Long-term effect of subacute ruminal acidosis on the morphology and function of rumen epithelial barrier in lactating goats

HU Hong-lian ,YANG Shu-qing ,CHENG Meng ,SONG Li-wen ,XU Ming ,GAO Min ,YU Zhong-tang

1 Institue of Animal Nutrition and Feed,Inner Mongolia Academy of Agriculture &Animal Husbandry Sciences,Hohhot 010031,P.R.China

2 College of Animal Science,Inner Mongolia Agricultural University,Hohhot 010018,P.R.China

3 Department of Animal Sciences,The Ohio State University,Columbus 43210,USA

Abstract Grain-induced subacute ruminal acidosis (SARA) impairs rumen epithelial barrier function,but it is yet to be determined if SARA can cause persistent damage to the morphology and function of the rumen epithelial barrier. The objective of the present study was to investigate if SARA has persistent effects on the morphological structure and permeability of ruminal epithelium and the expression of the genes involved in epithelial barrier function using a lactating goat model. Twelve mid-lactating Saanen goats with rumen cannulas were randomly assigned to 1 of 2 groups: control group (Ctrl,n=4) fed a basal diet with a non-fiber carbohydrate (NFC) to neutral detergent fiber (NDF) ratio of 1.40,and SARA group (SARA,n=8) fed the same basal diet but with increasing NFC to NDF ratio from 1.4 to 1.79,2.31,and 3.23 overtime to induce SARA. At the end of the SARA challenge (post-SARA),4 goats were randomly selected from the SARA group and fed only hay mixture ad libitum for another 4 weeks to allow for restitution (post-SARA). Ruminal pH was continuously recorded to monitor the severity of SARA. Samples of the ventral ruminal epithelium were collected after slaughter to examine the structural and functional changes of the ruminal epithelium using transmission electron microscopy (TEM),Ussing chambers,qRT-PCR,and Western bolt analyses. Compared with the Ctrl group,ruminal papilla length,width,surface area and thickness of stratum corneum increased (P＜0.05),while stratum spinosum and basale thickness,and total depth of the epithelium decreased (P＜0.05) in the SARA group. These changes diminished or tended to return to the levels of the Ctrl group in the post-SARA group (P＞0.05). The SARA challenge also decreased cellular junction and widened the intercellular space between epithelial cells. Rumen transepithelial short-circuit current (Isc),tissue conductance (Gt),and mucosa-to-serosa flux of paracellular horseradish peroxidase (HRP) all increased (P＜0.05) both in the SARA and post-SARA groups,which indicates that SARA can induce a sustained increase in epithelial permeability and barrier dysfunction. Moreover,the mRNA and protein expressions of CLDN1,OCLN and ZO-1 were down-regulated(P＜0.01) in both the SARA and post-SARA groups. The results of this study showed that SARA could result in sustained epithelial barrier dysfunction,at both structural and functional levels,which is associated with decreased expression of rumen epithelial tight junction proteins,and the restitution of rumen epithelial barrier function is slower than that of its morphology.

Keywords: subacute ruminal acidosis,epithelial morphology,permeability,tight junction proteins,restitution

1.lntroduction

Ruminants are of major worldwide interest and economic importance because of the increasing demand for meat and milk. It has become common to use readily fermentable (i.e.,high-grain) diets to maximize the productivity and performance of high-yielding cattle,sheep,and goats. However,this feeding strategy creates a challenge to the rumen microbiome and especially for the ruminal epithelium with respect to the adaptation to the resultant high acidity,and it can place the animals at a greater risk of developing metabolic diseases. The most economically important severe metabolic disease of ruminants is subacute ruminal acidosis (SARA),which impairs gastrointestinal functions,while inducing inflammation and other health-related problems (Khafipouret al.2009;Zebeli and Metzler-Zebeli 2012;Steeleet al.2016). To date,SARA has been one of the most prevalent animal welfare issues in intensive ruminant production systems,especially in regions where high-quality forages are in short supply.

Several studies conducted in cows and goats demonstrated that grain-rich diets or SARA induced thereby lead to disruption of the rumen epithelial barrier as indicated by disrupted morphological and histological integrity of the ruminal papillae (Steeleet al.2009;Zhanget al.2017,2020),increased lesions(Steeleet al.2011) and paracellular permeability of ruminal epithelium (Klevenhusenet al.2013;Meissneret al.2017;Sunet al.2018). It is well recognized that elevated levels of lipopolysaccharides (LPS),high acidity,and hyperosmolality induced by SARA decrease the barrier function and enhance the permeability of the ruminal epithelium. The increased rumen epithelial permeability following the acidotic insult may be caused by impaired adhesion and organization of cells of the stratum granulosum (Steeleet al.2011),increased cell necrosis (Liuet al.2013),and delay of the return of the swollen epithelial cells to normal cell volume,all of which further opens the paracellular space jointly with the damaged tight junction (TJ) (Aschenbachet al.2019).At the molecular level,the enhanced rumen epithelial permeability induced by grain-rich diets is caused by down-regulated expression of genes encoding critical barrier function proteins,including the TJ proteins (e.g.,CLDN4 and OCLN),and by redistribution of CLDN1,CLDN4,and OCLN (Liuet al.2013). Our recent study demonstrated that grain-induced SARA increased rumen epithelial permeability and down-regulated the intercellular junction proteins including CLDN1,CLDN7,OCLN,ZO-1,Cx43,and DSG1,simultaneously (Sunet al.2018).However,the underlying mechanisms of the disruption of the barrier functions of ruminal epithelium in response to SARA are still not completely understood.

Although grain-induced SARA has been reported to impair ruminal barrier function (Steeleet al.2011;Zhanget al.2017,2019),most of the studies reported so far have focused on the short-term effects on the structure and function of ruminal epithelium,leaving the likelihood of persistence of the damaging changes unknown. A recent study reported that the recovery of rumen epithelial function bears a high degree of uncertainty because only a few studies have investigated the functional recovery of the ruminal epithelium after an acidotic insult (Aschenbachet al.2019). Therefore,it remains largely unknown if SARA results in compromised barrier function of ruminal epithelium in a sustained manner. It was hypothesized that SARA might cause sustained rumen epithelial barrier dysfunction,which might not recover. Thus,the objective of this study was to investigate the long-term effect of grain-induced SARA on the lactation performance,rumen epithelial morphological structure and permeability,and the expression of TJ proteins in lactating goats and to elucidate the molecular mechanisms orchestrating these changes.

2.Materials and methods

2.1.Animals,experimental design and diets

This study was a part of a series of studies designed to evaluate the effects of SARA on the gastrointestinal barrier function and health of dairy goats. Twelve rumencannulated mid-lactating Saanen goats ((36.50±2.13) kg of body weight) aged 2-3 years were used in this study.Balanced for body weight and age,the goats were assigned to 1 of 2 groups in a completely randomized design: control group (Ctrl,n=4) that was fed a basal diet with a non-fiber carbohydrate (NFC) to neutral detergent fiber (NDF) ratio of 1.40 (concentrate:forage,51:49)until slaughter at day 60,and SARA group (SARA,n=8)that was fed the same basal diet but with increasing NFC:NDF ratio from 1.40 to 1.79,2.31,and finally 3.23 overtime to induce SARA,with each NFC:NDF ratio being fed for 15 d including 12 d for adaption and 3 d for sampling. At the end of the SARA challenge,4 goats were randomly selected from the SARA group and slaughtered for sampling,while the other 4 goats were fed only a hay mixture consisting of alfalfa hay and Chinese wildrye hay in a 31:19 ratioadlibitumfor an additional 4 weeks to allow for restitution (post-SARA,n=4),as adapted from Wu (2013). The 4 increasing NFC:NDF ratios (the corresponding concentrate:forage ratios were 51:49,60:40,68:32,and 79:21,respectively) were chosen according to prior SARA-induced trials in dairy goats (Cheng 2016;Sunet al.2018). These diets were formulated to meet all nutrient requirements of 40 kg dairy goats producing 2 kg of milk per day according to the recommendations for Chinese dairy goats (Jin 1989) and ensure that nutrient and energy content remained constant during the SARAinduce period. The ingredients and nutrient composition of the diets are presented in Table 1. The goats were fed twice daily at 08:30 and 18:30 during SARA induction,and the feed offered and refused were manually recorded daily.These goats were kept under the same environmental conditions,with the temperature in the goathouse kept at 15 to 26°C and the air humidity maintained at 47 to 55%.All goats were housed in individual pens with free access to drinking water.

Table 1 Ingredients and composition of the experimental diets with different NFC:NDF ratios

2.2.Ruminal pH measurement

The ruminal pH was continuously monitored during the last 3 d of each SARA induction period using an indwelling pH probe (S651CD,Sensorex Corporation,Stanton,CA) as described by Wanget al.(2020). Briefly,each pH probe was placed through the rumen cannula and suspended in the ventral sac of the rumen. The pH probe was protected by a wire shield and attached to a 600 g stainless-steel ball to keep the probe positioned in the rumen ventral sac. The pH probes were connected to a Jenco Digital pH Transmitter (model 692,Jenco Inc.,La Jolla,CA). The output of the pH transmitter was captured by a universal analogue input multiplexer AI-12 (model R4100,SUPCON Technology,Inc.,Zhejiang,China)and a DO-12 analogue input board (R4100,SUPCON Technology Inc.). The software used for data capturewas Inscan HIS Version 6.30 (SUPCON Technology Inc.).The pH was measured once every minute and averaged over each 10 min interval,then stored digitally for 72 consecutive hours. Daily lowest,highest,and mean ruminal pH,as well as the duration below pH 5.8 and 5.5 for each 72 h period was calculated. The threshold of incidence of SARA was set at pH 5.5.

2.3.Milk sampling and analysis

The goats were milked at 07:30 and 17:30 daily. Daily milk production was recorded using weighing scales.Daily milk samples were taken from both milkings and mixed at a ratio of 6:4 (morning milking:afternoon milking)for each goat and were analyzed for milk fat,protein and lactose contents using a milk composition analyzer(Master Classic,LM2,Europe).

2.4.Rumen papilla sampling

The goats in the Ctrl and SARA groups were slaughtered at d 60,while the post-SARA goats were slaughtered after the 4-week restitution (at d 88) following SARA induction.Rumen epithelial tissue samples were collected from the ventral sac of each goat immediately after slaughter and the collected tissues were subsequently divided into 3 sections. The first section was used immediately to determine permeability using Ussing chambers (see permeability measurements below). The second section was washed,cut into pieces (approximately 1.0 cm×1.0 cm each),rinsed with ice-cold PBS buffer (pH=7.4),and then immediately fixed in 4% paraformaldehyde (Sigma-Aldrich,St.Louis,MO,USA) or 2.5% glutaraldehyde for evaluation of rumen epithelial morphology (see morphological measurement below). The third section was cut into small pieces (approximately 0.5 cm×0.5 cm each),snapfrozen in liquid nitrogen,and then stored at -80°C until mRNA isolation (see quantitative reverse transcription PCR analysis below) and protein expression analysis (see Western blotting analysis below).

2.5.Morphological measurement

Ruminal epithelium samples fixed in 4% paraformaldehyde solution were dehydrated in a graded series of ethanol (50,70,85,95,and 100%) baths,embedded in paraffin,and sectioned. The length and width of the ruminal papillae were determined using the Image-Pro Plus Software(Media Cybernetics,Inc.,Silver Spring,MD). The density of papillae was measured as the number of papillae per cm2of the epithelial surface. The total surface area of papillae per cm2of the epithelial surface was calculated as average papilla length×average papilla width×2×papilla density.

The total epithelial thickness and individual strata thickness within the ruminal epithelium were measured using the Image-Pro Plus Software (Media Cybernetics Inc.) at a magnification of 40× according to the method reported previously (Liuet al.2013).

Ruminal epithelium samples fixed in 2.5%glutaraldehyde were placed in a 1% osmium solution for 2-3 h and dehydrated in a graded series of acetone solutions (50,70,85,95,and 100%). The dehydrated samples were placed in a mixture of Spurr resin and acetone (1:1,v/v) overnight and then in 100% resin for 10 h. The samples and the resin were placed in molds and let still at 45°C for 12 h and then at 60°C for 24 h for the resin to polymerize. Semi-thin sections (0.25-0.5 μm)were cut and stained with 1% toluidine blue-O in 1%sodium borate. Ultrathin (60-80 nm) sections were also cut and then stained with uranyl acetate and then lead citrate. The ultrastructure of the ruminal epithelium was examined using a transmission electron microscope(Hitachi H-7650,Hitachi Technologies,Tokyo,Japan). Six specimens were prepared for each animal,and 5 fields of view were analyzed for each of them.

2.6.Permeability measurements

The rumen epithelial permeability was measured by transepithelial electrophysiological properties (i.e.,shortcircuit current,Isc;and tissue conductance,Gt) and paracellular flux of horseradish peroxidase (HRP,Sigma-Aldrich,St.Louis,MO) as a marker in Ussing chambers as described previously (Klevenhusenet al.2013;Yang 2014). Briefly,rumen epithelial tissue (approximately 50 cm×50 cm) from the ventral sac was excised and rinsed by immersion in 37°C buffer solution. The removed ruminal epithelium was then stripped from the muscular layer in an oxygenated (95% O2and 5% CO2) buffer solution,cut into a 1 cm×1 cm squares and mounted in an Easy Mount Ussing Chamber System (VCC MC6,Physiologic Instruments,San Diego,CA). Both halves of the chamber were rapidly filled with buffer solution and gassed with carbogen gas (95% O2and 5% CO2)at 37°C. After equilibration for 20 min with the buffer solution (open-circuit condition) in the Ussing chambers,the ruminal epithelium was short-circuited and HRP was added to the mucosal side of each chamber to a final concentration of 2 μmol L-1. After 20 min of equilibration,200 μL of the solution from the serosal side was collected every 1 h for 3 h. To maintain the assay volume,200 μL of buffer solution was added to the mucosal side. In total,6 incubation chambers were used per animal,and they were considered as repeated measurements. The concentrations of HRP collected from the serosal side were measured at a wavelength of 380 nm using a microplate reader as described previously (Yang 2014;Cheng 2016;Chenget al.2016). Both Isc and Gt were recorded at 10-min intervals over a 3-h period with the aid of an automatic computer-controlled voltage-clamp device and the Acquire and Analyze Software (Physiologic Instruments Inc.,Reno,NV,USA).

2.7.qRT-PCR

Total RNA was isolated from rumen epithelial samples of each goat using the TRIzol Reagent(Invitrogen Corporation,Carlsbad,CA) according to the manufacturer’s protocol. The RNA was purified following degradation of DNA using RNase-free DNase I.The qRT-PCR was performed using specific primers(Table 2) and an SYBR Green Master Mix (Promega Corporation,Madison,WI) with theβ-actinserving as the housekeeping gene. The mRNA expression of each genewas normalized against theβ-actinlevels,and the data were analyzed according to the 2-ΔΔCtmethod (Livak and Schmittgen 2001).

Table 2 Primer sequences and length of PCR amplicons

2.8.Western blotting

Approximately 100 mg of each frozen rumen epithelial tissue sample was homogenized in 1 mL of ice-cold radioimmunoprecipitation assay protein isolation buffer(Sunshine Biotechnology Co.,Nanjing,China) for total protein extraction. Protein concentration was then measured using the BCA protein assay (Thermo Fisher,USA). A total of 50 μg of total protein extract from each sample was subjected to electrophoresis on a 10% SDSPAGE gel,and the separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore Sigma,Bedford,MA). The membranes were incubated for 12 h at 4°C with specific primary antibodies: rabbit anticlaudin-1,mouse anti-occludin and mouse anti-ZO-1,then washed and incubated in corresponding HRP-conjugated secondary antibodies. The membranes were incubated at 25°C for 2 h and then were washed and visualized using an Enhanced Chemiluminescence (ECL) Kit (Amersham Biosciences Corp.,Arlington Heights,IL). The signals were recorded using a Bio-Rad Imaging System (Bio-Rad Laboratories,Hercules,CA),and the results were analyzed with the Quantity One Software (Bio-Rad). β-actin was used as the reference protein for normalization. The protein expression of each target protein was expressed as the ratio of the densitometry units of TJ protein and β-actin.

2.9.Statistical analysis

The data were analyzed using the General Linear Model Procedure (GLM) in the SAS 9.2 Software (SAS Institute Inc.,Cary,NC). The treatment was included as fix effect and animal was included as random effect in the model. Normality of distributions of residuals was tested using PROC UNIVARIATE of SAS (SAS Institue Inc.). Time with ruminal pH below 5.8 was not normally distributed and was therefore log-transformed to alleviate heterogeneity of residual variances. One-way analysis of variance was used to determine the effects of treatments on morphological structure,permeability and tight junction proteins expression of ruminal epithelium from the lactating goats. Each individual animal was considered as an experimental unit for all data. The Duncan multiple comparison test was carried out to test for significant difference between treatments. Results were expressed as the mean±standard error. Significance was declared atP＜0.05.

3.Results

3.1.Feed intake,milk production and composition

Compared with the Ctrl group,the SARA group had significant decreases (P＜0.05) in feed intake (by 13.38%),milk yield (by 25.73%),milk protein percentage (by 8.33%),and milk fat percentage (by 19.40%),but milk lactose percentage remained unaffected (P＞0.05). These parameters were similar (P＞0.05) between SARA and post-SARA,except for milk protein percentage (Table 3).

3.2.Alterations in ruminal pH during SARA induction

As the NFC:NDF ratio increased,the minimum,maximum,and mean ruminal pH all descended (P＜0.05),and the durations of ruminal pH＜5.8 or pH＜5.5 elongated (P＜0.05)in an NFC:NDF-dependent manner (Table 4). At the end of the last induction period at NFC:NDF ratio of 3.23,the daily rumen pH fell below 5.8 for nearly 11.33 h and below 5.5 for nearly 3.83 h. The nadir of the ruminal pH fell to 5.5,while the peak ruminal pH was below 6.0. Based on the pH threshold for SARA (Kleenet al.2003;Penneret al.2007),SARA was inducted in all the goats during the last induction period at NFC:NDF ratio of 3.23.

3.3.Alterations in morphology and ultrastructure of ruminal epithelium in response to SARA

Compared with the Ctrl group,the SARA group had increases (P＜0.01) in the length (by 111.2%),width (by 9.0%),and surface areas of papillae (by 130.1%),but the papilla density remained unaffected (P＞0.05) (Table 5).The ruminal epithelium was thinner (by 18.8%,P＜0.05)in the SARA group than in the Ctrl group,but the stratum corneum was thicker (by 53.1%,P＜0.05) and the stratum granulosum tended to be thicker (by 8.7%,P＞0.05).The SARA group also had a thinner (by 36.7%,P＜0.01)stratum spinosum and basale than the Ctrl group. At the end of the 4-week restitution (the post-SARA group),papilla width,total epithelial thickness,stratum corneum thickness,and stratum spinosum and basale thickness all reversed to values not significantly different from those of the Ctrl group. The length and surface area of papillae,however,did not reverse to the values of the Ctrl group,remaining to be longer (by 46.6%,P＜0.01) and larger(by 54.6%,P＜0.01),respectively,than those of the Ctrl group.

Table 3 The feed intake,milk yield and milk composition of the lactating goats

Table 4 Ruminal pH in lactating goats during subacute ruminal acidosis (SARA) induction

Table 5 Morphological structure of ruminal epithelium of the lactating goats

The ruminal epithelium of the Ctrl goats appeared normal in both structural integrity and intercellular junctions(Fig.1-A and D),while apparent damages were observed in the SARA group,including peeling,irregular edges,vague intercellular boundaries,damaged TJ and increased intercellular space between epithelial cells (Fig.1-B and E).The compromised integrity of the ruminal epithelium showed some signs of recovery in the post-SARA group after the 4-week restitution,especially increased numbers of TJ and diminished peeling (Fig.1-C and F).

3.4.Permeability of the ruminal epithelium

Compared with the Ctrl group,an increase (P＜0.05) was noted in rumen transepithelial short-circuit current (Isc),tissue conductance (Gt),and paracellular mucosal-toserosal flux of HRP in the SARA group (Fig.2). At the end of the restitution for 4 weeks,the measurements of these 3 indicators of epithelial permeability decreased numerically (P＞0.05) compared to the SARA group,but they continued to remain higher (P＜0.05) than those of the Ctrl group.

3.5.mRNA and protein expressions of tight junction proteins involved in rumen epithelial barrier function

As shown in Table 6,all the analyzed genes encoding tight junction proteins were down-regulated (P＜0.01) in response to SARA. After the restitution,all the down-regulation was moderated,but they remained to be altered (P＜0.01)compared to the Ctrl and similar (P＞0.05) to those of the SARA group. The protein expression of the three tight junction proteins responded to SARA and restitution similarly to the mRNA expression of their coding genes (Fig.3).

Table 6 The mRNA expression of tight junction proteins of the ruminal epithelium in the lactating goats

4.Discussion

In the present study,we investigated if SARA could affect lactation performance,rumen epithelial morphological structure and permeability,and the expression of TJ protein involved in epithelial barrier function. The hypothesis that SARA might cause sustained rumen epithelial barrier dysfunction,which might not recover,was supported in part by the results of the presentstudy. Overall,our results showed that SARA resulted in compromised rumen epithelial integrity,persistent excessive permeability,and down-regulated expression of TJ proteins,CLDN1,OCLN,and ZO-1,of the ruminal epithelium. Moreover,the compromised rumen barrier structure could be restored only to some extent,and barrier function was not recovered 4 weeks after the SARA challenge,which corresponded to a persistent decrease in lactation performance of goats. Our study provided new insight into the mechanistic underpinning of the slow recovery of impaired lactation performance and barrier function weeks after SARA insults.

Previous studies on the effects of SARA on rumen epithelial barrier function were limited to the short-term effects (refs),but little information is available on the longterm effects,particularly whether compromised rumen epithelial barrier can recover after SARA insults. In the present study,we evaluated how SARA could damage the ruminal epithelium and to what extent the compromised ruminal epithelium could recover following SARA. As revealed by histological and ultrastructural analyses of the ruminal epithelium,SARA increased the dimensions(both length and width) of ruminal papillae,decreased the thickness of the epithelium strata,and widened the TJ between epithelial cells. Based on the magnitude of the alterations,the increased papillae surface area was primarily attributed to the elongation of the papillae.Similarly,the SARA-induced decrease in epithelial thickness was largely attributed to the decrease in the thickness of stratum spinosum and basale. Evidently,SARA compromised the structural integrity of the ruminal epithelium. These damaging alterations of the ruminal epithelium are consistent with those reported in dairy cows and goats (Steeleet al.2011;Liuet al.2013;Zhanget al.2017). Not analyzed in the present study,transepithelial translocation of LPS might have increased while the integrity of ruminal epithelium was disrupted.

We continued the feeding experiment to examine how the rumen epithelial integrity and structures restitution after the acidotic insult. All the measured papillae and epithelial stratum features returned to what was measured in the Ctrl group,except for the length and surface area of papillae,which remained to be longer (by 47%,P＜0.01)and larger (by 55%,P＜0.01) than those of the Ctrl group.These findings suggest that the papillae both grew and swelled in length during SARA,and the papillae elongation was not reversible after the SARA insult. These results are in general agreement with a study conducted in dairy cattle by Steeleet al.(2011),who reported that the reintroduction of the high-forage diet during the SARA challenge was marked by increased (P＜0.01) thickness of all living strata to levels not significantly different from that of pre-acidosis. The similarity findings of that study and the present study suggest that most of the compromised epithelial structure caused by SARA can be restituted to some extent after SARA insults. Such selfrestitution was attributed to likely alteration of the rate of mitosis and differentiation of epithelial cells in a ruminal VFA concentration-dependent manner (Goodlad 1981).However,the molecular underpinning of this cellular regulatory mechanism remains unknown. Interestingly,Steeleet al.(2011) found that when dairy cattle were fed a high-grain diet (65% grain) for 3 weeks and then all forage for another 3 weeks,the thickness of the stratum granulosum in the ruminal epithelium was significantly reduced,while the thickness of stratum corneum was not significantly affected during the high-grain feeding,especially during week 1 when SARA occurred. In contrast,the present study showed that the thickness of the stratum corneum was significantly increased,and the thickness of stratum granulosum was not significantly altered when SARA occurred. The discrepancy may be explained by the differences in experimental design,the physical nature of the diet,and most importantly,the duration of the grain challenge and the severity of the SARA insults. Overall,the above results indicated that SARA resulted in compromised rumen epithelial barrier structure,which could be restored naturally to some extent.

The compromised integrity and structure of the ruminal epithelium were accompanied by increased permeability across the ruminal epithelium. As expected,the acidotic insult associated with SARA significantly increased the Isc and Gt across the ruminal epithelium. A concurrent increase in both Isc (a measurement of active electrogenic electrolyte transport) and Gt (a measurement of passive ion permeability) reflects an increased capacity of charge transfer across the ruminal epithelium through the transcellular and/or paracellular route. Both Isc and Gt decreased only slightly after 4 weeks of restitution in the post-SARA group. The elevated Isc and Gt in the post-SARA group indicate that the restitution of the capacity of charge transfer across the ruminal epithelium was much slower than the restitution of the rumen epithelial structures. A long-term adaptation of the epithelium may have occurred in dairy goats,which may have balanced the ruminal concentration of the total VFA (Klevenhusenet al.2013). Several studies found that such adaptation can be achieved by increasing the epithelial surface area coupled with increased activity of ion exchangers for enhanced VFA absorption (Gaebelet al.1987;Lodemann and Martens 2006),which is in agreement with the increased ruminal papillae dimensions during the SARA challenge observed in the present study. The increased flux of HRP,which is a nonspecific fluid-phase endocytosis marker,observed in the SARA group is consistent with the finding of a previous study(Klevenhusenet al.2013). Similar to the restitution of Isc and Gt,the flux of HRP across the ruminal epithelium only decreased by a small margin after the 4-week restitution. The increased electrophysiological properties and permeability of the ruminal epithelium suggest that SARA may compromise epithelial permeability and barrier function for a much longer period,at least longer than 4 weeks. Compromised epithelial permeability can increase translocation of microbes and LPS from the rumen milieu into the portal circulation (Gozhoet al.2005;Emmanuelet al.2007),and thus SARA can increase the risk of liver abscesses,laminitis,and whole-animal inflammatory responses (Steeleet al.2011). Such risk may remain for some time after animals are switched from high-to lowconcentrate diets due to the slow and delayed recovery of rumen epithelial permeability following SARA.

To better understand the mechanistic underpinning of the elevated permeability of the ruminal epithelium,we quantified the expression,at both the transcriptional and translational levels,of 3 key epithelial TJ proteins. As expected,the expression of these 3 proteins was affected similarly at both the transcriptional and translational levels. The expression of all the analyzed proteins was down-regulated by SARA. These results are in accordance with twoinvitroandinvivofindings (Meissneret al.2017;Zhanget al.2019) and suggest that acidotic insult in combination with high VFA concentration during SARA has an extensive impact on TJ proteins.

The expression of the 3 TJ protein genes did not return to levels similar to those of the Ctrl group after the 4-week restitution. Evidently,the effect of the acidotic insult on these genes persisted for a much longer time,at least 4 weeks. The slow and delayed recovery of gene expression,at both the transcriptional and translational levels,of these TJ proteins mirrors that of the compromised permeability of the ruminal epithelium. Future research is needed to elucidate this underlying mechanism of the slow and delayed recovery of expression of the TJ proteins.

Claudins are identified as the key molecules in the barrier function of TJ protein (Tsukitaet al.2001),and OCLN plays both a functional and structural role in defining the paracellular barrier (Matter and Balda 2003). It has been proposed that down-regulation of intestinal OCLN expression may be an important mechanism leading to an increase in intestinal epithelial TJ permeability (Ciccocioppoet al.2006;Markovet al.2015). A study also demonstrated that OCLN depletion in intestinal epithelial cells,bothinvitroandinvivo,increased paracellular flux of larger molecules,suggesting that OCLN plays a crucial role in the maintenance of intestinal epithelial TJ barrier function,through the largechannel pathway (Al-Sadiet al.2011),which we also detected in the present study. ZO-1 is an important linker protein in TJ that affiliates with transmembrane protein,such as OCLN,and other cytoplasmic proteins such as ZO-2,ZO-3,and actin. TJ barrier formation disintegrated when ZO-1 was depleted in cultured epithelial cells(Umedaet al.2006). A variation in epithelial permeability can be related to a change in the abundance of TJ proteins. Our data revealed that decreased TJ protein CLDN1,OCLN,and ZO-1 during the SARA challenge coincided with the increased rumen epithelial permeability. Taken together,our findings suggest that SARA had a sustained profound effect on the expression of the genes related to barrier function in the ruminal epithelium and further confirmed that long-term downregulated expression of TJ proteins could contribute to the increased permeability and reduce the ruminal epithelium barrier function of goats suffering from SARA.

The damaged morphological structures of the ruminal epithelium were restored 4 weeks after the switch from the high NFC:NDF diet to hay mixture,but the compromised rumen epithelial barrier function only recovered slightly,remaining not significantly different compared to the goats suffering from SARA. The lagging of the functional recovery behind the structural recovery was also manifested at the molecular level,both mRNA and protein.We have not found any study in the literature that has documented such a difference in recovery pace between the structure and function of the ruminal epithelium following SARA. However,one study found that the restitution of intestine (jejunum,ileum,and colon) barrier function in rats was slower than that of the morphology after hemorrhagic shock,and there was no direct correlation between them (Changet al.2005). Another study in piglets demonstrated that early weaning resulted in sustained impairment in intestinal barrier function and found the recovery of the intestinal morphology has priority over the recovery of intestinal barrier function (Huet al.2013). The results of the present study and the other studies using rats and pigs point towards a slower recovery process of epithelial barrier function.

In this study,we found that SARA did not only persistently increase permeability or impair barrier function of the ruminal epithelium,but also led to negative effects on feed intake,milk yield and milk component. Similar results have been observed by Zhangetal.(2020),who found dairy goats suffering from SARA showed a significantly reduced dry matter intake (DMI) and milk yield compared with the control goats. At the end of the restitution for 4 weeks,feed intake and milk yield decreased continuously compared with the SARA goats in this study. Decreased DMI might be a self-regulatory mechanism of the body to the acidic environment in the rumen,alleviating the depressed ruminal pH by reducing the intake of fermentable substrates,or it might also be due to focal and systemic inflammation induced by repeated bacterial or LPS entry into the ruminal epithelium and blood circulation.Previous studies reported that low feed intake could reduce SCFA absorption (Albornozet al.2013),papillae surface area (Pederzolliet al.2018),and increase permeability of the gastrointestinal tract when feed intake was restricted to 25% of voluntary intake (Zhanget al.2013). Whereby decreased feed intake had a sustained profound effect on compromised rumen epithelial barrier function.

5.Conclusion

As a common metabolic disorder in dairy production,SARA causes rather extensive damages to the structure of the ruminal epithelium and compromises its barrier function. In this study,we demonstrated that SARA could result in lingering impairment in the rumen epithelial barrier function in lactating goats after one month of feeding with mixed hay only. The slow recovery of the epithelial barrier function coincided with the laggard recovery of down-regulated expression of key TJ proteins in the ruminal epithelium. Future research is warranted to elucidate the effect of the reduced feed intake by SARA on the above aspects and mechanism underpinning the slow recovery of impaired regulation of expression of the genes encoding tight junction proteins.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31472124),the Natural Science Foundation of Inner Mongolia,China (2019MS03031),the earmarked fund for China Agriculture Research System (CARS-36),the Innovation Fund of Inner Mongolia Agricultural and Animal Husbandry,China(2021CXJJM02),and a China Scholarship Council(Beijing) fellowship to Hu Honglian for training at the Ohio State University,USA (201909150001).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All animal experimental procedures used in this study were approved by the Inner Mongolia Academy of Agricultural &Animal Husbandry Sciences’ Animal Care and Use Committee,China.