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0.05).Treating silages with AF supernatant reduced the pH by around 0.3 units (P=0.04). Besides, pH in grass silages was on a higher level than in straw silages (P<0.01), i .e.,4.72 vs. 3.87, respectively. The straw silages treated with AF supernatant had less acetic acid compared to its control,whereas acetic acid concentrations in grass silages were sim-ilar (P<0.01). Additionally, a substrate effect was observed (P<0.01), revealing that acetic acid was lower abundant in grass silages than in straw silages. Similarly, propionic acid concentration was also lower in grass silages than in straw s i lages (P=0.03). Regarding ethanol, a substrate effect was observed with higher concentrations in straw than in grass silages (P=0.04), whereas AF supernatant or its interac-tion with substrate had no impact. The ammonia proportion was generally i ncreased by the addition of AF supernatant (P<0.01) with higher values in S _AF than S _CON _AF (P=0.03). Besides, concentrations of ammonia were lower in grass silages than i n straw silages (P<0.01). No effects of AF supernatant treatment, substrate or their interaction were found for concentrations of ADL, WSC, lactic acid,and butyric acid (each P>0.05). Both the nutritional composition and fermentation pattern of grass and straw silages t reated with mixed ruminal fluid are presented in Table 3. Regarding the nutritional com-position, mixed ruminal fluid reduced the ADL concentra-tion compared to control silages, i.e., 64.4 vs. 118 g/kg DM (P<0.01), and tended to increase the ADFom concentration in silages (P=0.08). Moreover, the substrate affected several nutritional composition variables with higher concentrations of DM, ash, crude protein, and ether extract in grass silages compared to straw silages (each P<0.01), whereas al l f iber Table 3 Effect of mixed ruminal fluid on nutr i tional compos i tion, and fermentation pattern of silages Grass silage Straw silage SEM P-values CON_RF RF2 CON_RF RF Treatment Substrate Interaction DM'concentration, g/kg 409 386 342 348 0.80 0.31 <0.01 0.13 Ash, g/kg DM 105 111 68.1 63.6 2.84 0.78 <0.01 0.11 Crude protein, g/kg DM 187 190 36.1 45.4 3.56 0.14 <0.01 0.35 Ether extract, g/kg DM 26.7 28.7 13.0 10.8 1.38 0.94 <0.01 0.18 aNDFom", g/kg DM 553 576 832 842 8.78 0.10 <0.01 0.50 ADFom', g/kg DM 315 326 500 512 5.70 0.08 <0.01 0.94 ADL°, g/kg DM 93.1 47.3 142 81.4 6.70 <0.01 <0.01 0.30 WSC,g/kg DM 20.6 28.3 52.4 19.6 18.1 0.49 0.52 0.29 pH 4.60aB 4.29 4.19A 4.25 0.05 0.04 <0.01 0.01 Lactic acid, g/kg DM 22.7 84.87 8.85 46.0 11.7 0.01 0.07 0.30 Acetic acid, g/kg DM 16.2 29.6* 21.7 20.0 2.91 0.09 0.48 0.05 Butyric acid, g/kg DM 0.00 0.00 8.69 4.93 0.41 0.65 0.11 0.59 Propionic acid, g/kg DM 0.17 0.97B 1.27 4.13aA 0.35 <0.01 <0.01 0.03 Ethanol, g/kg DM 2.55B 4.60 8.44A 5.83 0.76 0.71 0.01 0.03 Ammonia, g/kg N 47.5 53.0 77.7b 104.8 4.59 0.01 <0.01 0.04 In each row, superscript capitalized letters indicate difference (P<0.05) between substrates within each treatment , i.e., CON _AF and AF, and superscript lowercase letters indicate di f ference (P <0.05) between t reatments wi t hin each substrate, i.e., straw and grass Silage prepared with 18 mL of heat-inactivated mixed ruminal fluid Silage prepared with 18 mL of freshly col l ected mixed ruminal fluid Dry matter “Neutral detergent f iber assayed with a heat stable o-amylase and expressed exclusive of residual ash Acid detergen t f i ber expressed exclusive of residual ash 6Acid detergent lignin Water-soluble carbohydrates fractions were higher in straw silages than in grass silages (each P<0.01). Regarding the silage fermentation pattern,mixed ruminal fluid lowered the pH in grass silages, whereas no differences were present within straw silages, but pH of S _CON _RF was lower than of G _CON _RF(P=0.01).Silage pH was also affected by main effects of mixed rumi-nal fluid treatment and substrate with higher pH in control silages (P=0.04) and grass si l ages (P<0.01), respectively.Similar to pH results , the acetic acid concentration in mixed ruminal f luid-treated grass silages was higher than in con-trol grass silages but not differing between straw silages (P=0.05). Contrastingly, the ammonia concentration was higher i n S _RF compared to S _CON _RF with similar values in grass silages (P=0.04). Besides, main effects of mixed ruminal fluid treatment and substrate revealed generally lower ammonia proportions in control silages (P=0.01)and grass silages (P <0.01), respectively. The addition of mixed ruminal fluid increased the lactic acid concentra-tion in both grass and straw silages (P=0.01). Moreover,grass silages tended to have more lactic acid compared to straw s i lages (P=0.07). The propionic acid concentration was higher i n mixed ruminal fluid-treated straw silages than control straw silages (P=0.03); plus within mixed ruminal fluid-treated silages, straw silage contained more propionic acid than grass silage (P=0.03). Besides, main effects of mixed ruminal fluid treatment and substrate were observed with higher propionic acid values for mixed ruminal fluid treatment than control (P<0.01) and straw silages than grass silages (P<0.01), respectively. The ethanol concentration was affected by the interaction of mixed ruminal f luid and substrate (P=0.03) with higher values in control straw silage than in control grass silage. Besides, a main substrate effect was observed for this variable and straw silages con-tained more ethanol than grass silages (P=0.01). Effects of mixed ruminal f luid treatment, substrate, or their interaction were not f ound for either WSC or butyric acid concentration (each P>0.05). In situ degradability The results of effective ruminal degradability regarding the use of AF supernatant as a silage additive are presented in Figs. 1 and 2; the respective data for constants a, b, and c and lag phase can be obtained from Table 4. The DM degradabil-ity was not different between G _CON _AF,G _AF, and fresh grass (Fig . 1A), whereas an interaction of substrate and AF supernatant treatment for DM degradabi l ity was observed (P<0.01) with higher values for S _CON _AF than S _AF and fresh straw (Fig. 2A ). Besides, also substrate affected DM degradability with higher values for grass than for straw (P<0.01). No main effect on DM degradability was observed for AF supernatant (P=0.55). An interaction of substrate and AF supernatant treatment was also present for A 60 50 20 10 0 F R E S H CON _A F A F Fig. 1 Ef f ective ruminal degradabi l ity (4%/h passage rate) of dry matter (A), neutral detergent fiber (B), and acid detergent fiber (C)of original fresh grass (FRESH) or grass silages prepared with heat-inactivated (CON _AF) or freshly prepared anaerobic fungi superna-tant (AF). Asterisk=P <0.05 in Tukey -Krame r post hoc test the aNDFom degradability (P<0.01) with lower values for S _AF than S _CON _AF and fresh straw (Fig. 2B), whereas for grass, aNDFom degradabil i ty was lower for G _CON _AF than G _AF and f resh grass (Fig. 1B). Moreover, main effects of substrate (P<0.01) and AF supernatant (P<0.01) were significant for ruminal aNDFom degradability. Regarding A 米 * 50 0 FRE SH CON _A F AF Fig.2 Effective ruminal degradability (4%/h passage rate) of dry matter (A), neutral detergent fiber (B), and acid detergent f i ber (C)of original f resh straw (FRESH) or straw silages prepared with heat-inactivated (CON _AF) or f reshly prepared anaerobic fungi superna-tant (AF). Asterisk=P <0.05 in Tukey-Kramer post hoc test the degradability of ADFom, interaction of substrate and AF supernatant was significant (P=0.01) with higher ADFom degradabi l ity for G _AF than for fresh grass with G _CON _AF being i ntermediate (Fig. 1C), whereas ADFom degradabil i ty of S _CON _AF was higher when compared to S _AF and fresh straw (Fig. 2C ). As found for aNDFom degradability, main effects of substrate (P<0.01) and AF supernatant (P=0.02) were also significant for ADFom degradability. The results of effective ruminal degradabil i ty regard-ing the use of mixed ruminal f luid as a silage inoculant are shown in Figs. 3 and 4; the respective data for constants a, b,and c and lag phase can be obtained from Table 5. Hereby,the effective ruminal DM degradabil i ty was influenced by substrate (P<0.01) with generally higher values for grass than for straw. The mixed ruminal fluid treatment (P=0.28)as well as the interaction of substrate and mixed ruminal fluid (P=0.45) showed no differences in DM degradability between incubated feedstuffs (Figs. 3A and 4A ). For aND-Fom degradabil i ty, a trend for the interaction of substrate and mixed ruminal f luid was found (P=0.08) with higher values for fresh grass compared to G _CON _RF and G _RF (Fig. 3B), but similar values for fresh straw, S _CON _RF,and S _RF (Fig. 4B ). Besides, substrate (P<0.01) and mixed rumina l fluid (P<0.01) affected the aNDFom degradabil-ity. As found for DM degradabi l ity, ADFom degradability was i nfluenced by substrate (P<0.01) with generally higher values for grass than for straw, whereas neither the mixed ruminal fluid treatment (P=0.41) nor its interaction with substrate (P=0.45) impacted the ADF degradability of incu-bated feedstuffs (Figs. 3C and 4C ). Discussion The present study investigated the impact of AF supernatant with active fungal enzymes on composition and fermenta-tion quality of grass and straw silages, i .e., a typical and a recalci t rant f iber-rich forage, and determined thei r ruminal DM and fiber degradability. We hypothesized a stronger lactic acid fermentation dur i ng ensiling as well as a higher ruminal fiber degradability due to enhanced cleavage of structural carbohydrates by AF supernatant in the silo. Regarding silage quality, the addition of AF superna-tant lowered the pH in all silages compared to respective controls, and in case of grass silage, the treatment reduced the silage pH to 4.54 and thus below the DM-dependent threshold for stable conservation (Muck 1988). Although the lactic acid level was not significantly affected, the numerical increase of 20.5 g/kg DM i n AF supernatant-treated grass silages seemed to - at least partly - explain this observed pH decline. Likewise, the AF supernatant decreased the acetic acid concentration i n straw silages without affecting the lactic acid concentration, indicating a shift towards a homolactic-dominated fermentation (Borreani et al . 2018).Consequently, these beneficial i nfluences on silage fermenta-tion characterist i cs revealed an improved silage quality and forage preservation with AF supernatant, which was also reflected by the trend for reduced DM losses in both grass fungi supernatant (S _AF) G_FRESH G_CON_AF G_AF S FRESH S_CON_AF S_AF Dry matter a 43.64 40.31 42.38 17.52 21.50 20.42 b2 43.46 48.63 48.10 57.67 58.34 54.45 c,%/h 0.06 0.05 0.06 0.03 0.03 0.03 Lag time 2.90 2.59 2.14 4.90 4.70 4.40 Neutral detergent fiber assayed with a heat stable o-amylase and expressed exclusive of residual ash C 37.59 23.28 30.32 10.59 11.64 9.79 b 51.16 63.96 58.20 71.16 67.59 69.60 c,%/h 0.05 0.04 0.05 0.03 0.03 0.02 Lag time 3.87 3.82 4.43 4.90 4.90 4.80 Acid detergent fiber expressed exclusive of residual ash C 16.47 20.15 23.12 7.50 16.83 13.00 b 81.41 66.00 62.74 62.16 61.16 52.75 c,%/h 0.03 0.04 0.05 0.02 0.03 0.02 Lag time 5.26 4.30 3.60 4.50 4.90 4.40 Fraction that disappears f rom t he bag immediately Insoluble but potent i ally rumen-degradable fraction 'Constant degradation rate of fraction b and straw silages. Therefore, our hypothesis of a stronger lactic acid fermentation may not be confirmed, but the overal l improved silage quality in response to AF superna-tant i ndeed suggest a beneficial effect of AF supernatant in silages. Interestingly, the addition of AF supernatant resulted ir an increased ammonia concentration in straw silages, sug-gesting higher protein degradation in the silo. I t is conceiva-ble that the AF supernatant as well i ncluded proteases as the applied AF supernatant was obtained from species of Neo-callimastix, a genus whose members partly show proteolytic activity, as well (Har t inger et al. 2018). Such AF-induced proteolysis is believed to be associated with the degrada-tion of structural proteins to sufficiently decompose fibrous plant structures, and i t may further modify the activities of other f ungus-derived CAZymes (Wal l ace and Jobl i n 1985).Therefore, a certain proportion of proteolytic activity i n AF cultures may be i nevitable, but in terms of f iber degrada-tion suppor t ive. However, i t has to be noted that ammonia proportion stil l amounted for less t han 10% of total nitrogen in AF supernatant-treated straw silages, which is deemed as a suf f icient t rue protein conservation (Kung et al. 2018).Similarly, no difference in ammonia concentrations between control and AF supernatant-t reated silages was observed for grass, meaning our positive assessment of AF supernatant for silage quality holds t rue. The proximate nutrients of the s i lages were mainly affected by the ensiled substrate, i.e., grass or straw,with expectedly higher concentrations of ash, crude pro-tein and ether extract , but lower concentrations of fiber fractions in grass than straw, as i t has also been found in the fresh substrates. The overall aNDFom concentrations were decreased after ensiling, which may be related to the acidic hydrolysis of hemicelluloses in the silo (Dewar et al.1963). The treatment with AF supernatant, however, showed no influence on the fiber fraction concentrations, meaning that a potential fiber tackling effect of AF enzymes was not reflected in silage composition. This is in contrast to prior studies inoculating rice straw or whole-plant corn with vari-ous viable AF species before ensi l ing, which observed less NDF and ADF concentrations compared to controls (Lee et al. 2015; Wang et al. 2019). The ruminal degradabi l ity of f iber fractions, however,was i ndeed affected by this treatment. Accordingly, our most i mportant finding here was t he improved fiber degra-dabi l ity of grass silages when ensiled with AF supernatant:The aNDFom degradability of AF supernatant-treated grass si l ages was higher t han of control grass silages and similar to fresh gras s , while ruminal ADFom degradabi l ity, i.e.,ruminal l i gnocellulose degradation, was even higher for AF supernatant-treated grass silages when compared to fresh grass. Thus, our hypothesis was confirmed and it can be assumed that the enzymes present in the AF supernatant pre-cleaved l ignocellulosic complexes in t he silos, thus allow-ing a higher fiber degradability, especially cellulose, and eventually higher energy exploitation from grass silage in the rumen. As outlined in our companion paper (Hartinger and Zebeli 2021), AF comprise a large enzymatic spectrum,and apart from p-glucosidase and endoxylanase, which have been considered i n our study, more enzymes or cellulosomes have likely been active in the applied AF supernatant. For instance, using transcriptomics and proteomics, Wang et al . A FRES H CO N _RF R F Fig.3 Effective ruminal degradability (4%/h passage rate) of dry matter (A), neutral detergent f iber (B), and acid detergent f iber (C) of fresh grass (FRESH) or grass si l ages prepared with heat-inac t ivated (CON _RF) or freshly prepared mixed ruminal fluid (RF). Aster-isk=P<0.05 in Tukey-Kramer post hoc test (2011) identified several novel f iber-degrading enzymes in Neocallimastix patriciarum W5. Thus, similar omics-based approaches can help to characterize the AF enzymes and consequently also to understand the modes of action in the silo. In case of straw silages, the beneficial effects of AF supernatant seen for silage quality could not be transferred to ruminal degradation. Surprisingly, the control straw silages, i.e., prepared with heat-inactivated AF supernatant, A 50 0 FRES H CO N _RF R F Table 5 Constants of ruminal degradation for fresh grass (G _FRESH), grass silage treated with heat-inactivated mixed ruminal f luid (G _CON _ RF), grass silage t reated wi t h freshly collected mixed ruminal f l uid (G _RF), fresh straw (S _FRESH), straw silage t reated with heat-inactivated mixed ruminal f luid (S _CON _RF),and straw silage treated with freshly collected mixed ruminal fluid (S _RF) G_FRESH G_CON_RF G_RF S_FRESH S_CON_RF S_RF Dry matter a 43.64 42.52 41.00 17.52 15.76 17.04 b2 43.46 45.70 49.47 57.67 55.91 50.59 c,%/h 0.06 0.06 0.05 0.03 0.02 0.04 Lag time 2.90 3.48 3.50 4.90 4.90 3.50 Neutral detergent fiber assayed with a heat stable o-amylase and expressed exclusive of residual ash C 37.59 22.55 19.50 10.59 6.71 10.96 b 51.16 66.61 69.28 71.16 76.03 62.14 c,%/h 0.05 0.04 0.05 0.03 0.02 0.03 Lag time 3.87 4.90 4.00 4.90 4.90 4.50 Acid detergent fiber expressed exclusive of residual ash C 16.47 21.68 20.08 7.50 6.94 11.17 b 81.41 68.66 65.38 62.16 59.91 48.97 c,%/h 0.03 0.04 0.05 0.02 0.01 0.03 Lag time 5.26 4.90 3.48 4.50 4.90 3.80 Fraction that disappears from the bag immediately Insoluble but potent i ally rumen-degradable fraction 'Constant degradation rate of fraction b et al. (2015), who observed higher ruminal fiber degrada-bility when ensi l ing rice straw with viable AF cultures. In contrast, wheat straw was ensi l ed in the present study, and apart f rom a di f ference between the application of viable AF cultures or their supernatant, a straw type-specific influence is possible and may be taken into account. Focusing only on the application of AF supernatant as a silage addi t ive, our results clearly showed a substrate-spe-cific influence on the efficacy of AF supernatant in silages,i.e., grass vs. straw. The AF supernatant i mproved the qual-ity of both grass and straw silages, but beneficial effects on ruminal f iber degradability were only present for grass silages. Thus, our hypothesis of a higher ruminal degrada-bility of forages ensiled with AF supernatant was confirmed for grass silages only. Studies investigating the impact of AF supernatant in other common forages, such as corn and alfalfa, can provide further information on substrate-speci f icities and therefore better define the potential areas of application. Likewise, refinements in the culturing and preparation of AF or their supernatant are required to further increase the efficacy of this novel silage additive. Hereby,co-culturing of AF and methanogens may consti t ute an option to additionally increase the f ibrolytic enzyme yield as suggested by the observed upregulated transcription of CAZymes in such co-cultures (Swift et al. 2019). Likewise,as previous data i ndicate di f ferences in AF species regarding their effectiveness as silage inoculants (Wang et al. 2019;Lee et al. 2015), research on applying supernatants from further AF species or genera seems rationale - although it may be noted that the present AF supernatant was obtained from a culture of three Neocallimastix species, a genus that is considered to express highest enzyme activities among AF cultures (Dagar et al . 2018), par t icularly after sequential sub-culturing (Ekinci et al, 2006). In regard to esterase activity,being mainly responsible for the breakup of l ignocellulosic complexes, a collection of AF supernatant after 1 or 2 days of cultivation may enhance its fiber cleaving impact as a silage inoculant, especially in straw silages, since esterase activities were observed to be highest during early fungal growth, whereas cellulase and xylanase peak at a later time point of incubation (Dagar et al.2018). As a second part, our study further assessed the use of mixed ruminal fluid as a silage additive in grass and straw silages, which may inoculate the silo with f i brolyt i c microbes and their enzymes that in consequence tackle f iber components during silo storage. Provided a similar efficacy as when using AF supernatant for ensi l ing, the approach of directly applying mixed rumina l f luid would mean a reduc-tion in complexity, time, and labor compared to the produc-tion of AF supernatant (Dollhofer et al. 2015). Consequently,we analyzed the same parameters as for AF supernatant-treated silages and expected an improved silage fermentation and subsequent ruminal fiber degradability in response to the microorganisms and enzymes deriving from the mixed ruminal fluid. The higher lactic acid concentrations in silages treated with mixed ruminal fluid indeed demonstrated an enhanced lactic acid fermentation. Likewise, addition of mixed rumi-nal fluid reduced the pH to 4.29 i n grass silages and there-fore indicated a stable forage conservation (Muck 1988).Compared to control, the acetic acid concentration was dou-bled in grass silages wi t h mixed ruminal f luid, i .e., 16.2 Vs. 29.6 g/kg DM, which suggested a higher activity of heterol-actic lactobacil l i or ruminal fluid-derived acetate producers.However , as shown by the lack of differences in DM losses,an acetic acid level of around 30 g/kg DM may not cause energy losses, but can actually be interpreted as beneficially in terms of aerobic stability due to yeast i nhibitory effects (Danner et al . 2003). Likewise, the overall low ethanol lev-els indicate a general reduced presence and suppression of epiphytic yeasts in al l si l ages (Kung et al . 2018), therefore supporting the assumed sufficient aerobic stability. The slightly higher ammonia concentrations observed in treated straw silages may have been caused by proteolytic microbes brought into the silo via mixed ruminal fluid (Hartinger et al. 2018; Puniya et al. 2015). Thus, the effects of mixed ruminal fluid on ammonia formation in straw silages were the same as those of AF supernatant and appear to have a similar rationale. In parts, the higher ammonia may have also originated from the mixed ruminal fluid i tself (Puniya et al. 2015) and vaporized during heat inactivation, thus co-explaining the lower ammonia level i n control straw silages Sti l l, the ammonia concentration of 10.5% of total nitrogen in mixed ruminal fluid-treated straw silages appears uncriti-cal in regards to true protein conservation (Kung et al. 2018)and, consequently, our hypothesis of an improved silage fermentation with the addition of mixed ruminal fluid was confirmed. Worth of notice is the apparently high l ignin reduction with mixed ruminal fluid inoculation in both grass and straw silages , which has not been observed for the treatment of silages with AF supernatant . Since the rumen microbiota is incapable or only minimally able to degrade lignin (Susmel and Stefanon 1993), this observation was indeed very sur-prising and lacks a direct explanation. It might be conjec-tured that the mixed ruminal f luid-induced ADL degradation led to a higher availability of fermentable sugars and there-fore would explain the increased lactic acid concentrations but absence of an effect of mixed ruminal fluid on WSC concentration - but this seems actually highly specula tive. In consequence, ruminal f iber degradabil i ty should be increased if lignin was truly degraded (Zabel and Morrell 2020). However, a beneficial effect of such a lignin reduc-tion was not reflected in ruminal degradabi l ity. In fact,the DM and ADFom degradability was not influenced by mixed ruminal fluid in grass and straw silages and,for aNDFom, ruminal degradability was even lower after ensiling grass with fresh or heat-inactivated mixed ruminal fluid when compared to fresh grass. Thus, our hypothesis of an improved f i ber degradation due to a pre-cleaving effect by ruminal fluid-derived microbes during ensi l ing could not be confirmed. However, as the DM degradability remained similar between fresh grass and grass silages,degradabil i ty of other nutrients must have compensated the reduction in aNDFom degradability, which may be investigated in future studies. Consequently, inoculation of grass and straw silages with mixed ruminal fluid may be suitable for improving silage quality, but for a f iber-cleaving activity in the silo, the targeted application of AF supernatant cannot be replaced by directly using mixed ruminal f luid . However, as demanded for AF supernatant,further research may help to optimize this approach, as well. For instance, a concentration of microorganisms and their enzymes via ultra f iltration of mixed ruminal fluid,like i t has been performed during processing of the AF supernatant, could increase the fiber cleaving potential during ensiling. Since the present straw silages were pre-pared of pure wheat straw, applying mixed ruminal f luid as a silage inoculant in mixed silages of straw and a second substrate, e.g., grass or WSC-rich by-products, may be worth of further investigation. In conclusion, thi s is the f irst study to investigate AF supernatant and mixed ruminal f luid as novel s i lage addi-tives for ensil i ng grass and wheat straw and we provide strong evidence that both candidates improve silage quality. The treatment with AF supernatant addi t ionally enhanced ruminal fiber degradability of grass silage,which should be associated with a f iber-cleaving fungal enzyme activity in the silo. Therefore, the application of AF supernatant in silages represents a promising strategy to support forage utilization by ruminants and should be pursued . Differences in ruminal degradabil i ty between wheat straw and grass silages suggest substrate-specific effects that need consideration in future research Acknowledgements The authors thank Anita Dockner, Suchitra Sharma, Sabine Leiner, and Manfred Hollmann from the Institute of Animal Nutr i tion and Functional Plant Compounds at the University of Veterinary Medicine, Vienna, for the assistance during the chemical analyses of samples, as well as Lenka Strosova from the I nstitute of Animal Physiology and Genetics, Czech Academy of Sciences, for t he AF supernatant preparation. Author contribution TH and QZ designed the research and acquired the funding. TH and KF conducted experiment and analyses . TH ana-lyzed the data and wrote the manuscript. KF and QZ reviewed and edited the manuscript . All authors read and approved the manuscript. Funding Open access funding provided by University of Veterinary Medicine Vienna . This study was funded by the Austrian Federal Min-istry of Agriculture, Regions and Tourism, Grant Number 101623. Data availability All data generated or analyzed during this study are included in this published ar t icle. Declarations Ethical statement All applicable international , national, and/or i nstitu-t i onal guidelines for t he care and use of animals were followed . Competing interests The authors declare no competing interests Open Access This article is licensed under a Creative Commons Attri-bution 4.0 Internationa l License, which permits use, sharing, adapta-tion, distribution and reproduction in any medium or format , as long as you give appropriate credi t to the original author(s) and the source,provide a link to the Creative Commons licence, and indicate i f changes were made. The images or other t hird party material in t his article are included in the article's Creative Commons licence , unless indicated otherwise in a credi t line to the material . If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds t he permitted use, you will need to obtain permission direct l y f rom the copyright holder . To view a copy of this l i cence , visi t http://creativecommons.org/licenses/by/4.0/. 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