Journal of Animal Science and Technology (JAST) is a peer-reviewed, open access journal publishing original research, review articles and notes in all fields of animal science. Topics covered by the journal include: genetics and breeding, physiology, nutrition of monogastric animals, nutrition of ruminants, animal products (milk, meat, eggs and their by-products) and their processing, grasslands and roughages, livestock environment, animal biotechnology, animal behavior and welfare.

This study aimed to characterize the non-starch polysaccharides (NSP) content of 23 macroalgae and to evaluate how algal polysaccharides are degraded in the rumen to be able to evaluate their potential as feed for dairy cattle. Selected macroalgae species were incubated for 48 h in an in vitro system simulating rumen fermentation. The raw macroalgae and undegraded macroalgae residue (UDMR) were analyzed for NSP content. Chemometric analysis was performed on the data sets using principal component analysis. The UDMR clustered according to either their degradability or content in undegraded sugars rather than their phylogenetic group. The highest degradability was observed in Palmaria palmata with 78.1 % dry matter degradability which can be attributed to its high content in xylans. Macroalgae with lower degradability had higher contents of insoluble galactose, rhamnose and glucose, all present in less degradable algal polysaccharides such as kappa-carrageenans and ulvans. In conclusion, algal polysaccharides determined with the NSP method were in accordance with the phylogenetic group and the literature which validates the use of the NSP method for macroalgae. P. palmata would be of interest as an ingredient for ruminant nutrition as well as other macroalgae with the help of exogenous enzymes to increase their degradability.

There is a need for rigorous and scientifically-based testing standards for existing and new enteric methane mitigation technologies, including antimethanogenic feed additives (AMFA). The current review provides guidelines for conducting and analyzing data from experiments with ruminants intended to test the antimethanogenic and production effects of feed additives. Recommendations include study design and statistical analysis of the data, dietary effects, associative effect of AMFA with other mitigation strategies, appropriate methods for measuring methane emissions, production and physiological responses to AMFA, and their effects on animal health and product quality. Animal experiments should be planned based on clear hypotheses, and experimental designs must be chosen to best answer the scientific questions asked, with pre-experimental power analysis and robust post-experimental statistical analyses being important requisites. Long-term studies for evaluating AMFA are currently lacking and are highly needed. Experimental conditions should be representative of the production system of interest, so results and conclusions are applicable and practical. Methane-mitigating effects of AMFA may be combined with other mitigation strategies to explore additivity and synergism, as well as trade-offs, including relevant manure emissions, and these need to be studied in appropriately designed experiments. Methane emissions can be successfully measured, and efficacy of AMFA determined, using respiration chambers, the sulfur hexafluoride method, and the GreenFeed system. Other techniques, such as hood and face masks, can also be used in short-term studies, ensuring they do not significantly affect feed intake, feeding behavior, and animal production. For the success of an AMFA, it is critically important that representative animal production data are collected, analyzed, and reported. In addition, evaluating the effects of AMFA on nutrient digestibility, animal physiology, animal health and reproduction, product quality, and how AMFA interact with nutrient composition of the diet is necessary and should be conducted at various stages of the evaluation process. The authors emphasize that enteric methane mitigation claims should not be made until the efficacy of AMFA is confirmed in animal studies designed and conducted considering the guidelines provided herein.

Condensed tannins (CT) are plant polyphenols that can affect feed digestibility and are potentially able to reduce enteric methane emissions in ruminants. In this in vivo trial with 8 lactating goats, we investigated the effects of 4 levels of inclusion of a commercial CT extract from quebracho (0, 2, 4, 6% on DM basis; C, Q2, Q4, Q6, respectively). The experimental design was a repeated 4 × 4 Latin square with 28-d periods (24 d of diet adaptation and 4 d of sample collection) using metabolic cages and 4 open circuit respiration chambers. The inclusion of CT in the diets did not affect the dry matter intake (DMI) but caused a linear decrease in diet digestibility, with reductions up to −11% for dry matter (DM), −21% for crude protein (CP), −23% for neutral detergent fiber (aNDFom), and −13% for gross energy, when comparing the Q6 and C diets. However, ruminal total volatile fatty acids (VFA) concentration was not affected by CT, although there were changes in VFA proportions. Milk yield (g/d) was highest for Q4 (3371) and lowest for Q6 (3066). In terms of milk composition, CT induced a linear reduction of fat and CP concentrations. The reduction in CP digestibility resulted in a linear reduction in the milk urea level, up to −37% with Q6. Positively, CT linearly reduced the somatic cells count expressed as linear score. The feed efficiency was linearly decreased by CT inclusion. Furthermore, a shift from urinary to fecal nitrogen excretion was observed with CT. The retained nitrogen was always negative (on average −1.93 g/d). The methane yield (on average 19.2 g CH4/kg DMI) was linearly reduced by CT inclusion, up to −18% with Q6. Regarding the CH4 intensity, CT induced a linear reduction when expressed per kg of milk, but not per kg of fat and protein corrected milk. Moreover, the CH4 production per kg of digestible aNDFom was linearly increased by CT. The metabolizable energy intake (MEI) was not affected by the treatments, but the metabolizability (q = MEI/gross energy intake) was reduced as CT inclusion increased. From the results of the present study, it turned out that CT have a negative impact on feed digestibility and feed use efficiency. Condensed tannins can lower CH4 emissions from ruminants; however, the main mechanism of action is likely the decrease in feed digestibility. Furthermore, CT did not improve the N use efficiency. According to these findings, the positive environmental impacts of CT are only related to the shift from urinary to fecal N excretion.

The administration of Bacilli to dairy cows exerts beneficial effects on dry matter intake, lactation performance, and milk composition, but the rationale behind their efficacy is still poorly understood. In this work, we sought to establish whether cellulases and xylanases, among the enzymes secreted by B. subtilis, are involved in the positive effect exerted by Bacilli on ruminal performance. We took advantage of two isogenic B. subtilis strains, only differing in the secretion levels of those two enzymes. A multi-factorial study was conducted in which eight feed ingredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two strains conveniently grown in a growth medium based on inexpensive waste. Feed degradability and gas production were assessed. Fiber degradability was 10% higher (p < 0.001) in feeds treated with the enzyme-overexpressing strain than in the untreated control, while the non-overexpressing strain provided a 5% increase. The benefit of the fibrolytic enzymes was maximal for maize silage, the most recalcitrant feed. Gas production also correlated with the amount of enzymes applied (p < 0.05). Our results revealed that B. subtilis cellulases and xylanases effectively contribute to improving forage quality, justifying the use of Bacilli as direct-fed microbials to increase animal productivity.

The 2 brown seaweeds, Fucus serratus and Fucus vesiculosus, have demonstrated anti-methanogenic properties in vitro with reductions in CH4 production ranging from 53 to 63%. This study aimed to investigate the effects of the 2 Fucus seaweeds on enteric CH4 emissions, DMI, ECM, and nutrient digestibility when fed to dairy cows. The experiment was conducted using 4 multi-cannulated lactating Danish Holstein dairy cows, which over 3 experimental periods received either: 1) basal diet (CON; diet without any seaweed), 2) basal diet diluted with 4% (DM basis) Fucus serratus (SER), or 3) basal diet diluted with 4% (DM basis) Fucus vesiculosus (VES); resulting in one complete 3 × 3 Latin square and one incomplete 3 × 3 Latin square. Each period lasted 21 d and consisted of 14 d of adaptation, followed by 3 d of digesta sampling, and 4 d of gas exchange measurements using respiration chambers. Milk yield and feed intake were recorded daily. Blood samples were collected on d 15 and 17. All parameters were statistically analyzed using a mixed procedure of R. Opposite to what we had expected, neither of the 2 Fucus seaweeds reduced CH4 emissions from the dairy cows as daily CH4 production was significantly higher for both Fucus treatments compared with CON. Additionally, CH4 yield (g CH4/kg DMI) and intensity (g CH4/kg ECM) were significantly higher for SER compared with CON. Milk yield, DMI, and total-tract digestibility were unaffected by the treatments; however, SER resulted in lower milk protein yield (kg/d) and lower milk and blood plasma urea concentrations compared with CON. In conclusion, neither Fucus serratus (SER) nor Fucus vesiculosus (VES) showed potential as methane-mitigating feed additives when fed to dairy cows at an inclusion level of 4% of DM. The inclusion of the 2 brown seaweeds had no effects on DMI, milk yield, or total-tract digestibility.

AbstractA large body of literature has accumulated on the beneficial impact of the administration ofBacillito dairy cows, particularly on dry matter intake, lactation performances and milk composition. In this work we sought to establish whether the ability ofB. subtilisto secrete cellulase and xylanase enzymes could be involved in the positive effect exerted by these bacteria.Several feed ingredients were treated with twoB. subtilisstrains only differing for the amount of secreted cellulosolytic and xylanolytic enzymes, and feed quality was assessed. We found thatin vitrofibre digestibility correlated with the activity of those enzymes. Our results revealed thatB. subtiliscellulases and xylanases can effectively improve forage quality, providing a scientific rationale on the use ofBacillias forage supplements to improve animal productivity. Moreover, a particular care was taken in designing a sustainable and economically viableBacillus-based additive preparation process.

The administration of Bacilli to dairy cows exerts a beneficial effect on dry matter intake, lactation performances and milk composition. In this work, two isogenic strains, only differing in the se-cretion level of cellulolytic and xylanolytic enzymes, were compared to establish whether B. subtilis cellulase and xylanase enzymes are involved in the positive effect exerted by these bacteria on animal nutrition. Conveniently, bacteria were grown in a newly designed growth medium based on an inexpensive waste. A multi-factorial study was conducted in which eight feed in-gredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two B. subtilis strains. Feed digestibility and gas production, a parameter linked to feed fermentability, were assessed. Fiber degradability was significantly higher in feeds treated with the en-zyme-overexpressing strain (10% and 5% higher than control treatment with B. subtilis-free media and the parent strain, respectively). This effect was accentuated for the most recalcitrant feeds, in which the benefits of cellulolytic and xylanolytic enzymes were maximized. Fermentability also benefited by the treatment with the enzyme-overexpressing strain, although the trend observed in gas production didn’t reach statistical difference between the two strains, and only minor changes in fermentation profiles were detected. Our results revealed that B. subtilis cellulases and xylanases effectively contribute to improving forage quality, supporting the use of Bacilli as fodder sup-plements to increase animal productivity.

Plant secondary metabolites (PSMs) can potentially reduce ruminal methane formation. However, related to differences in their molecular structures, it is not yet clear what causes an anti-methanogenic effect. In an in vitro system simulating rumen fermentation, we investigated the impact of eight compounds with distinct chemical characteristics (gallic and salicylic acids, tannic acid, catechin, epicatechin, quercetin, rutin, and salicin) when added to a basal feed (maize silage) at a concentration of 12% of the feed dry matter. After 48 h of incubation in buffered rumen fluid, methane production was significantly lowered by quercetin (43%), tannic acid (39%) and salicylic acid (34%) compared to the control (maize silage alone) and without changes in total volatile fatty acid production during fermentation. No other PSM reduced methane formation as compared to control but induced significant differences on total volatile fatty acid production. The observed differences were related to lipophilicity, the presence of double bond and carbonyl group, sugar moieties, and polymerization of the compounds. Our results indicate the importance of distinct molecular structures of PSMs and chemical characteristics for methane lowering properties and volatile fatty acid formation. Further systematic screening studies to establish the structure-function relationship between PSMs and methane reduction are warranted.

Gastrointestinal nematodes (GINs) is a limiting health factor for dairy goats, and the integration of the diet with fodder containing condensed tannins (CT) is becoming increasingly important to control GINs. To preliminary evaluate their potential role as part of GIN control in goat breeding, the in vitro anthelmintic efficacy of the CTs of Silvafeed BYPRO (SBP), Silvafeed Q powder (SQ), and sainfoin hay (SH) was evaluated, and the untargeted metabolomics profiling of the selected formulations was performed. CTs were extracted in water and in ethanol, their concentration was determined, and their chemical characterization was conducted using High-Performance Liquid Chromatography (HPLC) coupled to High-Resolution Mass Spectrometry (HRMS) platform. The in vitro anthelmintic activity of the extracts was evaluated using the Eggs Hatch Test (EHT) and the Larval Migration Inhibition Test (LMIT) using different extract concentrations (150, 300, 600, and 1200 μg/mL). The metabolomic profile of the ethanol extract showed a high number of flavonoids, while the water extract showed higher levels of hydrolysable tannins. The ethanol extracts were effective on both eggs hatching and larvae migration at low concentrations (150 μg/mL) for the three analyzed samples, while the water extracts showed more varied results: SH showed the greatest ovicidal efficacy (concentration 150 μg/mL, %IH = 40.9), while SBP and SQ were more effective against the larvae migration (concentration 600 μg/mL, %LMI = 69.7% and 88%), respectively. The integration of CT-rich fodder into the diet may be considered for the control of GIN infection in goats.

Ruminants contribute to global warming by emitting greenhouse gasses, particularly methane (CH4) which is a product of rumen fermentation. The use of feed additives able to modulate rumen fermentation is a promising strategy to reduce enteric CH4 and ammonia (NH3) emissions. Among the various strategies investigated, plant secondary metabolites (PSMs) have attracted attention due to their apparent potential to reduce enteric CH4 and NH3 emissions, and it would be possible to use such compounds as feed additives in organic production systems. In an in vitro system simulating rumen fermentation, we have tested the impact of different classes of naturally occurring PSMs; catechin and quercetin (flavonoids), salicylic acid (phenolic acid) and tannic acid (hydrolysable tannin). The PSMs were added to two different basal feeds (maize and grass silages) at three inclusion doses 1.5, 3 and 6% of the feed dry matter (DM). CH4 production was significantly lowered upon addition of quercetin to two basal feeds at doses of 3 and 6%, and this without changes in concentrations of total volatile fatty acid (VFA) produced during fermentation. Quercetin, as the only tested additive, reduced CH4 production, and when added to maize silage and grass silage, the reduction increased linearly with increasing dose, ie., by 51 and 43%, respectively, at a dose of 3% of feed DM and by 86 and 58%, respectively, at a dose of 6% of feed DM. Moreover, quercetin significantly reduced NH3 concentration by >12% at doses of 3 and 6% in feed DM irrespective of the basal feed used as compared to when the basal feeds were incubated alone. Although none of the other additives affected CH4 formation, several additives had significant impacts on concentrations of NH3 and VFAs in the incubated fluid after fermentation. This study demonstrated a dose-dependent ability of quercetin to reduce CH4 emission from rumen fermentation, however, the magnitude of the suppression of CH4 depended on the basal feed. Furthermore, quercetin reduced NH3 concentration irrespective of the basal feed type. These findings encourage to in vivo studies to verify whether quercetin can reduce CH4 emission also in cows.

A large body of literature has accumulated on the beneficial impact of the administration of Bacilli to dairy cows, particularly on dry matter intake, lactation performances and milk composition. In this work we sought to establish whether the ability of B. subtilis to secrete cellulase and xylanase enzymes could be involved in the positive effect exerted by these bacteria.Several feed ingredients were treated with two B. subtilis strains only differing for the amount of secreted cellulosolytic and xylanolytic enzymes, and feed quality was assessed. We found that in vitro fibre digestibility correlated with the activity of those enzymes. Our results revealed that B. subtilis cellulases and xylanases can effectively improve forage quality, providing a scientific rationale on the use of Bacilli as forage supplements to improve animal productivity. Moreover, a particular care was taken in designing a sustainable and economically viable Bacillus -based additive preparation process.### Competing Interest StatementThe authors have declared no competing interest.

Abstract Condensed tannins may reduce enteric methane emissions in dairy goats by acting on feed digestibility and promoting microbial community composition shifts. We assessed the impact on the ruminal and fecal microbiota of a dietary integration with condensed quebracho tannins (CT) at three different levels (2%, Q2; 4%, Q4; 6% on dry matter, Q6) compared with a control diet (C), in a repeated 4 × 4 Latin square feeding scheme with four goats per square. The ruminal and fecal bacterial, archaeal, fungal, and protozoan microbiota were assessed at the end of each feeding period. The results were then evaluated in relation to CH4 and short chain fatty acid (SCFA) production, nitrogen excretion, and feed efficiency. In the rumen, a reduction in prokaryotic alpha-diversity was observed. Beta-diversity was also significant, with the strongest impact for the Q6 diet. Increasing CT concentrations induced more significant community shifts. The phylum Bacteroidota were enriched by all three CT levels, leading to inversion of the Bacteroidota/Firmicutes ratio. Methanobrevibacter with the corresponding phylum (Euryarcheota) and family (Methanobacteriaceae) were reduced by Q6. Ruminococcaceae, Synergistaceae and Flexilinea and Fretibacterium decreased, while Prevotellaceae, Acidaminococcaceae, Succiniclasticum and Fibrobacter increased. The diet did change the overall fungal biodiversity of the rumen, dominated by Neocallimastix, Feramyces, and Caecomyces. In the protozoal microbiota, dominated by Entodinium, only Diplodinium decreased, while Polyplastron and Isotrichia increased. In the feces, alpha and beta-diversity did not change significantly. In feces, the genus Methanobrevibacter and the corresponding phylum and family (Euryarcheota and Methanobacteriaceae, respectively) were increased by CT. The Q6 diet reduced fecal Arcanobacterium, Anaerococcus, and Megasphaera, while it enriched Alistipes and Corynebacterium. Malassezia dominated the fecal fungal microbiota with an average relative abundance of over 95%. Significant changes were observed in the feces for different fungal genera. In conclusion, increasing levels of CT in the diet induced increasingly relevant changes in the rumen microbiota of goats, including a reduction in the abundance of microbial taxa responsible for methane production. On the other hand, CT affected feed conversion efficiency, and this should be taken into account as a contributing factor in the total reduction of methane emissions.

Both condensed and hydrolysable tannins (CTs and HTs, respectively) have the ability to reduce enteric CH4 production in ruminants. However, the precise mechanism of action is not fully understood. Among the proposed hypotheses are the reduction of ruminal digestibility, direct control action on protozoa, reduction of archaea, and a hydrogen sink mechanism. In this in vitro study, which simulated rumen fermentation, two additives, one containing CTs (70% based on DM) from quebracho and one with HTs (75% based on DM) from chestnut, at four levels of inclusion (2, 4, 6, 8% on an as-fed basis) were added to the fermentation substrate and tested against a negative control. Both types of tannins significantly reduced total gas (GP) and CH4 (ml/g DM) production during the 48 h of incubation. The lower GP and CH4 production levels were linked to the reduction in dry matter digestibility caused by CTs and HTs. Conversely, no significant differences were observed for the protozoan and archaeal populations, suggesting a low direct effect of tannins on these rumen microorganisms in vitro. However, both types of tannins had negative correlations for the families Bacteroidales_BS11 and F082 and positive correlations for the genera Prevotella and Succinivibrio. Regarding the fermentation parameters, no differences were observed for pH and total volatile fatty acid production, while both CTs and HTs linearly reduced the NH3 content. CTs from quebracho were more effective in reducing CH4 production than HTs from chestnut. However, for both types of tannins, the reduction in CH4 production was always associated with a lower digestibility without any changes in archaea or protozoa. Due to the high variability of tannins, further studies investigating the chemical structure of the compounds and their mechanisms of action are needed to understand the different results reported in the literature.

Enteric methane (CH4) from cattle constitutes a large proportion of the emission of anthropogenic greenhouse gasses. The objectives of this study were to investigate interspecies and seasonal variations in macroalgae, procured from the Northern Hemisphere, on CH4 production and rumen feed degradability in vitro. Corn silage or grass/clover silage (0.5 g), were incubated separately in buffered rumen fluid with addition of one of 22 different macroalgae (0.1 g), including both red, green, and brown species, hereof four brown species harvested at different seasons, resulting in an inclusion rate of 17 %. Rumen degradability of the pure macroalgae was determined by incubating macroalgae (0.5 g) without any basal feed. Accumulated gas production during 48 h of incubation was determined using the AnkomRF system equipped with airtight gasbags. Basal feeds without macroalgae addition served as controls, while the red macroalgae Asparagopsis taxiformis was included as a methane-inhibiting control. Each sample type was tested in triplicate in two separate runs. Amount of undegraded dry matter (DM) and organic matter (OM) after incubation was determined by filtration. Gas composition and concentration of volatile fatty acids (VFA) were determined by gas chromatography. Both A. taxiformis and Fucus serratus significantly reduced CH4 production, expressed per g DM, OM, degraded DM, and OM, when added to corn silage. Similar CH4 mitigating effect was observed when A. taxiformis was added to grass/clover silage, while F. serratus only significantly reduced the CH4 production expressed per g DM. None of the species included in the investigation of seasonal variations decreased CH4 production compared to the basal feeds. Palmaria palmata had the highest rumen degradability and associated VFA production, while F. vesiculosus and F. serratus had the numerically lowest rumen degradability and VFA production, respectively. In conclusion, F. serratus was the only Northern procurable macroalga showing CH4 mitigating potential, which depended on the nature of the basal feed.

The high reliance of the European livestock sector on imported soybean meal (SBM), especially from South America, poses environmental problems, like greenhouse gas emissions for transportation and land-use change with the loss of carbon stock and biodiversity. Aim of the present study was to evaluate the partial substitution of SBM with whole-plant soybean silage in the diet of dairy cows. Thirty-six lactating Holstein cows were arranged according to a change-over design, with 2 weeks of adaptation and 5 days of sampling per period. A control diet (CON) was based on maize silage and SBM, representing 10.7% of total dry matter (DM). In a soybean silage diet (SBS) 35% (on DM basis) of SBM was replaced by soybean silage. The dietary treatment did not affect DM intake, milk production, and dairy efficiency while cows fed SBS resulted in lower milk crude protein (3.43 vs. 3.55%, p < .001) and higher milk urea (30.5 vs. 28.7 mg/dL, p = .002), in comparison with CON. Nutrients digestibility was lower for SBS than CON; particularly fibre digestibility was 31.5 vs. 38.8% (p < .001). The efficiency of nitrogen utilisation was higher for CON than SBS (32.7 vs. 31.3%, p = .003). Soybean silage did not penalise feed intake and milk production. However, to fully exploit this forage, digestibility, and nitrogen utilisation efficiency should be improved.Highlights Soybean silage can substitute one-third of soybean meal in dairy cow diet Soybean silage inclusion in the diet did not affect milk yield and DMI Soybean silage inclusion in the diet reduced N use efficiency

The weaning period is a stressful period for the gastrointestinal tract (GIT) of piglets. This work aims to evaluate the effects of the commercial polyphenol-based product GreenFIS® on: (1) GIT health and performance of 60 weaned piglets; (2) digestibility in 18 growing pigs. Three diets were tested: a control diet (C), C plus 2.5 g of GreenFIS®/kg C (T1), and C plus 5 g of GreenFIS®/kg C (T2). After the post-weaning trial three piglets per treatment were sacrificed for the GIT histological analysis. No differences between diets were recorded in terms of growing performance or clinical and biochemical blood parameters. The GIT histological analysis did not show any indicators of inflammation for any of the groups. The feces of the two extreme treatments (C and T2) were analyzed for microbiota, revealing a greater presence of the Ruminococcus bromii group, positively associated with starch degradation, in T2. In the second experiment six pigs per treatment were randomly chosen for the digestibility study. The inclusion of GreenFIS® at both levels led to a higher fecal digestibility of gross energy (86.2%, 89.1%, and 89.5%, for C, T1, and T2, respectively) and crude protein (87.0%, 90.2%, and 90.0%). In conclusion, the additive did not improve, in the excellent experimental hygienic conditions, the gut health, but it did increase nutrient digestibility.

The effect of whole linseeds or hemp seeds on milk production, energy and nitrogen balance, and methane emission was studied in 12 Alpine goats using respiration chambers. Diets tested were a control diet (C) and two diets supplemented with whole linseeds (L) or hemp seeds (H) at 9.3% on a dry matter (DM) basis. DM intake was similar among treatments, whereas DM and organic matter digestibility were lower for L compared to C. Milk yield (2.30 kg/d on average) and rumen fermentation profile were not affected by treatments. Treatment also did not affect the milk composition, with the exception of fat, which was higher in H and L compared to C (4.21, 3.94, and 3.20%, respectively). Oilseed supplementation caused a reduction in the concentration of de novo fatty acids (FA) (41.1, 48.8, and 64.1% of FA, for L, H, and C, respectively). Moreover, L and H diets reduced the sum of saturated FA, and increased monounsaturated FA, whereas only the L diet increased the concentration of polyunsaturated FA. Regarding methane production, and nitrogen and energy balances, no differences were registered among the diets. Our research indicates that including whole linseeds and hemp seeds in the dairy goat diet is an effective strategy for increasing milk fat content and positively modifying the milk FA composition, without a change in nitrogen and energy balances, but also without a reduction in enteric methane emission.

Undigested neutral detergent fibre (uNDF) is commonly used as an internal marker for the estimation of diet digestibility in ruminants. This work aimed to verify (i) whether the in vivo method with uNDF could be used to evaluate diet digestibility in growing pigs, and (ii) whether pre-treating the samples with neutral detergent solution (NDS) and α-amylase improves the accuracy of the estimates. Samples from a previously published work of two diets with known in vivo digestibility values estimated by the total faecal collection method and 16 individual samples of faeces were used. For each sample, four Ankom F57 bags were weighed. Before the incubation, two F57 bags were pre-treated with NDS and α-amylase. All the samples were incubated for 240 h in the Ankom DaisyII incubator and then analysed for their uNDF contents. Dry matter, organic matter, and neutral detergent fibre digestibilities were estimated using the uNDF contents, and the results were compared with those of the former study. The digestibility values obtained using the uNDF method with pre-treatment were not statistically different from those determined with the total faecal collection. On the contrary, the uNDF method without the pre-treatment could not satisfactorily predict the digestibilities of pig diets.