GUO Yun-xia ,YANG Ruo-chen ,DUAN Chun-hui ,WANG Yong ,HAO Qing-hong,Jl Shou-kunYAN HuiZHANG Ying-jie#,LlU Yue-qin#

1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, P.R.China

2 College of Life Sciences, Hebei Agricultural University, Baoding 071000, P.R.China

3 Engineering Research Center for Utilization of Agricultural Waste Resources, Baoding 071001, P.R.China

Abstact This study investigated the effects of dioscorea opposite waste (DOW) on the growth performance,blood parameters,rumen fermentation and rumen microbiota of weaned lambs.Sixty healthy weaned Small-Tailed Han lambs (male,(22.68±2.56) kg initially) were used as the experimental animals.Four levels of concentrate: 0 (control,CON),10%(DOW1),15% (DOW2) and 20% (DOW3),were replaced with DOW in the basal diet as experimental treatments.The results showed that lambs fed the DOW2 diet had a higher (P<0.05) dry matter intake (DMI) than the other groups.There was no significant difference (P>0.05) among DOW groups in average daily weight gain (ADG),and replacing concentrate with DOW linearly or quadratically increased (P<0.05) the ADG,while lambs fed the DOW2 diet showed greater (P<0.05) ADG than the CON group.The relative plasma concentration of growth hormone (GH),insulin like growth factor-1 (IGF-1) and insulin were affected by DOW,replacing concentrate with DOW linearly or quadratically(P<0.05) enhanced the plasma concentration of GH,IGF-1 and insulin,which was significantly higher (P<0.05) in the DOW2 group than in the CON,DOW1 and DOW3 groups.In addition,the DOW treatment showed a lower (P<0.05)concentration of blood urea nitrogen (BUN) than the CON group.Replacing concentrate with DOW quadratically decreased (P<0.05) the ruminal ammonia nitrogen (NH3-N) and increased (P<0.05) the total of volatile fatty acids (TVFAs)at 0 and 4 h after feeding as well as linearly decreased (P<0.05) the NH3-N at 8 h after feeding.Replacing concentrate with DOW linearly decreased (P<0.05) the propionate and increased the aceate before feeding,and linearly decreased(P<0.05) propionate and quadratically increased (P<0.05) the aceate at 4 and 8 h after feeding.Lambs fed the DOW2 diet increased the phylum Firmicutes and genera Succiniclasticum and Ruminococcus_1 groups,whereas decreased(P<0.05) the relative abundance of phylum Deferribacteres and genera intestinimonas and Ruminiclostridium.In summary,replacing the concentrate with 15% DOW was beneficial for improving the rumen fermentation and ADG by increasing the DMI and modulating the rumen microbial community.

Keywords: lamb,dioscorea opposite waste,growth performance,blood parameters,rumen fermentation,rumen microflora

1.lntroduction

Feeding agricultural by-products is a viable option to improve the sustainability of animal production systems by decreasing the feeding cost (Munnichet al.2017).Therefore,by-product feeds and crop residues are frequently used as fiber sources in sheep diets.ChineseDioscoreais a well-known vegetable used as food and traditional medicine for thousands of years in East Asia,especially in China (Amatet al.2014).Dioscorea opposite waste (DOW) is a relatively inexpensive and available fiber source that is produced in China as a byproduct of the Dioscorea opposite process,most of which was discarded,causing environmental pollution and waste of resources.Dioscorea usually contains 79.9%carbohydrate,12.6% crude protein,1.7% ether extract and 5.7% crude ash on a dry matter basis (Bitschet al.2003).The dry rhizome of Dioscorea opposite thumb is one of the traditional Chinese medicine and food homologous plants which have pharmacological activities of hypoglycemic,hypolipidemic,antioxidant,anti-tumor,immune regulation,and spleen and stomach function regulation (Kong X Fet al.2009;Luoet al.2016;Leeet al.2019),therefore,it is crucial to utilize the DOW as animal feed resource.

A previous study showed that Dioscorea could inhibit bacterial pathogens and enrich beneficial intestinal bacteria in SD rat cecum (Kong Xet al.2009).Jeonet al.(2006) have reported that a 40% ethanol extract of Chinese Dioscorea flour improved digestive capability and enriched lactose-fermenting bacteria in rats.Previous work indicated that dietary supplementation of Dioscorea skin has a high immunity activity of carp (Menget al.2019).One report showed that dietary supplementation of Dioscorea peel could effectively increase the feed intake of broilers (Ukonuet al.2018).Therefore,modulation of growth performance in ruminants production by using DOW deserves more attention and further investigation.Previous studies have shown that Dioscorea can improve the average daily gain of goats and pre-weaning lambs(Dokoet al.2013;Yashimet al.2016).Data from several studies suggest that the Chinese Dioscorea by-product can be well preserved by making silage,and it is a good potential dietary source of energy for ruminant diets (Xuet al.2009;Afolabiet al.2012).However,few studies about the effect of DOW on weaned lamb were reported.It is hypothesized that the replacement of concentrate with DOW in lambs ration could improve the growth performance and nutrient metabolism through modulating the ruminal microbial community.

The specific objective of this study was to evaluate the effect of DOW on growth performance,blood parameters and rumen microflora of weaned lamb,which can provide the technical guide for DOW usage.

2.Materials and methods

2.1.Animals,experiment design,treatments and diets

The animal trial was carried out over a two-month period(October to December 2021) on Hengshui Zhihao Animal Husbandry Technology Co.,Ltd.,in Hebei Province,China (37°32′14′′–37°41′25′′N,115°28′59′′–115°41′40′′E).The temperature was maintained at 1.2–13.5°C throughout the study period,and the mean relative humidity was maintained at 40.6%.Sixty Small-Tailed Han male lambs,at 60 d of age and with an initial body weight of (22.68±2.56) kg,were allocated to one of four experimental treatments according to the completely randomized design,with 15 lambs in each treatment.The experiment lasted for 63 d,including a 15-d preliminary feeding period for adaptation and a 48-d experimental feeding period.

The experiment treatments consisted of a basal control diet (CON),10% concentrate replaced with DOW per lamb per day (DOW1),15% concentrate replaced with DOW per lamb per day (DOW2) and 20% concentrate replaced with DOW per lamb per day(DOW3).The DOW was obtained from Dengfeng Ma Yam Professional Cooperative in China.The ingredient and chemical composition of diets are reported in Table 1,and the chemical composition of DOW is shown in Table 2.During the experimental period,lambs were reared in separate individual pens and fed twice a day at 0600 hand 1400 h withadlibitumaccess to total mixed ration and freshwater.The basal ration was formulated based on NRC (2007) recommendations with a forage to concentrate ratio of 55:45.The daily allowance of feeds to the animals was determined according to theadlibitumfeed intake measured in a preliminary feeding trial,ensuring the rejected feed accounted for 3–5% of the provided feed.The concentrate in each treatment’s diet was replaced with the corresponding DOW at the same time,while the concentrate in the control group’s diet remained unchanged.The management of the lamb was kept constant throughout the trial.

Table 1 Ingredient and chemical composition of diet (% of dry matter)

Table 2 Ingredient and chemical composition of dioscorea opposite waste (DOW,% of DM)

2.2.Growth performance

The daily dry matter intake (DMI) of each lamb was determined by taking the difference between providing feed and rejections on a dry matter (DM) basis,and the DMI was recorded daily for each lamb.The live body weight (BW) of each lamb was measured before morning feeding at the beginning and end of the experiment to calculate the daily weight gain (ADG) of the lambs.The feed conversion ratio (FCR) was calculated by dividing DMI by BW gain during the experimental period.

2.3.Feed sample collections and analysis

The daily amount of feed supplied and rejected was manually recorded using a weigh scale.Diet samples were collected weekly and stored at–20°C for further moisture and chemical composition analysis.The samples of the diet were ground to pass through a 40-mesh sieve.The DM of diet was estimated by drying at 105°C for 4 h(AOAC 1990;method 930.15).The neutral detergent fiber (NDF) and acid detergent fiber (ADF) concentrations were determined according to the method described by Van Soestet al.(1991) in an ANKOM 2000 Fiber Analyzer(ANKOM Technologies,Macedon,NY) without sodium sulfite,with heat-stable α-amylase being used in the NDF evaluation.The N was determined by the Kjeldahl method,and the crude protein content was calculated as N×6.25(AOAC 1990;method 981.10).Starch concentration was determined according to the method described by Oliveiraet al.(2016).The ether extract (EE) was detected using an automatic extractor (ANKOM XT101,ANKOM Technology Corp.,Macedon,NY,USA).The crude ash was measured by combustion in an electric muffle furnace at 550°C for 6 h(AOAC 1990;method 942.05).Calcium concentration was determined using a flame atomic absorption spectrophotometer (WFX-320,BRAIC,Beijing,China),and phosphorus was measured with a UV spectrophotometer(UV-VIS 8500,Shanghai Tianmei Scientific Instrument Co.,Ltd.,Shanghai,China).The polysaccharides of DOW were extracted using an Ultrasonic Cleaner (KQ3200E,Kun Shan Ultrasonic Instruments Co.,Ltd.,China) and then analyzed through the method described by Doet al.(2021).

2.4.Plasma collection and determination

The blood samples were collected before morning feeding on day 48 in the experimental period.A total of 10 mL of blood was harvested from six lambs per group with a vacuum tube by jugular vein punctures,which was then centrifuged at 8 000×g and 4°C for 10 min to separate the plasma and stored at–80°C refrigerator until further analyses.The growth hormone (enzyme-linked immunosorbent assay (ELISA,MM-34621O2,Jiangsu Meimian Industrial Co.,Ltd.,China),IGF-1 (ELISA,MM-0630O2,Jiangsu Meimian Industrial Co.,Ltd.) and Insulin(ELISA,MM-80043O2,Jiangsu Meimian Industrial Co.,Ltd.) were analyzed on a semiautomatic biochemical analyzer (Rayto,RT-6100) with commercial test kits following the manufacturer’s instructions.The blood urea nitrogen (BUN) was measured using a fully automated clinical chemistry analyzer with commercial test kits,as directed by the instructions.

2.5.Rumen fluid sampling,measurements and analysis

On experimental day 48,the rumen fluid was collected individually at 0,4 and 8 h after the morning feeding from the same animals used for blood collection by a stomach tube.To avoid saliva contamination,the first 200 mL rumen fluid was discarded;the subsequently 30 mL rumen fluid was detected pH value and then strained through four-layer cheesecloth and divided into three 10 mL of centrifugal tubes as the representative sample.The two portion samples of 10 mL from each group were frozen at–20°C for analyzing the volatile fatty acids (VFAs)and ammonia nitrogen (NH3-N),and the third sample from CON and DOW2 groups was frozen at–80°C for detecting rumen microbial community.

The pH value of rumen fluid was measured with a pH meter (Portable PHS-3D,Thermo Fisher Scientific,USA).The concentration of NH3-N in rumen fluid was determined using the phenol-sodium hypochlorite colorimetric method described by Broderick and Kang (1980) on the UV/vis spectrophotometer (UV-1801;Beifen Ruili Analytical Instrument Co.,Beijing,China).The volatile fatty acids(VFAs) of rumen fluid samples were determined using gas chromatography (7890B-7000D,Agilent Technologies,Santa Clara,CA,USA),which was equipped with a hydrogen flame detector and a capillary column (Agilent,Technologies;25-m long,0.20-mm diameter,0.40-μm film thickness) according to the method described in a previous study (Sunet al.2020).

2.6.Ruminal microbiota analysis

Quantitative real-time PCR (qPCR)Microbial DNA was extracted using the HiPure Soil DNA Kits (or HiPure Stool DNA Kits) (Magen,Guangzhou,China) according to the manufacturer’s protocols.DNA concentration and purity were determined by NanoDrop 2000 (Thermo Fisher Scientific,Waltham,MA,USA),and the quality was evaluated by 1% agarose gel electrophoresis.The V3 and V4 hypervariable regions of the bacterial 16S rRNA gene were amplified by PCR (95°C for 5 min,followed by 30 cycles at 95°C for 1 min,60°C for 1 min,72°C for 1 min and a final extension at 72°C for 7 min) using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R(5′-GGACTACHVGGGTWTCTA-AT-3′).PCR reactions were performed in a triplicate 50 μL mixture containing 10 μL of 5×Q5 reaction buffer,10 μL of 5×Q5 high GC enhancer,1.5 μL of 2.5 mmol L–1dNTPs,1.5 μL of each primer (10 μmol L–1),0.2 μL of Q5 high-fidelity DNA polymerase,and 50 ng of template DNA.

16S rRNA sequencing and analysisAmplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences,Union City,CA,USA) according to the manufacturer’s instructions and quantified using ABI StepOnePlus Real-Time PCR System (Life Technologies,Foster City,USA).Purified amplicons were pooled in equimolar and pairedend sequenced (PE250) on an Illumina platform according to the standard protocols.

To obtain high-quality clean reads,raw reads were further filtered according to the following rules: reads containing more than 10% unknown nucleotides and reads containing less than 50% of bases with quality were removed.Paired-end clean reads were merged as raw tags using FLASH (version 1.2.11) with a minimum overlap of 10 bp and mismatch error rates of 2% (Mago?et al.2011).Noisy sequences of raw tags were filtered under specific filtering conditions using the QIIME pipeline(version 1.9.0) to obtain high-quality clean tags (Caporasoet al.2010).The clean tags then underwent chimera detection using the UCHIME algorithm to remove all chimera,and effective tags were obtained (Edgaret al.2011).A total of 2 506 417 effective tags were kept,with over 80 342 for each sample.Filtered sequences were clustered into operational taxonomic units (OTUs) of≥97% similarity using UPARSE (version 9.2.64) pipeline(Edgar 2013).Then the representative sequences were categorized into organisms by comparing them to the SILVA Database (Pruesseet al.2007) using the Ribosomal Database Project Classifier (version v132,Wanget al.2007).

2.7.Statistical analyses

The difference in the alpha diversity index was analyzed by QIIME Software (version 1.9.0).The R Software (version 3.5.1) packages ‘ggplot2’ and ‘reshape2’ were used to plot rarefaction curves.The vegan and ggplot2 packages in R were used for principal coordinate analysis (PCoA).

Firstly,a normal distribution of the growth performance,plasma samples index,pH value,VFAs and NH3-N were analyzed using the Shapiro-Wilk method SAS 9.4 (SAS Inst.Inc.,Cary,NC,USA).Then all data were analyzed by the Proc mixed of SAS (Steel and Torrie 1980),with results presented as the least squares mean and standard error of the mean.The analytical model was as follows:

where trt is a fixed effect.Each replicate served as an experimental unit.Differences among treatment means were tested for significance using leastsignificant difference (LSD) multiple range test.Using the CONTRAST procedures of SAS to examined the effects of increasing DOW supplementation through linear and quadratic orthogonal contrasts (Manet al.2020).The residual normality test was performed using Proc Univariate with the Normal and Plot options.When significant (P<0.05),differences were detected.

3.Results

3.1.Effects of DOW on growth performance

The ADG,DMI and feed conversion rate are shown in Table 3.Replacing concentrate with DOW affected DMI and ADG.As the DOW level increased,the ADG increased (P<0.05) linearly or quadratically,and lambs assigned to the DOW2 showed a higher DMI (P<0.05)compared to the lambs distributed to CON treatments.In addition,although the ADG did not differ (P>0.05)between the DOW1,DOW2 and DOW3 groups,the DMI was higher (P<0.05) in the DOW2 group than in the DOW1 and DOW3 groups.There was no difference in feed conversion ratio between all DOW and CON groups.

Table 3 Growth performance of lambs supplemented with dioscorea opposite wastes (DOWs)

3.2.Effects of DOW on blood parameters

The plasma physiological characteristics of lambs fed various experimental diets are summarized in Table 4.Replacing concentrate with DOW increased (P<0.05) the plasma concentration of GH,IGF-1 and insulin in a linear or quadratic manner,while these parameters were higher(P<0.05) in the DOW2 group than in the DOW1 andDOW3 groups.In addition,replacing concentrate with DOW decreased (P<0.05) the concentration of BUN in a quadratic manner.

Table 4 Effects of dietary supplementation of dioscorea opposite waste (DOW) on the plasma biochemical indices in lambs

3.3.Effects of DOW on rumen fermentation parameters

Effects of replacing concentrate with DOW on rumen fermentation parameters are shown in Table 5.At 0 h after feeding,the ruminal pH,MCP and the molar proportions of isovaleric acid and valeric acid were not affected (P>0.05) by DOW,however,the DOW significantly decreased (P<0.05) the ratio of propionate in a linear manner and decreased (P<0.05) the concentration of NH3-N in a quadratic manner,while the diets containing 15 and 20% DOW significantly increased(P<0.05) the TVFAs and the ratio of acetate.The acetate:propionate (A:P) was significantly higher (P<0.05)in a quadratic manner,which was higher in the DOW2 group than in other groups.At 4 h after feeding,the DOW had no evident effect (P>0.05) on the ruminal pH,isovaleric acid and valeric acid.Replacing concentrate with DOW quadratically increased (P<0.05) the proportion of acetate,butyric acid and isbutyric acid,quadraticallydecreased the content of NH3-N,tend to linearly increased(P<0.05) the A:P and decreased (P<0.05) the proportion of propionate,and these results were more pronounced in DOW2 group than other groups.At 8 h after feeding,the CON and DOW treatment groups exhibited no difference (P>0.05) on ruminal pH and TVFAs.Replacing concentrate with DOW significantly increased (P<0.05)the valeric acid percentage and decreased (P<0.05) the concentration of NH3-N and the ratio of propionate in a linear manner,while the diet containing DOW significantly increased (P<0.05) the proportion of butyric acid and isovaleric acid in a quadratic manner.

3.4.Effects of DOW on rumen microbiota profiles

In total,1 606 852 effective tags were kept,with over 80 342 for each sample.Rarefaction curves were generated using observed OTUs for each feed type to assess the adequacy of the sampling depth.Rarefaction curves indicated a diminishing rate of new OTU identification as the number of reads per sample increased,with all rarefaction curves of OTU tending to plateau at 65 000 tags,implying that the sequencing depth and amount were saturated (Fig.1).Similarly,the Good’s coverages for all samples exceeded 98%,which demonstrated the accuracy and reproducibility of the sequencing.Chao1 value and Shannon index were used to measure the richness and diversity of microorganisms(Fig.2-A and B).PCoA plots were performed using the Bray_curtis,where bacterial communities were clustered by feedstuff type,clearly showing the distinct bacterial community structure in the different groups (Fig.2-C and D).

Fig.2 Differences in bacterial diversity and richness between the dioscorea opposite waste (DOW) and control group (CON)groups.A,bacterial richness estimated by the Chao1 value.B,bacterial diversity was estimated by Shannon index.C and D,principle coordinate analysis (PCoA) profile of rumen bacteria diversity at phylum (C) and genus (D),respectively.

Taxonomic analysis of the reads revealed the presence of more than 13 bacterial phyla;the predominant phyla were Bacteroidetes and Firmicutes,accounting for 55.08 and 27.34% of total reads,respectively (Fig.3-A).Among these phyla,the relative abundance of Deferribacteres was significantly lower (P<0.05) in the DOW2 group before and after 4 h of feeding (Fig.4-A and C),but that of Firmicutes was significantly increased (P<0.05) after 4 h of feeding (Fig.4-C).At the gene level,the top 10 genera of abundance in rumen samples were identified.The predominant genera werePrevotella1(29.38%),SuccinivibrionaceaeUCG-001(10.15%),Succiniclasticum(5.13%) andRikenellaceae_Rcggutgroup(3.69%)(Fig.3-B).The relative abundances ofSucciniclasticum,Ruminococcus_1,Blautia,Lachnospiraceae_NK3A20_group,saccharofermentans,Ruminococcaceae_UCG_001andLachnoclostridium_1were significantly increasing (P<0.05) as well as theErvsipelotrichaceae_UCG_004,Lachnoclostridium,Lachnospiraceae_NK4A136_group,intestinimonas,AligtipesandRuminiclostridiumsignificantly decreased (P<0.05) in the DOW2 group compared to the CON group after 4 h of feeding.The ruminal microbiome of the DOW2 group had a higher (P<0.05) abundance ofSucciniclasticumand lower (P<0.05) abundance ofBlautiaandMucispinllumthan the CON group after 0 h of feeding (Fig.4-B and D).

Fig.3 Classification of the bacterial community composition across the dioscorea opposite waste (DOW) and control group (CON)groups.A,phylum level.B,genus level.

Fig.4 The significantly difference Bacteria between the concentrate and feed groups.A and B,the bacteria at the phylum (A) and genus (B) level that had significant differences between the DOW and CON groups after fed 0 h,respectively.C and D,the bacteria at the phylum (C) and genus (D) level that had significant differences between the DOW and CON groups after fed 4 h,respectively.

4.Discussion

The present study partially supported the hypothesis that the replacement of concentrate with DOW in lambs ration could improve the growth performance and nutrient metabolism through modulating the ruminal microbial community.It indicated that replacing of concentrate with 15% DOW increased the ADG,total VFA production and the proportion of acetate,probably due to an increase in DMI and regulation of the rumen microbiota,but decreased the BUN concentration and increased the growth hormone,IGF-1 and insulin concentration in the plasma.These results provided a theoretical basis for the further application of DOW in ruminant production.

Factors related to ADG are affected by the feed types,animal breeds and management levels (Duthieet al.2017).Few studies have reported the effect of DOW on ruminant growth performance.Yashimet al.(2016)observed that ADG was increased for red Sokoto goats fed diets containing Dioscorea as a replacement for maize offal.In a similar study,Dioscoreaand cassava peels prevented lamb’s weight loss and allowed expressing zootechnical performances across the season (Dokoet al.2013).Our study indicated that the ADG in all treatments ranges from 239.82 to 288.17 g,which showed that all diets have sufficient nutritional contributions to growth and production.The ADG and DMI were affected by DOW,with increasing levels of added DOW,ADG increased in a quadratic or linear manner,and the lambs assigned to the DOW2 showed a higher ADG and DMI than the lambs distributed to the CON group,which was probably due to some of the health benefits ingredients such as starch and polysaccharide in DOW (Epping and Laibach 2020).Many medical reports indicated that the Dioscorea polysaccharide possesses a variety of biological activities,such as anticoagulant,antitumor,immune regulation and antioxidant activities (Luoet al.2008;Juet al.2014;Chen Y Fet al.2015).Ekweet al.(2020) reported that yam peel meal replacement for maize could improve the DMI of rabbits,and the same trend was observed in weanling rats by Chinese yam polysaccharide (Kong Xet al.2009).Ekenyemet al.(2006) also found that adding 15% Dioscorea peel meal to the ration increased the DMI of broilers,probably due to the improved palatability by supplementation of Dioscorea peel meal to the diets.Therefore,we speculate that the elevated DMI was due to the 15% DOW that provided a source of polysaccharides and thus improved the palatable diet.The other important concentition of increasing DMI improved the ADG (Marín-Garcíaet al.2020).However,the observed difference in ADG between CON and DOW3 in this study was not significant,which might be due to the higher NDF (Salinas-Chaviraet al.2013) and resistant starches (Epping and Laibach 2020) concentration in the DOW3 group.Feed conversion ratio values were lower in the DOW2 group than in the CON group,which indicated that replacing 15%concentration with DOW could be beneficial for the growth performance of lambs.

Metabolites could be changed by the dietary nutrition levels,which are used as a signal indicating the growth and development of the body.Therefore,the lamb’s growth performance can be observed indirectly by detecting the alters of plasma metabolites (Liet al.2018).Prior studies showed that Dioscorea could regulate blood sugar levels and control cholesterol,fat uptake and hypertension (Amatet al.2014;Zhanget al.2016).However,few studies have measured the effect of DOW on blood GH,IGF-1 and BUN.In this study,replacing concentrate with DOW linearly or quadratically increased the concentration of GH and IGF-1 while it was higher in the DOW2 group than other groups.The plasma level of somatomedins (GH and IGF-1) may represent the growth capacity of young bulls(Lund-Larsenet al.1977).Some researchers reported that GH could promote the accretion of protein,and IGF-1 mediates certain GH activity (Eisemannet al.1986),which is related to the increase of cartilage and muscle growth in non-ruminant animals (Etherton and Kensinger 1984).The plasma metabolism was influenced by both dietary energy intake and rumen fermentation (Graugnardet al.2012).Liuet al.(2019) reported that the positive response of blood GH,INS and IGF-1 were associated with the increase in DMI and ruminal total VFA concentration.Shenet al.(2004) also found that the plasma levels of IGF-1 were increased in goats consuming a high protein and high energy diet.Thus,a possible explanation for the fluctuation of plasma metabolites might be attributed to the increase in DMI.The BUN concentration is positively correlated with the rumen ammonia concentration (Davidsonet al.2003),while the ammonia could be accumulated in the rumen fluid,absorbed into the blood,transported to the liver and converted to urea;then,BUN produced during the ammonia in urine is eliminated through the urea cycle(Kimet al.2012).The present study showed that replacing concentrate with DOW decreased the concentration of BUN in a quadratic manner,which might be due to the lower concentration of NH3-N caused by altered rumen fermentation.Results of the present study indicated that dietary supplementation DOW increased the relative plasma concentration of GH,IGF-1 and insulin as well as decreased the relative concentrations of BUN.

Ruminal pH is regulated through behavioral and physiological mechanisms under different feeding regimes(NASEM 2016).The ruminal pH value usually fluctuates between 5.5–7.5 (Colmanet al.2010);when the pH is at 6.2–6.8,the rumen microbial activity is the strongest(Zhenget al.2020),in which most feed fiber can be effectively degraded.In the present study,no differences in pH (range 6.31–6.86) between treatments were found.As the main nitrogen source for rumen microbial growth,NH3-N reflects the balance between protein degradation and synthesis under specific dietary composition (Aguerreet al.2011).There was a linearly or quadratically decrease of the NH3-N with increasing dietary DOW,in accordance with the decreased plasma concentrations of BUN,which might be due to an imbalance or antinutritional factor in DOW,which resulted in a lower protein degradation rate.

Carbohydrates were fermented by a variety of bacteria and enzymes in the rumen and then transformed into volatile fatty acids (VFAs) (Wanget al.2020).Changes in feed types not only alter the available fermentation substrates but also affect the ruminal environment,including pH and VFAs profiles (Ghimireet al.2017;Huaet al.2017;Zhanget al.2018).The amount and profiles of VFA formed in the rumen have consequences for the efficiency of energy utilization,production of methane,risks of ruminal acidosis and composition of animal products (Noziereet al.2011).In the present study,higher TVFAs and acetate concentrations were obtained in a quadratically manner at 0 and 4 h after feeding with the DOW level increased,while the proportion of propionate was decreased in a linearly manner,with the DOW2 group had a higher proportion of acetate than the other groups.Interestingly,we observed that replacing concentrate with DOW enhanced the proportion of butyric acid and isbutyric acid after 4 h feeding and increased valeric acid percentage after 8 h after feeding.VFAs are the main energy source for ruminants,could promote gastrointestinal movement and inhibit the growth of pathogenic microorganisms (Zhenget al.2020).Saleemet al.(2013) reported that VFAs are the endproducts of microbiota and could potentially indicate the shifts in bacterial community composition.On the one hand,the fluctuations of VFAs observed after a period of feeding could be mediated by changes in the rumen bacterial populations (Hungateet al.1975).On the other hand,the VFAs content is closely related to feeding intake,and an increased DMI probably promotes VFAs production,resulting in lower rumen pH in ruminants(Nakamuraet al.2018;Tomczaket al.2019).The effect of DOW on rumen VFAs production has not been reported yet.In the present trial,the DMI of lambs fed the diet with 15% DOW was significantly higher,so the changes in rumen VFAs content can be attributed to the change in DMI and rumen microbes.Acetate was produced by fiber degradation in the diet,a process in which the fiber-decomposing bacteria and starchdecomposing bacteria of rumen promote the deposition of propionate.Acetate:propionate (A:P) is often used as a marker to evaluate the type of rumen fermentation,where A:P<3 indicates the propionate fermentation and>3 is the acetate fermentation (Chenget al.2019).Our study results showed that the A:P was less than 3,the concentration of acetate was increased,and propionate decreased significantly in the DOW groups compared with the CON group,indicating a propionate fermentation type and DOW shift in the rumen fermentation.The linearly increased of acetate concentrations also could be associated with improved digestion of structural carbohydrates (Gutierrezet al.2017).In rumen,branchchain VFAs (valerate,isovalerate,and isobutyrate)originate primarily from ruminal oxidative deamination and decarboxylation of valine,leucine,and isoleucine (Owens and Basalan 2016).Therefore,the fluctuations of valeric acid and isobutyric acid in the DOW group may be due to the unbalanced content and ratio of amino acids in the DOW.In summary,the ruminal environment was affected by both DMI and the rumen bacterial community,and supplementing DOW could decrease the propionate and increase the proportion of acetate.

Although rumen bacterial communities were relatively stable,they were highly responsive to changes in host genetics (Huanget al.2017),feeding paradigms (Xueet al.2017),diet (Linet al.2018),age (Jamiet al.2013)and environmental factors (Uyenoet al.2010).Of these,the diet was the key factor in determining microbial community structure (Maet al.2019).Menget al.(2019)have reported that Chinese Dioscores peel can improve the microbiome of the fish gut.Additionally,a previous study has reported that polysaccharides from Chinese Dioscores enriched beneficial intestinal bacteria in SD rat cecum (Kong Xet al.2009).Results of the present study showed that the main microbiome was occupied by the phyla Bacteroidetes,Firmicutes,and Proteobacteria in the DOW2,which was consistent with previous studies(Chen Y Bet al.2015;Lyonset al.2017;Liet al.2020).There were no significant differences in alpha diversity metrics (Shanno index and Chao1 value) after lambs were fed DOW.Taxonomic analysis showed that at the phyla,the relative abundances of Deferribacteres was significantly lower in the DOW2 group before and after 4 h of feeding,while Firmicutes was significantly increased after 4 h of feeding.Such discrepancy may be caused by the polysaccharides from DOW (Kong Xet al.2009).The phylum Deferribacteres was a distinct lineage within the bacteria based on phylogenetic analysis of 16S rRNA sequence;at present,the members of this phylum were organized into a single class,order,and family (Garrityet al.2001).However,the information on the function and metabolic biochemical pathways of Deferribacteres were scarce.Firmicutes were the major rumen microorganisms in degrading structural carbohydrates (Khafipouret al.2016),mainly comprised of Gram-positive,low-G+C-content bacteria (Voset al.2011).Previous studies have shown that the proportion of Firmicutes-related sequences detected in 16S rRNA gene clone libraries from rumen samples of cattle fed different hay-based diets considerably varies between 25 and 90% (Tajimaet al.2000;Kocherginskayaet al.2001).Firmicutes could increase forage fermentation and cellulose decomposition,which potentially improves plant biomass breakdown,enhancing the growth performance of lambs (Liuet al.2020).At the genus level,SucciniclasticumandRuminococcus_1were found to be more abundant in the DOW2 group than in the CON group.Succiniclasticumhas been reported as the main participant in the fermentation of succinate to propionate,which was the most important precursor of glucose in ruminants (Van Gylswyket al.1995).The increased relative abundance ofSucciniclasticumcould explain the increase in the molar proportion of propionate of rumen fluid in the DOW2.Liuet al.(2020) have revealed that the generaSucciniclasticumwas significantly positively correlated with the valerate,indicating that this genus was sensitive to an increase in the valerate of the DOW2.A previous study showed that theRuminococcus_1plays an important role in degrading fibers which can produce cellulases to degrade cellobiose and reduce the number ofRuminococcus_1and will reduce the fiber digestibility(Koike and Kobayashi 2001).A previous study has evaluated that the relative abundance ofRuminococcus_1is the strongest negative correlative with feed conversion ratio (McLoughlinet al.2020),which could explain the lower feed conversion ratio in the DOW2.Zhaoet al.(2020) reported that the increase in the ruminal concentration of total VFAs could be attributed to the increased relative abundances ofRuminococcus.In addition,these changes in rumen microbiota may be explained by the fact that the increased DMI provides sufficient substrate for the growth and development of specific rumen microorganisms.Although there were significant correlations between rumen bacteria and VFAs,NH3-N,and growth performance,the actual mechanisms by which the rumen microorganism influences growth performance remain unknown and should be researched further in future studies.Then,the actual mechanisms by which the DOW impact the rumen fermentation and growth performance need to be investigated in the future.

5.Conclusion

The replacement of Dioscorea opposite waste at 15%per lamb per day was an effective way of promoting the growth performance of lamb by enhancing the DMI.This could lead to an improvement in total VFA and the proportion of acetate of rumen,meanwhile,it also positively affected the plasma concentrations of metabolites,including IGF-1,GH and Insulin.However,the substitution of Dioscorea opposite waste at 10% or 20% in the lamb diet showed no significant difference in DMI and ruminal metabolism in this study.

Acknowledgements

This research was supported by the Key R&D Project of Hebei Province of China (21322907D and 21322910D),the Natural Science Foundation of Hebei Province,China(C2022204174),and the China Agriculture Research System (CARS-38 and CARS-39-23).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures used in this study were approved by the Laboratory Animal Ethics Committee of Hebei Agricultural University,China (permit number 2018082).