Browsing by Subject "Faecal microbiome"

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Evaluation of fresh and preserved sheep faeces as an inoculum source in in vitro gas production assays
(2025) Rippstein, Lena; Rodehutscord, Markus
In order to meet the animals’ requirements of energy and nutrients, knowledge of the feed value of individual feed components is essential. In this context, information on ruminal degradability of feeds is crucial for formulating rations for ruminants. Information of this kind can be obtained using in vitro methods, such as the Hohenheim gas test (HGT). This method allows for the estimation of the organic matter digestibility and the energy value, as well as the protein value of ruminant feeds when applying the extended HGT (eHGT). In vitro methods provide a cost-efficient, rapid, and standardisable alternative to in situ and in vivo approaches, while contributing to reducing animal burden and the number of experimental animals. However, the HGT currently depends on using rumen fluid, which is commonly obtained from rumen-cannulated animals. Due to ethical concerns related to animal welfare and practical considerations, there is growing interest in replacing rumen-cannulated animals for the in vitro feed evaluation in the HGT and eHGT systems. However, the use of individual enzymes or enzyme mixtures for in vitro evaluation of feeds has not yet proven suitable for adequately representing the complex microbial activity of a rumen fluid inoculum (RI). In contrast, several findings in the literature indicate the potential of faeces as an alternative inoculum source to rumen fluid. However, to date, this approach has not been established in the routine analysis of ruminant feeds. The overarching aim of the present thesis was to systematically evaluate the suitability of sheep faeces as an alternative inoculum source to rumen fluid in the HGT. To this end, in vitro gas production (GP) was compared between faecal inoculum (FI) and RI. Additionally, the potential of using preserved faeces as an alternative inoculum source to fresh faeces, as well as the application of FI in the eHGT system, was evaluated. To compare faeces and rumen fluid as inoculum sources in the HGT and eHGT, RI was prepared according to the standard procedure using rumen fluid obtained from two rumen-cannulated lactating dairy cows and FI was prepared from rectally collected faeces of three adult wether sheep. The objective of Manuscript 1 was to examine whether FI and RI generally follow similar GP kinetics and to assess whether feed-specific variation could be observed. Furthermore, there was considerable interest in determining whether FI-GP and RI-GP are related to each other, as this could provide the basis for the future applicability of FI. A total of 90 currently relevant ruminant feeds from various categories and differing nutrient compositions were incubated in vitro with both FI and RI for 72 h, with multiple readings in the HGT. By using FI, lower GP kinetics were observed across all feed categories compared to RI. On average of all feeds, the potential GP was 9 mL/200 mg dry matter (DM) lower and the GP rate was 3.1%/h lower with FI than RI. Additionally, a lag phase of 1.51 h was estimated with FI, whereas no lag phase was observed for RI. The results indicate an overall lower fermentation activity of FI compared to RI. Despite these differences, the GP kinetic curves of the two inocula exhibited a very similar progression. Moreover, strong linear relationships were found between RI-GP at 24 h, the common incubation time of RI in the HGT, and FI-GP at both 24 h (Slope = 1.02, R² = 0.97) and 48 h (Slope = 1.1, R² = 0.97). Additionally, within the scope of this thesis, linear regression analyses were conducted based on a combined dataset from Manuscript 1 and previous studies. By using data from more than 400 different feeds in these analyses, the strong linear relationships between RI-GP at 24 h and FI-GP at 24 h (Slope = 0.98, R² = 0.93) as well as 48 h (Slope = 1.02, R² = 0.96) were confirmed. Dividing the dataset into the feed categories roughages and concentrates for the calculation of separate regression equations did not provide a clear advantage over using a single equation for all feeds. Despite the lower GP observed with FI, a consistent relationship was evident between the GP of both inocula across the different feeds, enabling a reliable estimation of RI-GP from FI-GP in the HGT. The use of preserved instead of fresh faeces would allow for a centralised housing of donor sheep, thereby reducing the number of animals required and improving standardisation. The aim of Manuscript 2 was to investigate the effect of differently preserved sheep faeces on the in vitro GP of nine different feeds and the microbiome in the HGT, intending to maintain a high level of microbial activity during incubations. Seven different freezing and freeze-drying treatments were applied. On average across all feeds, the potential GP of the frozen treatments (61 mL/200 mg DM) was comparable to that of the fresh faeces (62 mL/200 mg DM), whereas the freeze-dried treatments accounted for only 71-85% of the fresh faecal value. The results were confirmed by metaproteome analyses, as the microbiomes of the fresh and frozen treatments were significantly different from that of the freeze-dried treatments based on the relative abundance of the core proteins (p < 0.001). This demonstrated that stress factors associated with the freeze-drying process significantly impaired the microbiome, consequently affecting fermentation activity and GP. By contrast, the freezing process appeared more gentle on the microbiome, preserving a high microbial activity. Furthermore, strong relationships were found between RI-GP at 24 h and GP of the frozen treatments at 48 h of incubation (Slope = 1.27, R² = 0.96). Additionally, the effect of storage on freeze-dried and frozen treatments was investigated, revealing a considerable negative impact on GP and its relationship with RI-GP for both treatments. This limits the high potential for estimating RI-GP, which was particularly demonstrated with frozen faeces, and therefore requires further research. Manuscript 3 aimed to investigate the potential suitability of FI in the eHGT for estimating the protein value of ruminant feeds. The eHGT is used to estimate ruminally undegradable crude protein (RUP) and microbial crude protein. Ammonia-nitrogen (NH3-N) is a key parameter in this context, as it is released during microbial crude protein degradation and provides a nitrogen source for the microbes. FI and RI were therefore compared based on NH3-N and calculated microbially bound nitrogen (mN) following in vitro incubation of six different feeds for 8, 24, and 48 h. The NH3 N content was 17 and 23% lower with FI than with RI after 24 and 48 h, respectively. With RI, mN values decreased over the incubation time for most feeds, whereas with FI, mN initially increased before declining at later incubation times. This suggests that crude protein degradation and microbial binding of nitrogen occur more slowly and to a lesser extent with FI. However, both inocula demonstrated a comparable response to an additional energy source and showed strong linear relationships for NH3-N, particularly after 24 h (Slope = 1.39, R² = 0.98), indicating similar microbial mechanisms in faeces and rumen fluid. The RUP was also estimated for both inocula in this thesis, and the results showed an inconsistent ratio between FI and RI incubations across the six feeds. Similarly, an inconsistent ratio between the two inocula was observed for the mN data. Therefore, further studies involving a larger number of feeds, as well as the testing of mathematical approaches, are necessary to better evaluate the suitability of FI for estimating the protein value of ruminant feeds with the eHGT. In conclusion, the use of sheep faeces as an inoculum source for the in vitro analysis of ruminant feeds can be considered suitable for replacing rumen fluid and thus rumen-cannulated animals in the HGT. By reliably predicting the RI-GP from FI-GP, the predicted RI-GP can be used in the official and validated equations to estimate organic matter digestibility and metabolisable energy of ruminant feeds. The methodological approach applied in this thesis, including sheep feeding, faeces collection, and inoculum preparation, appeared appropriate in achieving a high and consistent microbial activity in the FI. Furthermore, a high potential was demonstrated for using preserved, particularly frozen, sheep faeces for application in the HGT, as well as the use of FI for estimating the protein value of feeds in the eHGT. However, further investigations are required for the two application fields to assess the suitability of FI comprehensively.