Most of the fiber content is from cellulose and lignin as ADF makes up 26.1% of the leave’s total DM.Lignin content of leaves used here was lower compared to Corbin et al. , but this may be due to the differences in the maturity level of the leaves, whereas ash content had similar levels between the two studies. Crude protein and crude fat were low in agave . This is not surprising and low crude protein and fat concentrations also provide the foundation for why agave is categorized as lower quality forage. However, total digestible nutrients values can be used to determine the energy provided by feedstuff which accounts for carbohydrates, lipids, and protein content . Agave leaves had a TDN index of 58.8 and ME at 0.94 Mcal/lb, which can potentially be considered as a forage replacement especially in areas that require higher water requirements. The availability of studies on using agave leaf as animal feed is scarce, especially when looking at specifically those of the Agave tequilana cultivar. However, in a study by Dias et al., used the more common Agave americana, which happens to be reported as invasive in certain countries, to repurpose them as animal feed and observe rumen fermentation. However, they reported that adding A. americana did not affect in vitro rumen fermentation, plastic square planter pots which agrees with our current work as total gas production did not differ between any of the treatment groups with our negative control.

However, it is noteworthy that there was a weak trend towards CO2 production when 10% of agave replaced traditional feed. Since CO2 acts as a major H2 sink in CH4 production, if available CO2 is reduced, it could potentially lead to lower CH4 production from methanogenesis. However, longer studies including microbiome profiling may be needed to better understand the long-term effects of agave leaves on fermentation patterns.Almond hulls have gained popularity as cheap feed alternative for high producing cattle as almond hulls are low in fiber and energy rich. This is reflected in our chemical analysis with almond hulls having 12.4% crude fiber and a TDN of 65.8%. Diets containing higher soluble sugar and starches with lower fiber contents produce less methane during fermentation compared to high fiber diets making almond hulls a promising candidate for reducing methane. Even though the control diet, a diet commonly used in the dairy industry, contained small amounts of almond hulls , we wanted to investigate if increasing the non-structural carbohydrate concentrations influenced enteric methane production. Consistent with other studies that substituted dairy cattle diets with almond hulls no changes in TGP, CH4, or CO2, were observed, suggesting that raw almond hulls have no methane mitigation effect. However, it needs to be noted that the purity of almond hulls and subsequentially the chemical composition and nutritional value of almond hulls can vary significantly between charges, processing procedures and from year to year .

The impact of additional components in feed additives, intentional or unintentional, was highlighted by the observation that the mixture of almond hulls and almond shells, a combination that is natural intermediate in the almond processing process, possessing a lower ME and higher fiber content than pure almond hulls. We also tested almond shells which are considered low quality feed due to their high fiber content and a low lower energy index . When mixed with almond hulls, the TDN of the mixture decreased by 7.5% compared to pure hulls. However, since almond shells also contain bio-active compounds such as anthraquinones, flavonoids, and phenolic compounds that may contain antimicrobial properties we hypothesized that although the addition of shells to the cattle diet might reduce the nutritional value of the supplemented diet, a potential reduction of enteric methane caused by these bio-actives might compensate for this. We expected to see a decrease in fermentation rate when shells were added into the diet alongside hulls since NDF contributed to almost 50% of the mixed diet and providing less ME. Interestingly, no significant differences in the gas parameters between the treatment groups containing the different almond byproducts was observed. This may be due to the limited duration of the in-vitro rumen fermentation potentially not allowing differences in microbial responses to manifest themselves sufficiently.Onion waste was comprised of different components of the plant, including outer skin, peels, bulbs, and roots. Chemical analysis revealed that the mixture contained high levels of fiber but still had high total nutrient digestibility . Onion peels have also shown to contain bio-active compounds, mainly in the form of quercetin accounting for up to 80% of their flavonoid content which have been shown to have antimicrobial activity by inhibiting the formation of biofilms in bacteria . In a study by Lubberding et al. onion waste tested in fermenters inoculated with rumen fluid and found that ~60% of onion biomass was degraded within 60 h. Quercetin availability in onion waste can vary from 14.7 to 21.8 mg/g dependent on type of onion and the type of extraction .

The onion waste tested in-vitro had minimal processing as it was only grinded down to mimic chewing from the cow to represent the quality of onion waste when received from producers, which could have possibly resulted in a lower concentration of quercetin exposed to microbes in the tested rumen fluid compared to other studies where onion peels are extracted using different methods to increase the available amount .We did not observe any significant differences in the gas production parameters when onion waste was added, which corresponds with results from Alabi et al. looking at the effects of onion waste on rumen fermentation. There was a tendency of lower methane production when 1% of onion waste was added, however, longer exposure to onion waste is required to understand its effect on fermentation.Nitrogen that is not utilized by rumen microbes for ammino acid synthesis is converted to ammonia and eventually secreted by the ruminant animal as urea . The reduction of available nitrogen through microbial protein can no longer be absorbed in the lower digestive tract resulting in an decrease of CH4 production . Due to their high protein content, sunflower meal has been widely used as a protein replacement for both growing animals and lactating cows and some researchers suggested that sunflower meal has the potential to reduce enteric CH4 production. Although Haro and colleagues were able to confirm this with processed sunflower meal in 2018 and 2020 in two independent studies, no changes in CH4 production was observed in this study. Since our sunflower meal was added without any being processed, which usually changes the chemical composition, to the feed, it is feasible that the differences in chemical composition between processed and unprocessed sunflower meal might cause the difference in methane production. When analyzing for second order of effects, sunflower meal appeared to have a critical inclusion rate between 10 and 20% , that may have potential to inhibit methanogenesis at 24 h of exposure. However, further studies with processed and unprocessed sunflower meal at higher inclusion rates and for longer fermentation times will be required.Grape pomace contains bio-active compounds, such as phenolics, that can have antimicrobial activities also affecting the rumen microbiome . Since grape pomace is usually high in moisture, which makes it prone to spoilage, prolonged storage is a challenge and, in most cases, not economical. To avoid contamination, spoilage and subsequently changes in their phenolic profile , square pots for planting freezing grape pomace was shown to trigger no differences in rumen fermentation , hence grape pomace used in this study was immediately transferred into -20C after it was collected. Besides phenolics, lignin is also a significant component of grape pomace which results in decreased digestibility when added at increased inclusion rates to the animal’s diet. From the samples we evaluated grape pomace from Viognier had the lowest concentration and grape pomace from Pinot Noir grapes had the highest concentration of lignin , suggesting that grape pomace from Pinot Noir would reduce digestibility and CH4 production due to its increased lignin content per se if added in sufficient amount.

However, no differences in gas production or methane production were observed for all varieties of grape pomace. This may have been caused by an insufficiently low inclusion rate or rumen fermentation time. Evidence from Moate et al., who fed different varieties of grape pomace to lactating dairy cows over 28 days at an inclusion rate of 33% and found a reduction in methane by 15% when compared to a basal ryegrass diet, suggest that the latter is the case and prolonged in vitro rumen fermentation will be needed before differences in gas and methane production manifest itself and can be detected. In the same study, it was found that the red grape pomace had considerably less condensed tannins than white grape pomace, however, there were no differences in fermentation between the two groups. Many factors can affect the fermentability of grape pomace such as the composition and different ratios of skin, seed and stem. California plays in important role in providing food at the national and global level, generating a diverse repertoire of plant-based products. In addition to this California is also a leader in dairy and beef production, providing a significant portion of animal protein to the US market. Rerouting plant-based byproducts away from the landfill and utilizing them as animal feed is a promising strategy to decrease waste from agriculture, while also making animal production more economical. If these byproducts have the additional benefit of reducing the emission of greenhouse gases such as CH4 from enteric fermentation, the rerouting and upgrading of byproducts also provides an attractive strategy to reduce to environmental footprint of the livestock industry. Over recent years there has been a great interest in identifying feed additives that might upgrade conventional cattle feed and that could provide additional avenues for methane mitigation. However, in many cases results from these studies have been inconsistent at best often even contradicting each other. This is not surprising, since these studies varied greatly in the experimental design and utilized compounds without generating detailed chemical profiles that would enable to identification of potential bio-actives that drive rumen methanogenesis or its inhibition. The work presented here provides detailed chemical analyses of some common agricultural byproducts generated in California and therefore a valuable foundation for subsequent more refined in vitro rumen fermentation studies. Results obtained from the work presented here also suggest that a prolonged in-vitro rumen fermentation time might be essential to detect any methane mitigation effect from compounds that contain bio-actives with lower potency than bromoform or the red seaweed Asparagopsis taxiformis that trigger methane mitigation to the extent that it can be reliably detected after 24 h of rumen incubation. Byproducts from plant-based agriculture remain a promising option as feed supplement and replacement with the potential to reduce enteric methane emission, but more refined in-vitro screening protocols and detailed biochemical information of the byproducts will be necessary before they can employ reliably and at large scale.The alteration of natural habitats by human activities is generally acknowledged to reduce the abundance and diversity of organisms . However, our understanding of the consequences of anthropogenic disturbance remains incomplete because most studies that address the effects of disturbance pool or average data over time, ignoring the fact that most biological assemblages exhibit seasonal turnovers in the identity and abundance of species. Given that seasonal variation in diversity and abundance plays an important role in the structure and function of communities , the effects of disturbance may be greatly underestimated without explicit consideration of such seasonal dynamics. To account for how seasonal dynamics influence the response of an assemblage to disturbance, one may separate the assemblage’s diversity into temporal gamma, alpha, and beta components in a manner similar to the partitioning of spatial diversity . When diversity is examined in this temporal framework, temporal gamma diversity pertains to data pooled across individual temporal samples from a given locality . As such, temporal gamma diversity is equivalent to “site level diversity,” one of the most commonly reported measures of diversity in assemblage and community-level studies. Temporal alpha diversity pertains to the finest temporal scale in which sampling is conducted , providing insight into diversity at discrete points in time and allowing for analyses of temporal trends within a study site.