vigh et al., 2023). Addition of (Cu) to dairy cow diets was associated with increased populations of the genera Rumminococcus and Fibrobacter (Wang et al., 2021). Another study reported no change bacterial abundance in response to Cu supplementation from 0 – 60 mg/kg DM but reduced abundance at higher concentrations (Hernandez-Sanchez et al., 2019). Other studies have shown selenium (Se) supplements lead to a reduction in total bacteria in dairy cows and increased abundance of the genera Rumminococcus, Fibrobacter, and Butyrivibrio in dairy bulls (Du et al., 2019; Liu et al., 2019; Zhang et al., 2020). Our results showed no difference in bacterial community composition between mineral boluses and thus is not likely to impact rumen function or compensate for low quality forage.

Rumen bacterial communities were predominated by members of the phyla Bacteriodota and Fermicutes which is consistent with other studies (Myer et al., 2015; Liu et al., 2016; Gharechahi et al., 2021; Zhou et al., 2021). The genus Prevotella, a member of the phylum Bacteriodota, was strongly associated with the high-quality forage diet 8 hours post feeding (Figure 3). Similar results have been observed in dairy cattle where alfalfa hay increased the abundance of Prevotella and Selenomonas compared to cows fed cornstalks (Zhang et al., 2014). Prevotella is one of the most abundant rumen genera and is crucial for degradation and utilization of polysaccharides and protein (Bohra et al., 2019). The high-quality forage had a higher protein content which may have contributed to the greater abundance of Prevotella. This contrasts with other studies which have found Prevotella is the dominant genus regardless of diet (Myer et al., 2015; Cristobal-Carballo et al., 2021). These previous studies did not consider temporal changes in Prevotella abundance with measurements collected pre-feeding (Cristobal-Carballo et al., 2021) or at the end of the feeding experiment (Myer et al., 2015) which may explain why a diet associated effect was not observed. Previous work has shown Prevotella populations peak on the surface of fiber within six hours of material entering the rumen (Liu et al., 2016). Our results are consistent with this finding and suggest forage quality may have a significant but transitory effect on Prevotella abundance.

Multiple predicted functions were correlated to rumen factors (Figure 8). One limitation of this study is that functional predictions were limited to the genus level resolution of the sequencing data. Microbial function can vary even at the species or strain level and predictive functional profiling cannot resolve strain level variations or identify functions if the pathway annotations are poor (Langille et al., 2013). Predicted functions associated with methanogenesis were negatively correlated with forage quality. This is consistent with previous findings which reported higher methane associated with low quality feed (Johnson and Johnson, 1995).

Precision nutrition and/or the optimization of rumen function depends on increased knowledge of the response of the rumen microbiome to the nutrient composition of the diet. Although previous research has demonstrated methanogenesis is negatively correlated with diet quality (Johnson and Johnson, 1995; Chagunda et al., 2010), most of the research to date is limited to comparisons of concentrate and forage-based diets. Similarly, the present study found that methanogenesis is negatively correlated with forage quality. Additionally, in this study, the rumen microbiome reflected the nutritional environment where crude protein intake was 78% greater in the high-quality diet and Prevotella was strongly associated with the higher protein alfalfa grass hay diet, and Ruminococcus was strongly associated with the lower protein, high fiber mature wheatgrass hay. These results are consistent with the literature since numerous strains of Prevotella are associated with protein and polysaccharide digestion while members of the genus Ruminococcus are associated with cellulose and hemicellulose degradation (Griswold et al., 1999; Zhang et al., 2014; Wei et al., 2021). However, since only genus level taxonomic identification was obtained in our study, these functions cannot be definitively correlated to the presence of these genera. The addition of supplemental protein to protein deficient high fiber diets likely enhances the fiber digesting microbiota in the rumen, such as Ruminococcus (DelCurto et al., 2000) suggesting a need to provide supplemental protein to cattle grazing dormant senesced vegetation or consuming high fiber protein deficient diets (straw or mature hay). There was no response of the bacterial community to the trace mineral boluses, suggesting that trace minerals were not limiting in the rumen reticular environments for both alfalfa grass or mature wheatgrass hay-based diets.

Supplementation of diets for beef cows with different sustained release mineral bolus types did not result in any changes to the rumen microbiome composition or predicted function. In contrast, forage quality altered rumen bacterial community composition and predicted function. These findings suggest that livestock managers who use supplemental minerals to increase beef cattle performance in rangeland systems can do so with minimal impact to rumen bacterial community. However, these mineral supplements cannot substitute for low forage quality, which impact the rumen microbiome composition and function. Results from this research underscore the need to evaluate forage quality for potential impacts on rumen microbial community composition and function. Optimization of the rumen reticular environment can enhance microbial community function, resulting in more efficient use of forages and nutrients available to the host animal.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA945870.

The animal study was reviewed and approved by Montana State University Agricultural Animal Care and Use Committee (#2019-AA12).

JE: Investigation, Data curation, Visualization, Writing- original draft, Writing- reviewing & editing. SW: Conceptualization, Methodology, Data collection, Writing- reviewing & editing. TC: Methodology, Data collection, Writing- reviewing & editing. TD: Conceptualization, Methodology, Supervision, Resources, Writing- reviewing & editing. All authors contributed to the article and approved the submitted version.