Effect of tannins and cellulase on growth performance, nutrients digestibility, blood profiles, intestinal morphology and carcass characteristics in Hu sheep

Objective This study was conducted to evaluate the effects of tannins and cellulase on growth performance, nutrient digestibility, blood profiles, intestinal morphology, and carcass characteristics in Hu sheep. Methods A total of 48 three-month-old meat Hu sheep (25.05±0.9 kg) were blocked based on body weight, and randomly allotted to 4 treatments with 3 replicates of 4 sheep each. The experiment lasted for 80 d, and dietary treatments were as follows: i) CON, control diet; ii) TAN, CON+0.1% tannins; iii) CEL, CON+0.1% cellulase; iv) TAN+CEL, CON+0.1% tannins and 0.1% cellulase. Results Compared with CON, CEL, and TAN+CEL had greater (p<0.05) final body weight (FBW) and average daily gain but lower (p<0.05) feed conversion ratio, while FBW of TAN+ CEL was lower (p<0.05) than that of CEL. The apparent total tract digestibility (ATTD) of dry matter in TAN, CEL, and TAN+CEL groups were higher (p<0.05) than that in CON. CEL and TAN+CEL groups had greater (p<0.05) ATTD of crude fiber compared with TAN and CON, while TAN group had lower (p<0.05) ATTD of crude protein than other treatments. TAN, CEL, and TAN+CEL groups increased (p<0.05) serum globulin and alkaline phosphatase but decreased (p<0.05) albumin/globulin. Serum total protein was greatest for TAN+CEL, intermediate for TAN and CEL and least for CON (p<0.05). TAN+CEL group increased (p<0.05) dressing percentage compared with CON, while the backfat thickness of CEL was lower (p<0.05) than that of CON. The villus height of jejunum and ileum in CEL and TAN+CEL groups were greater (p<0.05) than that in CON, and the crypt depth and villus height: crypt depth of jejunum were increased (p<0.05) in TAN, CEL, and TAN+CEL groups. Conclusion The addition of tannins and cellulase together promoted nutrient digestion, liver protein synthesis and intestinal development and thus improved growth performance and carcass characteristics.


INTRODUCTION
Tannins are a group of polyphenolic compounds generally occurring in the forages con sumed by ruminants [1]. Researches have shown that tannins improved protein utilization in ruminants because they could complex with the proteins and protect them from deg radation, and then release the protein later in the intestine [2]. Therefore, the amount of highquality protein available to ruminants for absorption would be increased [3]. More over, feeding tannincontaining forages or tannins supplementation reduced CH 4 and N 2 O emissions and increased the amount of energy available to the ruminants. It is reported that tannins could reduce total methanogen populations and meth ane emissions [4].
In ruminants, fiber digestion was a limiting factor in nutrient utilization and ruminant production systems [5]. Exogenous cellulase supplementation has been proved to be an effec tive mean of improving nutrient utilization of forages and yielding more metabolizable energy for ruminants [5]. Pre vious studies demonstrated that although the results were inconsistent, the addition of cellulase could increase nutri ent digestibility and feed conversion [6].
However, tannins could interact with the cell wall and secrete catabolic enzymes and then inhibit cellulolytic microorgan isms in the rumen, and ultimately reduce the secretion of cellulase [7]. Moreover, previous study proved that tannins presented enzymespecific interactions with cellulase to in hibit its activity. As a result, fiber digestion would be interfered by tannins [8].
An in vitro experiment showed that cellulase addition de creased soluble tannins and condensed tannins with increasing enzyme inclusions [9], which might counteract the reduc tion of cellulase. However, reports about the effects of tannins or cellulase alone or together on sheep were very limited. Thus, we hypothesized that the addition of tannins and cellulase together might have a beneficial effect in sheep. Therefore, the objective of this study was to determine the effects of tannins and cellulase on growth performance, nutrients di gestibility, blood profiles, intestinal morphology and carcass characteristics in Hu sheep.

Animals, diets, and treatments
All animal experiment procedures in this study were approved by the Institutional Animal Care and Use Committee of Yan bian University. The experiment was conducted at Animal Husbandry Research Institute of Yanbian Academy of Agri cultural Sciences from May 2018 to September 2018. A total of 48 threemonthold meat Hu sheep (25.05±0.9 kg) were blocked based on body weight and randomly allocated into 4 treatments with 3 pens per treatment and 4 sheep per pen. Prior to the experiment, the animals were treated against in ternal and external parasites. Experimental treatments included: i) CON, control diet; ii) TAN, CON+0.1% tannins (95% pu rity); iii) CEL, CON+0.1% cellulase (15,000 IU/g); iv) TAN+ CEL, CON+0.1% tannins and 0.1% cellulase. After an ad aptation period of 14 days to the experimental conditions, the sheep stayed for 80d feeding trial. Diets were formulated to provide all the nutrients to meet or exceed NRC require ments [10]. All diets were kept at a concentrate: roughage ratio of 50:50, where the roughage component was maize straws and the concentrate consisted of corn, wheat bran, soybean meal and premix ( Table 1). The diets were offered to the animals in two equal halves at 7:30 and 15:30 daily. Animals were allowed ad libitum access to feed and water throughout the experiment.

Growth performance
The sheep were weighed individually on d 1 and 80 of the experiment. Average daily gain (ADG) was calculated by the difference between final body weight (FBW) and initial body weight. Feed offered and refused was quantified daily for each pen to calculate average daily feed intake (ADFI). Feed conversion ratio (F/G) was calculated as a ratio of ADFI to ADG.

Nutrient digestibility
Digestibility trial was conducted at the end of the feeding trial and feces were collected daily for 6 days from a fecal collec tion bag attached to the sheep. The feces were weighed and mixed, and 25% of representative samples were taken and frozen at -20°C. At the end of the collection period, all sam ples of each sheep were pooled and 25% of the composite sample was taken and dried at 65°C for 72 h. Daily feed per animal was also collected. The chemical analysis of feces and feed were performed to determined dry matter (DM), crude protein (CP), ether extract (EE), crude fiber (CF) and ash according to standard AOAC method [11]. Apparent total tract digestibility (ATTD) of DM and other nutrients was determined as a percentage of the nutrient intake not recov ered in the feces.

Blood profiles
At the end of feeding trial, blood samples (10 mL) via the jug ular vein were collected from three sheep from each treatment. The blood samples were stored at -4°C and then submitted for biochemical parameters analysis. The albumin (ALB), globu lin (GLB), albumin/globulin (A/G), total protein (TP), total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), alkaline phosphatase (ALP), glucose (GLU) concentrations were determined by an automatic biochemistry analyzer (Hita chi Model 7600, Tokyo, Japan).

Carcass characteristics
After blood collection, the same sheep were weighted and euthanized with an intravenous injection of 200 mg/kg of body weight (BW) sodium pentobarbital (Sigma Chemical Co., St. Louis, MO, USA) after 24 h fasting. Decapitation and skinning of the sheep were undertaken after removing legs below the hock and knee joints. The carcass weight was checked after removing thoracic, abdominal and pelvic contents, and the organs (heart, liver, lungs, and kidneys) were weighed. Dressing percentage was calculated as carcass weight divided by the live body weight. The carcass was split between 12th and 13th ribs perpendicular to the backbone to measure the cross section of rib eye muscle area and the backfat thick ness over the maximum muscle depth at same site was also recorded.

Tissue collection and measurement
After abdominal contents collection, 2 to 2.5 cm longitudinal sections of jejunum and ileum were taken from 5 cm distal to the duodenojejunal junction and jejunoileal junction sep arately. All samples were soaked in 4% paraformaldehyde fix solution immediately and then embedded in paraffin and sectioned (3 to 5 μm). Slides were stained with hematoxylin and eosin (H&E) as described in Holle et al [12], then digi tized and measured by a fluorescence microscope camera via DP2BSW software (Olympus, Tokyo, Japan). The crypt depth (CD), villus height (VH), and VH:CD ratio was mea sured according to methods described by McLeod et al [13]. For each section, 12 of the most representative villi and crypt were used for measurement.

Statistical analysis
Data were analyzed using the general linear model Procedure of SAS as a randomized complete block design (SAS Inst. Inc., Cary, NC, USA) with the pen being considered as the exper imental unit. The average initial BW was used as a covariate for ADFI and ADG. Differences among treatment means were determined using Tukey's range test. Variability in the data was expressed as the standard error means and a probability level of p<0.05 was considered statistically significant.

RESULTS
Growth performance CEL and TAN+CEL groups increased (p<0.05) FBW com pared with CON, and the FBW of CEL group was higher (p< 0.05) than that of TAN+CEL group (Table 2). ADG did not differ (p>0.05) between CEL and TAN+CEL group but both were higher than CON and TAN group (p<0.05). There were no significant differences in ADFI among treatments (p>0.05). F/G of CEL group was lower (p<0.05) than that of CON group. TAN and TAN+CEL groups had no significant effects on F/G compared with CON.
Nutrient digestibility ATTD of DM in TAN, CEL and TAN+CEL groups were great er (p<0.05) than that in CON (Table 3). CEL and TAN+CEL groups had higher (p<0.05) ATTD of CF compared with TAN and CON, while TAN group had lower (p<0.05) ATTD of CP than other treatments. ATTD of EE or ash did not differ (p>0.05) among groups.

Blood profiles
Compared with CON, TAN, CEL, and TAN+CEL groups increased (p<0.05) serum GLB and ALP but decreased (p< 0.05) A/G (Table 4). TP was greatest for TAN+CEL group, intermediate for TAN and CEL group, and least for CON (p<0.05). No differences were observed (p>0.05) in ALB, TC, TG, ALT, or GLU among treatments.

Intestinal morphology
The VH of jejunum and ileum in CEL and TAN+CEL groups was greater (p<0.05) than that in CON, and no differences were observed (p>0.05) between TAN and CON (Table 5). Compared with CON, the CP and VH:CD of jejunum were increased (p<0.05) in TAN, CEL, and TAN+CEL groups. How ever, no significant differences in the CD and VH:CD were found (p>0.05) for the ileum.

Carcass characteristics
The slaughter body weight of CEL group was higher (p<0.05) than that of CON and no differences were observed (p>0.05) among the other three groups ( Table 6). The carcass weight did not (p>0.05) differ between CEL and TAN+CEL group but both were greater than CON (p<0.05). TAN+CEL group significantly increased (p<0.05) dressing percentage com

DISCUSSION
It has been generally accepted that tannins might cause a re duction of daily feed intake and weight gain [14]. However, FBW, ADG, ADFI, and F/G were not affected by tannins sup plementation alone in current study. A metaanalysis study indicated that tannins did not have negative effect on DM intake or live weight change in growing sheep, which was consistent with results in the present study [15]. Compared with CON, the cellulase supplementation alone increased FBW and ADG, but reduced F/G in the current study. Simi larly, calves fed cellulase had higher FBW, ADG and feed efficiency [5]. The mixture of tannins and cellulase increased FBW and ADG compared with CON in the present study. However, the sheep fed tannins and cellulase together had lower FBW than those fed cellulase alone. The results sug gested that the cellulase supplementation alone improved growth performance but tannins may partly interfer. As is well known, tannins have an ability to complex with protein to protect it against ruminal degradation, and the protection was more effective for proteins than DM or other nutrients [16]. In the present study, tannins supplementation alone increased DM digestibility and decreased CP digesti bility. Similarly, it is noted that quebracho tannin extract in diets decreased apparent digestibility of CP in cattle [17]. However, pervious researches also showed that tannins sup plementation would decrease DM digestibility in ruminants [15]. These inconsistent results might be attributed to the different origins of tannins, which vary greatly in their ca pacity to bind carbohydrates [18]. The increased digestibility of DM and CF noted with cellulase supplementation alone in the current study agreed with the results of other studies. It is found that lambs fed exogenous cellulase diets had greater DM and CF digestibility than those fed noncellulase diets [5]. In vitro research also proved that exogenous cellulase supplementation alone increased substrate fiber degrada tion, volatile fatty acid production, and ruminal microbial growth to enhance rumen fermentation [19]. This improve ment could ultimately increase DM and CF digestibility and might partly explain the increase of ADG and reduction of F/G. The mixture of tannins and cellulase supplementa tion increased DM and CF digestibility compared with CON and had greater CP digestibility than tannins group. As men tioned above, the results indicated that tannins in the present study might not inactivate the cellulase, and the effect of cellulase could partially counteract the reduction of CP di gestibility by tannins.
Serum TP was a marker of nutritional level and was in creased by tannins and cellulase in present study [20]. The results revealed that liver protein synthesis and rumen mi crobial protein production might be improved by tannins and cellulase, even when the CP digestibility was decreased by tannins. Similarly, previous study found that supplement ing lambs' diets with fiberolytic enzymes significantly increased serum TP compared with CON [21]. Furthermore, the mix ture of tannins and cellulase had greater serum TP than other treatments. As mentioned above, tannins and cellulase to gether increased CP digestibility compared with tannins alone and the cellulase could eliminate the side effect of tan nins on the CP digestibility, which might contribute to the higher serum TP. Therefore, the results suggested that the improvement of serum TP may be enhanced by the interac tion of tannins and cellulase. Moreover, the increased serum GLB contributed to the increase of serum TP and decrease of A/G in the current study. Serum GLB played an important role in immune system and the increased GLB could enhance humoral immune response against pathogenic viruses and microorganisms [22]. Therefore, the increased serum GLB indicated that tannins, cellulase and the mixture could im prove immunity of sheep. Besides, the ALP was increased by tannins, cellulase and the mixture, which played an integral role in metabolism within the liver and development within the skeleton [23]. The results revealed that liver function and skeleton development might be improved by tannins and cel lulase. However, the GLB and ALP were not increased by the mixture compared with tannins or cellulase alone. The results indicated that there was not a synergy between tannins and cellulase on improving serum GLB and ALP.
In the present study, the mixture of tannins and cellulase increased carcass weight and dressing percentage compared with CON, which indicated that the addition of tannins and cellulase together could improve slaughter carcass character istics. On the contrary, feeding xylanase and cellulase had no effect on hot carcass weight and dressing percentage of steers [24]. Tannins in birdsfoot trefoil did not affect carcass weight or dressingout of lambs [25]. The differences might be due to different species and tannins origins. Besides, the backfat thickness was decreased by tannins alone. Similarly, finishing steers fed hightannin sorghum diets had lower backfat thick ness than CON [26]. The results illustrated that tannins had adverse effect on backfat and the cellulase could eliminate the side effect. The tannins and cellulase increased liver, heart and kidney weight numerically, which was consistent with previous study [21]. The results showed that the mixture of tannins and cellulase might promote organs development, which may have contributed to increased ADG and the changes in blood profiles.
The VH, CD, and VH:CD ratio are markers of bowel in tegrity and increase during development [27]. In the present study, the CD of jejunum was decreased and VH:CD was in creased by tannins. However, feeding 0.5% quebracho tannin induced ulceration and an increase in mucosal histiocytes in the jejunum and ileum of sheep [28]. The differences might be due to the higher tannins content in their diets compared with the present study. Our results indicated that 0.1% tan nins could promote jejunum development. The cellulase increased VH of jejunum and ileum and VH:CD of jejunum, but decreased VH of jejunum in the current study. The re sults indicated cellulase could promote jejunum and ileum development and ultimately improve nutrient digestion and absorption, which may contribute to the higher ADG and feed efficiency [27,29]. The mixture of tannins and cellulase also decreased VH of jejunum, but increased VH and VH:CD of jejunum, especially the VH of ileum. The results suggested that there was a synergy between tannins and cellulase on the development of ileum. However, no significant differences in the CD or VH:CD was found for the ileum, illustrating that the morphology of the jejunum was more easily affected by tannins and cellulase than that of the ileum during growth [27].

CONCLUSION
The tannins supplementation alone increased ATTD of DM, serum GLB and TP, CD, and VH:CD of jejunum, but de creased ATTD of CP and backfat thickness. The cellulase supplementation alone increased ADG, feed efficiency, ATTD of DM and CF, serum TP, GLB, and ALP, VH and VH:CD of jejunum and VH of ileum, while decreased serum A/G and CD of jejunum. The mixture of tannins and cellulase improved ATTD of CP, serum TP and CD of ileum, but decreased FBW compared with tannins or cellulase alone. The results illustrated that although CP digestibility and FBW may be negatively affected by tannins, the mixture of tannins and cellulase might promote nutrient digestion, liver protein synthesis and intes tinal development and thus improved growth performance and carcass characteristics.