Induction  of  subacute  acidosis  in  the  Rusitec  model  compared  to  in  vivo

4.1.3   Induction  of  subacute  acidosis  in  the  Rusitec  model  compared  to  in  vivo   methods  

The  Rusitec  system  is  often  criticized  in  literature  for  several  reasons.  In  an  in  vivo   study,  an  intense  relationship  between  the  feeding  schedule  and  the  daily  fluctuations   of  ruminal  pH  has  been  detected  (Soto-­Navarro  et  al.,  2000a).  Moreover,  the  Rusitec   is  fed  only  once  a  day  and  therefore  differs  from  the  in  vivo  feeding  scheme.  Mostly,  in   in  vivo  studies,  animals  are  fed  twice  a  day  (Mao  et  al.,  2013,  Arik  et  al.,  2019)  or  more   often  (Hook  et  al.,  2011).  However,  some  studies  reduce  the  feeding  scheme  to  once   a  day  (Khafipour  et  al.,  2009c)  or  provide  an  ad  libitum  ration  (Palmonari  et  al.,  2010).  

In  vitro,  it  is  a  common  procedure  to  feed  the  Rusitec  model  only  once  a  day.  The  study   of  Cerrato-­Sánchez  et  al.  (2007)  observed  the  severity  of  SARA  in  in  vitro  experiments.  

Authors  reported  that  the  severity  of  SARA  was  not  influenced  by  the  number  of  pH  

bouts,  but  by  the  actual  time  spent  beneath  SARA  thresholds.  The  Rusitec  feeding   scheme,   therefore,   is   not   a   major   limiting   factor   influencing   the   SARA   induction.  

Furthermore,  the  Rusitec  intends  to  mimic  the  rumen  fermentation,  a  strict  anaerobic   process.  The  need  to  supply  the  system  with  fresh  feed  material  consequently  leads   to  the  exposure  of  oxygen  when  the  fermentation  vessels  are  opened  for  the  feedbag   exchange.  It  is  recommended  to  keep  the  oxygen  exposure  as  short  as  possible,  as   oxygen   is   toxic   to   most   ruminal   bacteria.   An   intense   oxygen   exposure   may   inhibit   growth,   the   adhesion   of   cellulolytic   bacteria   and   fermentation   capacity   and   would   therefore  impair  and  affect  the  fermentation  pattern  (Roger  et  al.,  1990,  Martínez  et  al.,   2010).  For  future  experimental  set  ups,  it  would  be  desirable  to  observe  the  effect  of   multiple  feeding  repetitions  on  the  fermentation  pattern  in  the  Rusitec  model.    

The   induction   of   SARA   in   the  in   vitro   model   is   performed   by   decreasing   the   buffer   capacity,  which  leads  to  a  decline  in  pH.  This  procedure  is  a  common  method  to  induce   low  pH  values  in  Rusitec  models  (Colombatto  et  al.,  2003,  Eger  et  al.,  2017).  In  our   experiment,  the  decrease  of  pH  values  resulted  in  a  reduction  of  SCFA  production,   which   is   also   reported   in   the   Rusitec   study   of   Mickdam   et   al.   (2016).   The  in   vitro   approach  appears  to  contradict  the  in  vivo  ethology  of  SARA.  In  in  vivo  studies,  the   acidotic  conditions  are  induced  by  a  sudden  increase  of  concentrates  in  the  daily  ration   and   a   massive   accumulation   of   SCFA,   which   exceeds   the   ruminal   buffer   capacity   (Khafipour  et  al.,  2009b,  Fernando  et  al.,  2010,  Danscher  et  al.,  2015).  However,  it   remains   unclear   whether   the   accumulation   of   SCFA   alone   or   a   reduction   of   the   buffering  saliva  enhances  the  reduction  in  ruminal  pH  (Maekawa  et  al.,  2002a,  b,  Jiang   et  al.,  2017,  Van  Soest,  2018).  In  awareness  of  the  SARA  initiation  in  vivo,  we  decided   to  enhance  the  concentrate  ratio  in  certain  treatment  groups.  These  groups  received  

a  changing  ratio  of  hay  and  concentrate.  During  both  control  periods,  they  received  a   low  amount  of  concentrate  and  during  acidosis  period,  the  concentrate  ratio  increased   to  70%.  We  expected  to  observe  an  increase  in  the  total  SCFA  production  rate  with  a   consequent  pH  decrease.  However,  this  approach  was  not  successful  and  we  neither   observed   an   increase   in   SCFA   nor   a   reduction   in   pH.   A   few  in   vivo   studies   report   decreasing   ruminal   pH   values   accompanied   with   unchanging   total   SCFA   concentrations  after  SARA  induction.  Lettat  et  al.  (2010)  observed  a  decrease  in  total   SCFA  when  SARA  was  induced  by  feeding  a  high  ratio  of  wheat  in  sheep  and  Li  et  al.  

(2012)  induced  SARA  by  reducing  fiber  in  the  ratio  of  goats.  Even  though  the  in  vitro   acidosis  induction  alters  from  the  general  in  vivo  situation,  the  further  development  of   SARA   is   very   similar   to   the   acidosis   induced   in   the   Rusitec   model.   After   an   initial   increase   of   SCFA,   the   ruminal   concentration   of   SCFA   declines   (Colombatto   et   al.,   2003),  due  to  the  impairment  of  the  bacterial  community.  A  changing  osmolarity  leads   to  a  successively  increasing  fluid  influx  and  thereby  to  dilution  of  the  ruminal  content.  

This  enhances  the  declining  SCFA  concentrations  (Huber,  1976).    

Feed  intake  in  vivo  is  known  to  reduce  ruminal  pH,  resulting  in  diurnal  pH  alterations   (Palmonari  et  al.,  2010,  Mao  et  al.,  2013).  By  using  the  continuous  pH  measurement,   we  were  able  to  record  daily  pH  fluctuations  within  the  reaction  vessels.  It  was  clearly   visible,  that  the  feeding  process  had  an  impact  on  the  daily  pH  development.  However,   we  were  not  able  to  induce  SARA  by  an  increased  concentrate  ratio  up  to  70%  and  no   feeding  scheme  applied  had  a  significant  impact  on  pH  values.  High  concentrate  diets   are   often   reported   to   be   difficult   to   mimic   in   Rusitec   models   (Carro   et   al.,   1995,   Mansfield   et   al.,   1995).   However,   in   a   recent   Rusitec   trial,   Belanche   et   al.   (2015b)   supplied   Rusitec   fermentation   vessels   with   a   50:50   concentrate-­to-­forage   ratio   and  

observed  a  decrease  in  pH  of  up  to  0.6  pH  units  4  h  post  feeding.  It  has  to  be  noted,   that  in  the  afore  mentioned  study  the  feed  was  ground  to  pass  through  a  1  mm²  sieve.  

This   highly   grounded   feed   led   to   a   higher   diurnal   variation,   compared   to   our   study   where   hay   particles   were   as   long   as   1   cm   and   concentrate   pellet   sizes   were   kept   approximately   0.5   cm.   Obviously,   a   higher   grinding   grade   enhances   bacterial   fermentation,   as   it   provides   a   greater   surface   area   for   adhesion   and   degradation   processes  (McAllister  et  al.,  1994,  Duarte  et  al.,  2017).  However,  1  mm²  feed  particles   are  not  very  comparable  to  the  in  vivo  situation  and  feeding  schemes  and  most  Rusitec   studies  use  bigger  particle  sizes  (Duarte  et  al.,  2017,  Eger  et  al.,  2017).  Generally,   particles  in  the  rumen  are  found  to  be  larger  than  1.8  mm  when  they  leave  the  rumen   to  further  parts  of  the  forestomach  (Poppi  et  al.,  1980).  For  further  studies,  a  Rusitec   trial   with   a   different   concentrate   composition   and   a   reduced   particle   size   would   be   desirable  in  order  to  clarify  the  impact  of  the  concentrate  ratio  on  SARA  induction  in   vitro.  Furthermore,  the  implication  of  the  continuous  measurement  in  all  fermentation   vessels  would  be  beneficial.  The  punctual  pH  measurement  before  feeding,  as  it  has   been  performed  in  the  present  study,  may  not  exactly  mirror  the  pH  decrease  during   the   acidosis   period.   The   diurnal   pH   fluctuation   visible   in   the   continuously   observed   vessels  indicated  an  increasing  pH  in  the  reaction  vessels  right  before  feeding.  When   the   pH   is   exclusively   measured   only   once   before   feeding,   the   daily   nadir   pH   value   remains  unclear  and  the  exact  definition  of  SARA  cannot  be  proved.