Gastrointestinal Segments In uenced Fermentation End- Products, Microbiota and Microbial Abundances in Goats


 Purpose: Carbohydrate diets altered fermentation end-products and microbial community in the gastrointestinal tracts (GIT) of goats. Gastrointestinal contents used to determine the impact of carbohydrate feeds on fermentation end-products and microbial community in goats.Methodology: in the study goats were assigned to one of the two treatments corn meal (CM) or Corn gluten (CG) in a randomized block design (400 g/kg DM each). Goats were slaughtered, GIT liquids were used to determine dissolved gasses, fatty acids and microbial community.Results: Goats fed CG increased molar acetate (P < 0.05), lowered butyrate and propionate in the fore and hindgut comparing to those goats received CM. Goats received CM had higher (P < 0.05) dH2 while lowered dH2S in the fore and hindgut than those goats fed with CG treatment. The fore and hindgut had higher (P < 0.01) 16S rRNA gene copies of bacteria, protozoa, methanogens and 18S rRNA gene copies fungi than in the ileum and cecum. Goats fed CG diet had higher (P < 0.05)16S rRNA gene copies of bacteria, protozoa, methanogens, and 18S rRNA gene copies of fungi than those goats fed with CM diet. Conclusion fore and hindguts improved dissolved gasses, fatty acids and microbial community comparing with in the ileum and cecum. Goats fed CM had improved the Methanobacterials order and Methanobrevibacter genus as compared with those goats fed CG. The study suggested that hindgut segments have a reasonable contribution as foregut to methane emissions from goats.


Introduction
Fore and hindgut segments play a signi cant role for microbial digestion in ruminants. Ruminal carbohydrate fermentation produces VFA, H 2 , and CO 2 as end of fermentation products (Hungate 1967). In addition to foregut fermentation, microbial digestion of feeds is also takes place in the hindgut. Our study revealed that hindgut fermentation has signi cant contribution as that of foregut to fermentation products which is in line with the reports of Ulyatt et al (1975). Dihydrogen (H 2 ) is produced during feed fermentation in the fore and hindgut and eliminated by methanogens bacteria from the host animal in the form of methane (CH 4 ) for the normal physiological function and continuity of digestion in ruminants. Dietary interventions which stimulate production of alternative H 2 sink or inhibition of H 2 utilization is an option to ensure redirecting of H 2 away from methanogens to other bene cial pathways for animal productivity and of reduction of CH 4 emissions from ruminants. Unlike to brous carbohydrate, starchy carbohydrate can improve H 2 production, which stimulates reducing equivalents like propionate to sink H 2 than methane forming substrates (Bougouin et al. 2018). We hypothesized that Using a feedstuff with dietary high sulfur content could be used as H 2 reducing equivalent, since sulfate involves metabolic hydrogen in the reaction for the production of H 2 S (∆G0 = − 84.4 kJ), which can also favor alternative reducing equivalents than methanogenesis (∆G0 = − 67.9 kJ) (Ungerfeld EM 2006). Corn is predominantly utilized in ruminant diet and recently as an input for bioenergy production in various countries, thus its cost intensive to use as animal feed ingredient. Therefore, using alternative corn by-product is a feasible and can replace corn in ruminant diet (Kerr et al. 2008). Using corn byproducts in ruminant diet is underexplored, thus the study hypothesized that Carbohydrate diets could alter dissolved gas production and microbial community in the gastrointestinal tracts of goats. Therefore, the study investigated fermentation, dissolved hydrogen and microbial community in the gastrointestinal tract of goats fed corn meal or corn gluten (CG).

Experimental Design and Diets
Twenty-four male intact Xiangdong black goats (10 ± 0.2 months, 17.5 ± 2.67 kg (mean ± SD) were used for the study.
The experiment was conducted using a randomized block design with two dietary treatments (corn meal (CM) or corn gluten (CG); 400 g/kg DM each). The ration formulated to meet 1.3 times maintenance requirements of animals (Table 1). Goats were assigned to 12 blocks based on initial weight. Each block consisted of two animals and each animal within a block was randomly allotted to one of the two dietary treatments. Animals were held in individual pens equipped with feeding and watering trough. Animals in each pens had free access to water and fed individually equal meals at 0800 and 1700 h. Animals had 28 d of acclimatization to the diets and 2 d for GIT uid samples collection.

GIT uid Sampling
Goats were slaughtered, the abdomen was opened and each GIT segment (rumen, ileum, cecum, colon and rectum) were tied with a sterile thread at the start and end of each segment to avoid mixing of contents. Each GIT segment was longitudinally incised along the dorsal line using a sterile equipment. Samples of liquids from the rumen, ileum, cecum, colon and rectum were ltered through four layers of sterile cheesecloth and approximately 300 mL liquid from each segment was collected in air tight tubes according to Stevenson and Weimer (2007). One 100-mL liquid subsample from each GIT segment was directly chilled using liquid nitrogen and kept for one month at − 80°C for DNA isolation and subsequent quanti cation of microbial groups. Another subsample ~ 20 mL from each GIT segment used for immediate measurement of rumen pH and dissolved gasses.

Sample Analysis
Subsamples of the feed offered and refusals were dried (105°C, 24 h), crashed to pass through a 1-mm sieve and determined for dry matter (DM). Ash was dried in the furnace at 550°C for 8 h. Organic matter (OM) was computed by the difference between DM and ash content. Crude protein (CP) (N x 6.25) was determined using the Kjeldahl method (AOAC 1995). Gross energy (GE) was analyzed using an isothermal automatic calorimeter (5E-AC 8018, Changsha Kaiyuan Instruments Co. Ltd, Changsha, China). Neutral detergent ber (NDF) and acid detergent ber (ADF) values were analyzed and expressed inclusive of residual ash according to Van Soest et al. (1991), and NDF was determined with the addition of amylase and sodium sul te. The starch was analyzed according to Kartchner and Theurer (1981).
The pH of GIT samples was measured using a portable pH meter (Starter 300; Ohaus Instruments Co. Ltd, Shanghai, China). The dissolved hydrogen (dH 2 ) dissolved hydrogen sulfur (dH 2 S) of GIT samples was determined using microsensor with H 2 and H 2 S electrode, respectively, according to protocols of the manufacturer's manual (Unisense, Aarhus, Denmark). Concentrations of VFA were determined using 2 mL of GIT centrifuged at 12,000 x g for 10 min at 4°C. Aliquots of the supernatants (1.5 mL) was transferred into plastic tubes each containing 0.15 mL of 25% (wt/vol) metaphosphoric acid. Then individual VFA concentrations were analyzed by gas chromatography (GC, Agilent 7890A, and Agilent Inc.). Ammonia concentrations were measured according to Weatherburn (1967).
Gastrointestinal uid (GIT) samples were freeze-thawed prior to physical disruption using a bead beater. The total DNA were extracted from repeated beads beating plus column puri cation as described by Yu and Morrison (2004 Ash was determined in a mu e furnace at 550°C for 8 h. Organic matter (OM) was calculated by the difference between DM and ash content. Crude protein (CP) (N x 6.25) was analyzed using the Kjeldahl method (AOAC 1995).
Gross energy (GE) content was determined using an isothermal automatic calorimeter (5E-AC 8018, Changsha Kaiyuan Instruments Co. Ltd., Changsha, China). Neutral detergent ber (NDF) and acid detergent ber (ADF) contents were determined and expressed inclusive of residual ash according to Van Soest et al. (1991), and NDF was assayed with the addition of amylase and sodium sul te. Starch content was analyzed according to Kartchner and Theurer (1981).
The pH of GIT samples was measured using pH meter (Starter 300; Ohaus Instruments Co. Ltd., Shanghai, China).
The dissolved hydrogen (dH 2 ) and dissolved hydrogen sulfur (dH 2 S) of GIT samples were measured using microsensor with H 2 and H 2 S electrode, respectively, according to protocols of the manufacturer's manual (Unisense, Aarhus, Denmark). Concentrations of VFA were analyzed using samples of 2 mL from each GIT, centrifuged at 12,000 x g for 10 min at 4°C. Aliquots of the supernatants (1.5 mL) were transferred into plastic tubes each containing 0.15 mL of 25% (wt/vol) metaphosphoric acid. Then individual VFA concentrations were analyzed by gas chromatography (GC, Agilent 7890A, and Agilent Inc.). Ammonia concentrations were measured according to Weatherburn (1967).
The total DNA of GIT samples were isolated as described by Yu

Statistical analysis
Statistical analysis was analyzed using SPSS 19.0 software (SPSS Inc., Chicago, IL). The experimental model was a randomized blocks arranged in a factorial arrangement (diet by GIT). Data were analysed using a mixed linear model including diet and GIT segments as xed effect, and, block and interaction as a random effect. Tukey's adjustment was used to test mean separation. Differences of P ≤ 0.05 were considered as a signi cant and 0.05 < P ≤ 0.1 was considered as a trend.

Results
Gastrointestinal tract and diet in uenced the molar concentrations of volatile fatty acids and ammonia. Rumen (foregut), cecum, colon and rectum (hindgut) had higher (P < 0.05) ammonia than in the ileum (Table 3). However, total volatile fatty acids (VFA), molar acetate, propionate and butyrate were higher in the rumen, cecum and colon than in the ileum and cecum. Goats fed corn gluten (CG) had higher molar concentration of ammonia than those goats fed with CM. Goats fed corn gluten (CG) had higher concentration of acetate in the fore and hindgut than those fed with corn meal (CM; Fig. 1b; P < 0.05), while goats fed with CM improved the concentration of butyrate and propionate in the fore and hindgut than those fed with CG. (Fig. 1c and d; P < 0.05). Ace/Pro = acetate to propionate ratio; CE = cecum; CG = corn gluten; CM = corn meal; CO = colon; G*D = interaction (GIT x diet); IL = ileum; P = probability; RE = rectum; RU = rumen; VFA, volatile fatty acids The percentages of the individual volatile fatty acids were affected by the GIT segments, the percentages of acetate were higher (P < 0.05) in the fore and hindgut than in the ileum, while a lowered (P < 0.05) propionate was detected in the ileum than those in the fore and hindgut segments. A lowered trend (P = 0.07) of butyrate was also observed in the fore and hindgut than in the ileum (Table 4). Dietary treatments in uenced propionate, goats fed with CM increased (P < 0.05) propionate than those goats fed with CG treatment. However, no interaction effects GIT and diet noted for the proportion of individual fatty acids.  The concentrations of dissolved gasses, dissolved hydrogen (dH 2 ) and dissolved hydrogen sulfur (dH 2 S) were affected by the GIT segments and dietary treatments (Table 4) and, interaction of GIT and diet (Fig. 2). Fore and hindgut segments had higher (P < 0.01) dH 2 and dH 2 S than in the ileum. Goats fed CM had higher (P < 0.01) dH 2 than those goats fed with CG treatment, while goats fed CG had higher (P < 0.05) dH 2 S comparing with goats fed with CM treatment (Table 4). Goats fed CM had higher dH 2 in the fore and hindgut than those fed with CG dietary treatment ( Fig. 2a; P < 0.05). However, goats fed with CG had higher (P < 0.05) dH 2 S in the fore and hindgut than those fed with CG dietary treatment ( Fig. 2b: P < 0.05).
The gene copies of major microbial groups and bacterial species were in uenced by the GIT segments and dietary treatments (Table 5), with no interaction effects of GIT and diet detected for the gene copies of microbial groups. The fore and hindgut had higher (P < 0.01) 16S rRNA gene copies of bacteria, methanogens and, 18S rRNA gene copies of protozoa and fungi than in the ileum and cecum. Goats fed CG had higher (P < 0.05)16S rRNA gene copies of bacteria, methanogens and 18S rRNA gene copies of protozoa and fungi than those goats fed CM (Table 5). Likewise, fore and hindgut had higher (P < 0.05) 16S rRNA gene copies of Prevotella ruminicola, Selenomonas rumination, Ruminococcus amylophilus, Ruminococcus albus, Ruminococcus avefaciens, and Fibrobacter succinogenes than in the ileum. However, lowered gene copies of Selenomonas rumination, Ruminococcus amylophilus, Ruminococcus albus, Ruminococcus avefaciens, recorded in the hindgut than in the rumen. Goats fed CM had higher (P < 0.05) Ruminococcus amylophilus, and lowered Prevotella ruminicola, Selenomonas rumination, Ruminococcus albus, Ruminococcus avefaciens, and Fibrobacter succinogenes than those goats fed CG. Methanogens diversity indices of Chao1 (P = 0.06) and ace (P < 0.04) were in uenced by the dietary groups, goats fed CG tended to have lower chao1 and ace diversities than those goats fed with CM diet. The species diversity was also higher (P < 0.04) for goats fed CM diet than those goats fed CG. However, the rest of alpha diversity metrics were not affected by the dietary treatments (Table 6). Goats fed CG had higher abundances of the orders Methanosarcinales (P < 0.05), Methanomicrobiales (P = 0.09) and Thermoplasmalates recently called Methanomassiliicoccales (P = 0.08) than those fed with CM, while, less (P < 0.05) abundance of Methanobacteriales than those fed with CM diet. Regardless of the dietary regimens. Goats fed CG has tended to have higher abundances of the genera of Methanimicrococcus (P = 0.06) and Methanomicrobium (P = 0.09), and lowered abundance of Methanobrevibacter (P < 0.05) than those goats fed with CM diet (Table 7).

Discussion
The increased volatile fatty acids (VFA) production in the fore and hindgut segments can be associated with inhabitance of abundant microbes in the aforementioned segments that possibly hydrolyze the diet to organic acids. In addition, the higher volatile fatty acids in the rumen, colon and rectum over cecum and ileum caused by relatively higher extent of microbes and fermentation products noticed in the fore and distal hindgut (colon and rectum). The improved molar acetate in the fore and hindgut of goats fed corn gluten is associated with higher ber (NDF and ADF) contents of the diet, while decreased propionate and butyrate for those goats fed with CM caused by higher starch content of the diet.
Hydrogen regulates the production of volatile fatty acids (VFA), methane and other fermentation pathways in ruminant digestion. Enteric methane (CH 4 ) primarily produced using hydrogen (H 2 ) substrate in ruminants (Teklebrhan et al. 2018). Least ber carbohydrate fermentation in uenced volatile fatty acids production pathways, caused less e ciency of H 2 production per mol of glucose fermented than brous diet (Wang et al. 2016), consistent with improved propionate production pathway in goats fed with elevated starch in CM than those in CG treatment in the present study. in addition, digestible carbohydrate has faster rate and degree of fermentation than ber and protein feeds, resulting a fast accumulation of dH 2 in the rumen (Teklebrhan et al. 2020), agreed to this ndings, goats fed higher starch in CM diet improved dH 2 in the fore and hindgut than those goats received CG diet.
Thermodynamically, the higher dH 2 concentrations in the fore and hindgut segments of goats fed CM diet indicated less utilization of H 2 and thus facilitates fermentation pathways produced less H 2 over more H 2 per unit glucose fermented in the segments, which is supported by the positive correlations between dissolved dH 2 and molar percentage of propionate in the segments in the current study. The lower dH 2 in the fore and hindgut of goats fed CG treatment was resulted from lower starch, and higher protein content of the diet, increased ammonia concentration (+ 25 % vs. CM), indicating competence of amino acid consumption for microbial growth was lowered, causing to decrease H 2 . However, increased dH 2 S in the fore and hindgut of goats fed CG was caused by higher sulfur in the CG than CM dietary treatment because sulfate sinks H 2 and directed the electron away from methanogens towards dH 2 S production.
The increased16S rRNA gene copies of bacteria, protozoa, methanogens and 18S rRNA gene copies of fungi in the fore and hindgut segments indicate the segments are primarily evolved for microbial fermentation. However, the increased gene copies of the microbial groups of bacteria, protozoa, methanogens and fungi in the distal hindgut may indicate its importance for microbial fermentation of the feeds escaped ruminal degradation than cecum. Moreover, the increased bacterial species 16S rRNA gene copies of Prevotella ruminicola, Selenomonas rumination, Ruminococcus amylophilus, Ruminococcus albus, Ruminococcus avefaciens, and Fibrobacter succinogenes in the fore and hindgut associated with high rate of microbial fermentation in these segments than in the ileum. Whereas, the lowest gene copies of Selenomonas rumination, Ruminococcus amylophilus, Ruminococcus albus, Ruminococcus avefaciens, in the hindgut is primarily because these bacterial species are typically predominated in the rumen for cellulolytic functions. Dietary groups also in uenced the microbial groups, goats fed CG treatment increased the gene copies of the 16S rRNA gene copies of bacteria, protozoa, methanogens and 18S rRNA gene The study also evaluated the methanogen diversities between the dietary groups, goats fed CG had lower abundances of the Methanobacteriales order which is responsible for CH 4 production and it was consistent with higher methane production noticed in goats fed CM versus CG (Teklebrhan et al. 2020

Conclusion
Fore and hindguts improved dissolved gasses, fatty acids and microbial community comparing with ileum and cecum segments. Goats fed CG improved the production of acetate, while decreased propionate and butyrate in the fore and hindgut, then those goats fed CM. Goats fed CG reduced the production of dH 2 , but increased dH 2 S in the fore and hindgut segments than those goats fed CM. Fore and hindgut segments increased the populations of bacteria, protozoa, methanogens and fungi than in the ileum. Goats fed CG increased the16S rRNA gene copies of bacteria, methanogens, and 18S rRNA gene copies of protozoa and fungi than those goats fed with CM treatment. Goats fed CM had improved the Methanobacterials order and Methanobrevibacter genus as compared with those goats fed CG. The study suggested that hindgut segments have a reasonable contribution as foregut in terms of dissolved gasses and methane emissions from goats.

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Ethics approval and consent to participate All authors agreed to the content and publish the paper.

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Con icts of interests
Authors declared that there is no con ict of interest

Funding
There was no speci c fund received Authors' contributions TT designed study and wrote the manuscript. ZL revised and edited the manuscript. All authors read and approved the manuscript for the current submission.