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ubio	
  quickVET	
  RAPID	
  Pregnancy	
  Test	
  for	
  Cattle	
  
                                                                                                  u bi o B i ot e c h n ol ogy Sy s t e ms Pv t . Lt d.                           	
  
                                                                                                                                                                                  	
  
                                                                                                 Ph : +91-484-2532966, +91-9446438678
                                                                                                                                                                                  	
  
                                                                                W e b: h t t p: //www.u bi o.i n Emai l : c on t ac t @ u bi o.i n                                	
  
Accurate	
  diagnosis	
  of	
  pregnancy	
  is	
  of	
  crucial	
  importance	
  in	
  maintaining	
  optimal	
  reproductive	
  performance	
  of	
  
cattle	
  and	
  high	
  profitability	
  of	
  dairy	
  farms.	
  It	
  is	
  desirable	
  for	
  the	
  farmer	
  to	
  know	
  as	
  soon	
  as	
  possible	
  if	
  a	
  mated	
  
or	
  inseminated	
  cow	
  is	
  not	
  pregnant,	
  so	
  that	
  she	
  can	
  be	
  rebred	
  with	
  the	
  minimum	
  delay.	
  It	
  is	
  most	
  beneficial	
  if	
  
non-­‐pregnancy	
   can	
   be	
   detected	
   before	
   the	
   first	
   heat	
   after	
   insemination.	
   Conventional	
   methods	
   that	
   allow	
  
early	
   detection	
   of	
   pregnancy	
   are	
   not	
   accurate	
   enough,	
   and	
   reliance	
   on	
   these	
   methods	
   results	
   in	
   a	
   large	
  
number	
   of	
   non-­‐pregnant	
   cows	
   assumed	
   to	
   be	
   pregnant	
   and	
   some	
   pregnant	
   cows	
   being	
   inseminated	
  
inappropriately,	
   which	
   could	
   abort	
   them.	
   Conventional	
   pregnancy	
   detection	
   methods	
   that	
   are	
   accurate,	
   on	
  
the	
  other	
  hand,	
  can	
  be	
  performed	
  only	
  after	
  at	
  least	
  35	
  days	
  after	
  insemination,	
  leading	
  to	
  the	
  loss	
  of	
  at	
  least	
  
one	
  opportunity	
  to	
  inseminate.	
  
ubio’s	
   quickVET	
   pregnancy	
   detection	
   test	
   provides	
   a	
   perfect	
   tool	
   to	
   detect	
   non-­‐pregnancy	
   in	
   cattle	
  
accurately	
  within	
  21	
  days.	
  The	
  kit	
  provides	
  100%	
  detection	
  of	
  non-­‐pregnant	
  cases	
  and	
  allows	
  insemination	
  at	
  
the	
   first	
   opportunity.	
   The	
   average	
   savings	
   realised	
   through	
   the	
   use	
   of	
   ubio	
   quickVET	
   test	
   	
   is	
   as	
   high	
   as	
  
Rs.1300/-­‐	
  per	
  cow	
  per	
  lactation	
  period.	
  Moreover,	
  the	
  test	
  can	
  be	
  easily	
  performed	
  within	
  20	
  minutes	
  at	
  cow-­‐
side	
  without	
  any	
  instrumentation,	
  which	
  makes	
  the	
  tool	
  attractive	
  even	
  for	
  direct	
  use	
  by	
  dairy	
  farmers.	
  
The	
   test	
   employs	
   the	
   well-­‐tested	
   progesterone	
   assay	
   principle	
   to	
   detect	
   pregnancy.	
   It	
   measures	
  
progesterone	
  levels	
  in	
  milk	
  or	
  serum	
  (for	
  heifers)	
  using	
  a	
  Rapid	
  ELISA	
  technique.	
  
COMPARISON	
  OF	
  PREGNANCY	
  DETECTION	
  TECHNIQUES	
  

   Test	
                       Stage	
             Principle	
                              Advantages	
                  Disadvantages	
                      Accuracy	
  
   Non-­‐return	
  to	
         3	
  wks	
          Recording	
  dates	
  of	
               -­‐	
  Early	
                -­‐	
  Relies	
  on	
  good	
        About	
  
   service	
                                        service	
  and	
                         detection,	
                  oestrus	
  detection	
               50%	
  
                                                    observation	
  of	
                      without	
  missing	
  
                                                    symptoms	
  of	
  heat	
  	
             first	
  heat	
  after	
      -­‐	
  Large	
  variation	
  
                                                                                             insemination	
                within	
  a	
  population	
  
                                                                                                                           -­‐	
  Insemination	
  at	
  
                                                                                                                           ‘false	
  oestrus’	
  can	
  
                                                                                                                           lead	
  to	
  abortion	
  
   Early	
                      4	
  wks	
  +	
     Detects	
  pregnancy-­‐                  -­‐	
  Relatively	
           -­‐	
  Needs	
  blood	
              About	
  
   Conception	
                                     dependent	
  protein	
                   early	
  detection	
          -­‐	
  May	
  persist	
  after	
     90%	
  
   Factor	
                                         complex	
  using	
  ELISA	
                                            loss	
  of	
  pregnancy	
  
   Ultrasound	
                 4	
  wks	
  +	
     Visualization	
  of	
                    -­‐	
  Relatively	
           -­‐	
  High	
  capital	
  cost	
     About	
  
   Scan	
                                           images	
  of	
  the	
                    early	
  detection	
          -­‐	
  Need	
  good	
                100%	
  
                                                    conceptus	
  using	
                     -­‐	
  Accurate	
             technical	
  expertise	
  
                                                    realtime	
  ultrasound	
  
   Palpation	
                  5-­‐6	
             Feeling	
  the	
  presence	
             -­‐	
  Accurate	
             -­‐	
  Possible	
  damage	
      About	
  
                                wks+	
              of	
  a	
  fetus	
  swelling	
  in	
                                   to	
  cow	
  or	
  conceptus	
   100%	
  
                                                    one	
  of	
  the	
  uterine	
                                          -­‐	
  Need	
  good	
  skill	
  
                                                    horns	
  by	
  inserting	
  an	
                                       and	
  technical	
  
                                                    arm	
  into	
  the	
  rectum	
                                         expertise	
  	
  
                                                    of	
  the	
  cow.	
  
   Oestrone	
                   15	
                Detects	
  oestrone	
                    -­‐	
  Very	
  accurate	
     -­‐	
  Too	
  delayed	
  to	
  be	
   100%	
  
   Sulphate	
                   wks+	
              sulphate	
  in	
  milk.	
                                              useful	
  in	
  most	
  cases	
  
                                                    Concentration	
  is	
  
                                                    proportional	
  to	
  
                                                    oestrogens	
  produced	
  
                                                    by	
  conceptus.	
  
   Conventional	
               3	
  wks	
          Progesterone	
                           -­‐	
  Accurate	
             -­‐	
  High	
  capital	
  cost	
     100%	
  for	
  
Milk	
                                            concentration	
  at	
  19-­‐                    -­‐	
  Early	
                  -­‐	
  Requires	
  blood	
  in	
      non-­‐
   Progesterone	
                                    23	
  day	
  interval	
  from	
                 detection	
                     heifers	
                             pregnancy	
  
   plate	
  ELISA	
                                  last	
  heat	
  is	
  much	
                    -­‐	
  Non-­‐invasive	
         -­‐	
  Test	
  to	
  be	
             85%+	
  for	
  
                                                     higher	
  in	
  pregnant	
                                                      performed	
  on	
                     pregnancy	
  
                                                     cows	
  than	
  non-­‐                                                          specified	
  days	
  
                                                     pregnant	
  ones	
  	
  
   ubio	
                         3	
  wks	
         Progesterone	
                                  -­‐	
  Accurate	
               -­‐	
  Requires	
  blood	
  in	
      100%	
  for	
  
   quickVET	
                                        concentration	
  at	
  19-­‐                    -­‐	
  Early	
                  heifers	
                             non-­‐
   Rapid	
  Milk	
                                   23	
  day	
  interval	
  from	
                 detection	
                     -­‐	
  Test	
  to	
  be	
             pregnancy	
  
   Progesteron                                       last	
  heat	
  is	
  much	
                    -­‐	
  Non-­‐invasive	
         performed	
  on	
                     85%+	
  for	
  
   e	
  Assay	
                                      higher	
  in	
  pregnant	
                      -­‐	
  No	
                     specified	
  days	
                   pregnancy	
  
                                                     cows	
  than	
  non-­‐                          instrumentation	
  
                                                     pregnant	
  ones	
                              -­‐	
  Cow-­‐side	
  test	
  
ASSAY	
  PRINCIPLES	
  
The	
  ubio	
  quickVET	
  pregnancy	
  test	
  is	
  based	
  on	
  ELISA-­‐based	
  progesterone	
  assay	
  principle.	
  The	
  test	
  measures	
  
progesterone	
   concentration	
   in	
   milk	
   or	
   blood	
   using	
   Rapid	
   ELISA	
   technique.	
   A	
   progesterone-­‐specific	
  
monoclonal	
   antibody	
   is	
   coated	
   inside	
   the	
   test	
   cassette.	
   Milk/serum	
   and	
   labeled	
   progesterone	
   conjugate	
   are	
  
added	
  to	
  the	
  test	
  and	
  control	
  wells	
  during	
  the	
  test.	
  The	
  progesterone	
  from	
  the	
  milk	
  and	
  the	
  labeled	
  conjugate	
  
will	
   compete	
   to	
   bind	
   with	
   the	
   coated	
   antibody.	
   A	
   chromogen,	
   that	
   gives	
   color,	
   is	
   added	
   to	
   the	
   wells	
   and	
   it	
   will	
  
bind	
   only	
   to	
   the	
   conjugate.	
   If	
   the	
   sample	
   contains	
   progesterone	
   (animal	
   is	
   pregnant),	
   the	
   test	
   well	
   will	
   be	
  
lighter	
   than	
   the	
   control	
   well.	
   If	
   the	
   progesterone	
   level	
   is	
   very	
   low	
   (non-­‐pregnant	
   animal),	
   the	
   test	
   well	
   be	
  
bluer	
  than	
  the	
  control	
  well.	
  

   The	
   concentration	
   of	
   progesterone	
   in	
   the	
   milk	
   or	
                        In	
   the	
   case	
   of	
   a	
   pregnant	
   cow,	
   the	
   progesterone	
  
   blood	
   of	
   a	
   non-­‐pregnant	
   cow	
   varies	
   as	
   shown	
                           concentration	
  varies	
  as	
  given	
  below.	
  
   below,	
  with	
  Day	
  0	
  corresponding	
  to	
  the	
  occurrence	
  
   of	
   heat.	
   It	
   is	
   assumed	
   that	
   insemination	
   is	
  
   appropriately	
  performed	
  at	
  the	
  time	
  of	
  heat.	
  




                                                                                                                                                                                                 	
  
                                                                                                         	
  

                                                                                              	
         	
  

It	
  	
  is	
  obvious	
  that	
  there	
  is	
  a	
  significant	
  difference	
  between	
  progesterone	
  concentration	
  in	
  milk/blood	
  	
  samples	
  
taken	
  from	
  pregnant	
  and	
  non-­‐pregnant	
  cows	
  from	
  19th	
  to	
  23rd	
  day	
  after	
  heat	
  (and	
  insemination).	
  By	
  checking	
  
the	
  progesterone	
  level	
  during	
  this	
  time	
  frame,	
  we	
  can	
  accurately	
  identify	
  non-­‐pregnancy.	
  

This	
  principle	
  provides	
  100%	
  accurate	
  results	
  for	
  progesterone	
  levels,	
  which	
  translates	
  to	
  100%	
  accuracy	
  for	
  
non-­‐pregnancy.	
   However,	
   there	
   are	
   some	
   corner	
   cases	
   where	
   a	
   false	
   positive	
   may	
   occur	
   for	
   cases	
   marked	
  
‘pregnant’	
  by	
  the	
  test.	
  

              	
  

       1.     If	
  heat	
  is	
  incorrectly	
  interpreted,	
  and	
  insemination	
  is	
  done	
  at	
  the	
  wrong	
  time,	
  the	
  test	
  will	
  show	
  the	
  
              result	
   as	
   ‘pregnant’	
   as	
   we	
   are	
   actually	
   detecting	
   the	
   high	
   progesterone	
   level	
   during	
   mid-­‐heat,	
   instead	
  
              of	
  that	
  caused	
  by	
  pregnancy.	
  
 
   2.   A	
  cow	
  that	
  is	
  correctly	
  judged	
  pregnant	
  might	
  lose	
  her	
  conceptus	
  due	
  to	
  various	
  reasons.	
  In	
  this	
  case,	
  
        the	
   test	
   is	
   accurate,	
   but	
   the	
   pregnancy	
   does	
   not	
   result	
   in	
   calf-­‐birth.	
   It	
   is	
   suggested	
   that	
   the	
   test	
   be	
  
        repeated	
  if	
  any	
  sign	
  of	
  heat	
  is	
  seen	
  after	
  the	
  cow	
  is	
  judged	
  pregnant.	
  




                                                                                                                                                                       	
  
   3.   A	
  cow	
  with	
  luteinized	
  cystic	
  follicles	
  will	
  produce	
  progesterone	
  at	
  varying	
  levels,	
  and	
  the	
  test	
  result	
  is	
  
        not	
   reliable	
   in	
   such	
   animals	
   as	
   it	
   might	
   show	
   positive	
   or	
   negative	
   based	
   on	
   the	
   progesterone	
   level	
   on	
  
        the	
  day	
  of	
  test.	
  




                                                                                                                                                         	
  
CLINICAL	
  APPLICATIONS	
  

        1.     Ovulation	
  Test:	
  ubio	
  quickVET	
  rapid	
  pregnancy	
  test	
  for	
  cattle	
  can	
  be	
  used	
  for	
  detecting	
  ovulation	
  
               in	
   cows	
   that	
   do	
   not	
   show	
   regular	
   symptoms	
   of	
   heat.	
   The	
   test	
   	
   should	
   be	
   performed	
   during	
   the	
  
               suspected	
  heat	
  period.	
  A	
  dark	
  blue	
  spot	
  indicates	
  heat	
  and	
  insemination	
  can	
  be	
  performed	
  in	
  6-­‐
               18	
  hours.	
  A	
  clear	
  white	
  card	
  indicates	
  mid-­‐heat	
  and	
  the	
  test	
  needs	
  to	
  be	
  repeated	
  only	
  after	
  10	
  
               days.	
  A	
  light	
  blue	
  color	
  indicates	
  approaching	
  or	
  past	
  heat,	
  and	
  a	
  repeated	
  test	
  after	
  2	
  days	
  will	
  
               confirm	
  the	
  condition.	
  

        2.     Identification	
   of	
   follicular/luteal	
   cysts:	
   Ovarian	
   cysts	
   are	
   an	
   important	
   infertility	
   problem	
   in	
  
               dairy	
  cattle.	
  It	
  is	
  often	
  difficult	
  to	
  differentiate	
  ovarian	
  cysts	
  into	
  follicular	
  and	
  luteal	
  by	
  palpation	
  
               of	
  the	
  ovary	
  per	
  rectum.	
  However,	
  once	
  the	
  presence	
  of	
  an	
  ovarian	
  cyst	
  has	
  been	
  determined	
  by	
  
palpation,	
   differentiation	
   can	
   be	
   made	
   on	
   the	
   basis	
   of	
   progesterone	
   concentration.	
   Cystic	
   cows	
  
                              having	
  a	
  low	
  progesterone	
  concentration	
  are	
  considered	
  to	
  have	
  follicular	
  cysts,	
  and	
  those	
  with	
  
                              high	
   concentrations	
   are	
   classified	
   as	
   having	
   luteal	
   cysts.	
   For	
   practical	
   purposes,	
   differential	
  
                              diagnosis	
   of	
   ovarian	
   cysts	
   is	
   important	
   in	
   selection	
   of	
   endocrine	
   therapy.	
   The	
   cyst	
   can	
   be	
  
                              differentiated	
   by	
   performing	
   the	
   test	
   on	
   two	
   samples	
   taken	
   7	
   days	
   apart	
   –	
   a	
   dark	
   blue	
   spot	
   in	
  
                              both	
  tests	
  indicate	
  a	
  follicular	
  cyst	
  and	
  a	
  clear	
  white	
  card	
  in	
  both	
  tests	
  indicate	
  a	
  luteal	
  cyst.	
  

                    3.        Evaluation	
   of	
   endocrine	
   therapy:	
   	
   To	
   evaluate	
   treatment	
   of	
   follicular	
   cysts	
   using	
   GnRH,	
   ubio	
  
                              quickVET	
   rapid	
   pregnancy	
   test	
   can	
   be	
   used	
   to	
   evaluate	
   to	
   progesterone	
   levels	
   10	
   days	
   after	
  
                              administration	
  of	
  GnRH.	
  A	
  light	
  blue	
  spot	
  or	
  a	
  clear	
  white	
  card	
  indicates	
  response	
  to	
  treatment.	
  
                              Similarly,	
   the	
   test	
   can	
   be	
   used	
   to	
   evaluate	
   response	
   to	
   treatment	
   of	
   luteal	
   cysts	
   using	
  
                              Prostaglandin	
   compounds	
   as	
   unobserved	
   estrus	
   following	
   prostaglandin	
   administration	
   is	
   a	
  
                              common	
  occurrence.	
  Response	
  to	
  prostaglandin	
  is	
  dependent	
  upon	
  the	
  presence	
  of	
  a	
  functional	
  
                              corpus	
  luteum.	
  Therefore,	
  milk	
  progesterone	
  testing	
  prior	
  to	
  and	
  3	
  days	
  after	
  administration	
  of	
  
                              prostaglandin	
   can	
   accurately	
   assess	
   if	
   administration	
   is	
   justified	
   (high	
   concentration	
   prior	
   to	
  
                              administration)	
  and	
  if	
  luteolysis	
  has	
  occurred	
  (low	
  concentration	
  on	
  day	
  3).	
  

                    4.        Evaluation	
  of	
  fitment	
  for	
  embryo	
  transfer:	
  ubio	
  quickVET	
  rapid	
  pregnancy	
  test	
  for	
  cattle	
  can	
  be	
  
                              used	
   in	
   embryo	
   transfer	
   programs	
   to	
   check	
   if	
   there	
   is	
   adequate	
   progesterone	
   level	
   in	
   the	
  
                              recipient	
   to	
   support	
   the	
   embryo.	
   The	
   recipient	
   should	
   show	
   a	
   light	
   blue	
   test	
   result	
   before	
   an	
  
                              embryo	
  is	
  transferred.	
  

INTERPRETATION	
  OF	
  THE	
  RESULT	
  

The	
  control	
  well	
  is	
  usually	
  colorless	
  or	
  a	
  very	
  pale	
  blue.	
  A	
  test	
  well	
  that	
  is	
  considerably	
  bluer	
  than	
  the	
  control	
  
well	
   indicates	
   a	
   non-­‐pregnant	
   animal	
   with	
   low	
   progesterone	
   level.	
   A	
   test	
   well	
   lighter	
   than	
   or	
   of	
   the	
   same	
  
shade	
   as	
   the	
   control	
   well	
   indicates	
   a	
   pregnant	
   animal	
   with	
   higher	
   progesterone	
   concentration,	
   subject	
   to	
  
caveats.	
   Slightly	
   bluer	
   tinge	
   in	
   the	
   test	
   well	
   when	
   compared	
   to	
   the	
   control	
   well	
   indicates	
   non-­‐pregnant	
   result	
  
during	
  approaching	
  or	
  past	
  heat	
  period	
  –	
  in	
  this	
  case,	
  the	
  test	
  timing	
  and	
  heat	
  calculation	
  must	
  be	
  checked,	
  
and	
  the	
  test	
  should	
  be	
  repeated	
  if	
  necessary.	
  




                                                                      	
  	
  	
  	
  	
  	
                       	
  	
  	
  	
  	
  	
                                           	
  	
  	
  	
  	
  	
                                  	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  Not	
  Pregnant	
  	
  	
  	
  	
  	
  	
  	
  Not	
  Pregnant	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  Pregnant	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  Pregnant 	
  
TEST	
  PROCEDURE 	
                                                                      	
            	
            	
            	
            	
         	
                                     	
                             	
     	
     	
  
	
                                                                      1.	
  Collect	
  milk	
  sample	
  from	
  cow	
  in	
  a	
  clean	
  
                                                                        vessel	
  after	
  discarding	
  milk	
  from	
  the	
  first	
  few	
                                    KIT	
  COMPONENTS	
  
	
  	
  	
  	
  	
  	
  	
  	
  
                                                                        pulls.	
  For	
  pregnancy	
  test,	
  the	
  test	
  should	
  be	
                                      -­‐	
  One	
  Aluminium	
  pouch	
  containing	
  
	
                                                                      done	
  between	
  19th	
  to	
  23rd	
  day	
  after	
                                                   the	
  test	
  cassette.	
  
                                                                        insemination.	
                                                                                           -­‐	
  Three	
  droppers	
  for	
  transferring	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
                                                                                                                                                                                  milk	
  and	
  reagents.	
  
                                                                        2.	
  Keep	
  the	
  test	
  pack	
  at	
  26-­‐30ºC	
  for	
  1-­‐2	
  hours	
                           -­‐	
  One	
  transparent	
  vial	
  marked	
  ‘1’	
  
                                                                        to	
  bring	
  it	
  to	
  room	
  temperature.	
  Remove	
  the	
                                        containing	
  reagent	
  1.	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
           	
                     test	
  cassette	
  from	
  Aluminium	
  pouch	
  and	
  keep	
  
                                                                                                                                                                                  -­‐	
  One	
  dropper	
  bottle	
  marked	
  ‘2’	
  
                                                                        on	
  a	
  flat	
  surface.	
  There	
  are	
  two	
  wells	
  on	
  the	
  
                                                                                                                                                                                  containing	
  wash	
  buffer	
  (reagent	
  
                                                                        card,	
  marked	
  C	
  and	
  T.	
  
                                                                                                                                                                                  2).	
  
                                                                        3.	
  Add	
  three	
  (3)	
  drops	
  of	
  milk	
  to	
  both	
  the	
  wells	
                          -­‐	
  One	
  opaque	
  vial	
  marked	
  ‘3’	
  
	
  	
  	
  	
  	
  	
  	
                                	
            (C	
  and	
  T)	
  using	
  a	
  dropper	
  supplied	
  with	
  the	
                                     containing	
  reagent	
  3	
  
                                                                        test.	
  Dispose	
  the	
  dropper	
  immediately.	
                                                      	
  

                                                                         4.	
  Keep	
  the	
  cassette	
  on	
  a	
  flat	
  surface	
  for	
  5	
                  PRECAUTIONS	
  &	
  
                                                                         minutes.	
  Periodically	
  tap	
  the	
  side	
  of	
  the	
                              WARNINGS	
  
	
  	
  	
  	
  	
  	
                                                  	
  cassette	
  for	
  proper	
  mixing.	
  Take	
  care	
  not	
  to	
                     -­‐	
  Store	
  in	
  fridge	
  at	
  4-­‐8ºC,	
  do	
  not	
  
                                                                         splash	
  the	
  milk	
  in	
  the	
  wells.	
                                             freeze	
  

                                                                      5.	
  Using	
  a	
  fresh	
  dropper,	
  Add	
  three	
  (3)	
  drops	
                       -­‐	
  Do	
  not	
  reuse	
  test	
  kit.	
  
                                                                      of	
  reagent	
  into	
  both	
  the	
  wells	
  (C	
  and	
  T)	
  from	
  
                                                                                                                                                                    -­‐	
  Use	
  a	
  separate	
  dropper	
  for	
  each	
  
                                                                      the	
  bottle	
  marked	
  Reagent	
  1.	
  Let	
  milk	
  and	
  
                                                                                                                                                                    reagent.	
  
	
  	
  	
  	
  	
  	
  	
  	
                                   	
   reagent	
  mix	
  by	
  gently	
  tapping	
  the	
  cassette.	
  
                                                                                                                                                                    -­‐	
  Do	
  not	
  mix	
  components	
  from	
  
                                                                        6.	
  Keep	
  the	
  cassette	
  on	
  a	
  flat	
  surface	
  for	
  20	
                  different	
  kits.	
  
                                                                        minutes.	
  Gently	
  tap	
  the	
  cassette	
  at	
  4-­‐minute	
  
                                                                        intervals.	
                                                                                -­‐	
  Do	
  not	
  touch	
  the	
  test	
  wells.	
  
	
  	
  	
  	
                                                   	
  
                                                                        7.	
  Tip	
  the	
  cassette	
  and	
  empty	
  the	
  wells	
  into	
  a	
  
                                                                        sink.	
                                                                                     	
  

                                                                        8.	
  Fill	
  the	
  wells	
  with	
  the	
  wash	
  buffer	
  in	
  the	
                  	
  
                                                                        dropper	
  bottle	
  marked	
  ‘Reagent	
  2’,	
  tap	
  gently	
  
                                                                                                                                                                    	
  
	
  	
                                         	
  	
                   and	
  empty	
  the	
  wells	
  into	
  the	
  sink.	
  Repeat	
  this	
  
                                                                        washing	
  5-­‐6	
  times.	
  Shake	
  off	
  any	
  remaining	
                            	
  
                                                                        solution	
  from	
  the	
  wells.	
  
                                                                                                                                                                    	
  
                                                                      9.	
  Add	
  5	
  drops	
  of	
  Reagent	
  3	
  in	
  the	
  opaque	
  vial	
  
                                                                      to	
  both	
  the	
  empty	
  wells.	
  Keep	
  the	
  test	
  on	
  a	
  
	
  	
  	
                                                       	
   flat	
  surface.	
  The	
  result	
  appears	
  after	
  8	
  
                                                                      minutes,	
  and	
  can	
  be	
  read	
  till	
  15	
  minutes.	
  
                                                                                                                                                                    	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
                          	
  
                                                                        	
  

                                                                        	
  

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Ubio vet pregnancy_kit_technical_bulletin

  • 1. ubio  quickVET  RAPID  Pregnancy  Test  for  Cattle   u bi o B i ot e c h n ol ogy Sy s t e ms Pv t . Lt d.     Ph : +91-484-2532966, +91-9446438678   W e b: h t t p: //www.u bi o.i n Emai l : c on t ac t @ u bi o.i n   Accurate  diagnosis  of  pregnancy  is  of  crucial  importance  in  maintaining  optimal  reproductive  performance  of   cattle  and  high  profitability  of  dairy  farms.  It  is  desirable  for  the  farmer  to  know  as  soon  as  possible  if  a  mated   or  inseminated  cow  is  not  pregnant,  so  that  she  can  be  rebred  with  the  minimum  delay.  It  is  most  beneficial  if   non-­‐pregnancy   can   be   detected   before   the   first   heat   after   insemination.   Conventional   methods   that   allow   early   detection   of   pregnancy   are   not   accurate   enough,   and   reliance   on   these   methods   results   in   a   large   number   of   non-­‐pregnant   cows   assumed   to   be   pregnant   and   some   pregnant   cows   being   inseminated   inappropriately,   which   could   abort   them.   Conventional   pregnancy   detection   methods   that   are   accurate,   on   the  other  hand,  can  be  performed  only  after  at  least  35  days  after  insemination,  leading  to  the  loss  of  at  least   one  opportunity  to  inseminate.   ubio’s   quickVET   pregnancy   detection   test   provides   a   perfect   tool   to   detect   non-­‐pregnancy   in   cattle   accurately  within  21  days.  The  kit  provides  100%  detection  of  non-­‐pregnant  cases  and  allows  insemination  at   the   first   opportunity.   The   average   savings   realised   through   the   use   of   ubio   quickVET   test     is   as   high   as   Rs.1300/-­‐  per  cow  per  lactation  period.  Moreover,  the  test  can  be  easily  performed  within  20  minutes  at  cow-­‐ side  without  any  instrumentation,  which  makes  the  tool  attractive  even  for  direct  use  by  dairy  farmers.   The   test   employs   the   well-­‐tested   progesterone   assay   principle   to   detect   pregnancy.   It   measures   progesterone  levels  in  milk  or  serum  (for  heifers)  using  a  Rapid  ELISA  technique.   COMPARISON  OF  PREGNANCY  DETECTION  TECHNIQUES   Test   Stage   Principle   Advantages   Disadvantages   Accuracy   Non-­‐return  to   3  wks   Recording  dates  of   -­‐  Early   -­‐  Relies  on  good   About   service   service  and   detection,   oestrus  detection   50%   observation  of   without  missing   symptoms  of  heat     first  heat  after   -­‐  Large  variation   insemination   within  a  population   -­‐  Insemination  at   ‘false  oestrus’  can   lead  to  abortion   Early   4  wks  +   Detects  pregnancy-­‐ -­‐  Relatively   -­‐  Needs  blood   About   Conception   dependent  protein   early  detection   -­‐  May  persist  after   90%   Factor   complex  using  ELISA   loss  of  pregnancy   Ultrasound   4  wks  +   Visualization  of   -­‐  Relatively   -­‐  High  capital  cost   About   Scan   images  of  the   early  detection   -­‐  Need  good   100%   conceptus  using   -­‐  Accurate   technical  expertise   realtime  ultrasound   Palpation   5-­‐6   Feeling  the  presence   -­‐  Accurate   -­‐  Possible  damage   About   wks+   of  a  fetus  swelling  in   to  cow  or  conceptus   100%   one  of  the  uterine   -­‐  Need  good  skill   horns  by  inserting  an   and  technical   arm  into  the  rectum   expertise     of  the  cow.   Oestrone   15   Detects  oestrone   -­‐  Very  accurate   -­‐  Too  delayed  to  be   100%   Sulphate   wks+   sulphate  in  milk.   useful  in  most  cases   Concentration  is   proportional  to   oestrogens  produced   by  conceptus.   Conventional   3  wks   Progesterone   -­‐  Accurate   -­‐  High  capital  cost   100%  for  
  • 2. Milk   concentration  at  19-­‐ -­‐  Early   -­‐  Requires  blood  in   non-­‐ Progesterone   23  day  interval  from   detection   heifers   pregnancy   plate  ELISA   last  heat  is  much   -­‐  Non-­‐invasive   -­‐  Test  to  be   85%+  for   higher  in  pregnant   performed  on   pregnancy   cows  than  non-­‐ specified  days   pregnant  ones     ubio   3  wks   Progesterone   -­‐  Accurate   -­‐  Requires  blood  in   100%  for   quickVET   concentration  at  19-­‐ -­‐  Early   heifers   non-­‐ Rapid  Milk   23  day  interval  from   detection   -­‐  Test  to  be   pregnancy   Progesteron last  heat  is  much   -­‐  Non-­‐invasive   performed  on   85%+  for   e  Assay   higher  in  pregnant   -­‐  No   specified  days   pregnancy   cows  than  non-­‐ instrumentation   pregnant  ones   -­‐  Cow-­‐side  test   ASSAY  PRINCIPLES   The  ubio  quickVET  pregnancy  test  is  based  on  ELISA-­‐based  progesterone  assay  principle.  The  test  measures   progesterone   concentration   in   milk   or   blood   using   Rapid   ELISA   technique.   A   progesterone-­‐specific   monoclonal   antibody   is   coated   inside   the   test   cassette.   Milk/serum   and   labeled   progesterone   conjugate   are   added  to  the  test  and  control  wells  during  the  test.  The  progesterone  from  the  milk  and  the  labeled  conjugate   will   compete   to   bind   with   the   coated   antibody.   A   chromogen,   that   gives   color,   is   added   to   the   wells   and   it   will   bind   only   to   the   conjugate.   If   the   sample   contains   progesterone   (animal   is   pregnant),   the   test   well   will   be   lighter   than   the   control   well.   If   the   progesterone   level   is   very   low   (non-­‐pregnant   animal),   the   test   well   be   bluer  than  the  control  well.   The   concentration   of   progesterone   in   the   milk   or   In   the   case   of   a   pregnant   cow,   the   progesterone   blood   of   a   non-­‐pregnant   cow   varies   as   shown   concentration  varies  as  given  below.   below,  with  Day  0  corresponding  to  the  occurrence   of   heat.   It   is   assumed   that   insemination   is   appropriately  performed  at  the  time  of  heat.           It    is  obvious  that  there  is  a  significant  difference  between  progesterone  concentration  in  milk/blood    samples   taken  from  pregnant  and  non-­‐pregnant  cows  from  19th  to  23rd  day  after  heat  (and  insemination).  By  checking   the  progesterone  level  during  this  time  frame,  we  can  accurately  identify  non-­‐pregnancy.   This  principle  provides  100%  accurate  results  for  progesterone  levels,  which  translates  to  100%  accuracy  for   non-­‐pregnancy.   However,   there   are   some   corner   cases   where   a   false   positive   may   occur   for   cases   marked   ‘pregnant’  by  the  test.     1. If  heat  is  incorrectly  interpreted,  and  insemination  is  done  at  the  wrong  time,  the  test  will  show  the   result   as   ‘pregnant’   as   we   are   actually   detecting   the   high   progesterone   level   during   mid-­‐heat,   instead   of  that  caused  by  pregnancy.  
  • 3.   2. A  cow  that  is  correctly  judged  pregnant  might  lose  her  conceptus  due  to  various  reasons.  In  this  case,   the   test   is   accurate,   but   the   pregnancy   does   not   result   in   calf-­‐birth.   It   is   suggested   that   the   test   be   repeated  if  any  sign  of  heat  is  seen  after  the  cow  is  judged  pregnant.     3. A  cow  with  luteinized  cystic  follicles  will  produce  progesterone  at  varying  levels,  and  the  test  result  is   not   reliable   in   such   animals   as   it   might   show   positive   or   negative   based   on   the   progesterone   level   on   the  day  of  test.     CLINICAL  APPLICATIONS   1. Ovulation  Test:  ubio  quickVET  rapid  pregnancy  test  for  cattle  can  be  used  for  detecting  ovulation   in   cows   that   do   not   show   regular   symptoms   of   heat.   The   test     should   be   performed   during   the   suspected  heat  period.  A  dark  blue  spot  indicates  heat  and  insemination  can  be  performed  in  6-­‐ 18  hours.  A  clear  white  card  indicates  mid-­‐heat  and  the  test  needs  to  be  repeated  only  after  10   days.  A  light  blue  color  indicates  approaching  or  past  heat,  and  a  repeated  test  after  2  days  will   confirm  the  condition.   2. Identification   of   follicular/luteal   cysts:   Ovarian   cysts   are   an   important   infertility   problem   in   dairy  cattle.  It  is  often  difficult  to  differentiate  ovarian  cysts  into  follicular  and  luteal  by  palpation   of  the  ovary  per  rectum.  However,  once  the  presence  of  an  ovarian  cyst  has  been  determined  by  
  • 4. palpation,   differentiation   can   be   made   on   the   basis   of   progesterone   concentration.   Cystic   cows   having  a  low  progesterone  concentration  are  considered  to  have  follicular  cysts,  and  those  with   high   concentrations   are   classified   as   having   luteal   cysts.   For   practical   purposes,   differential   diagnosis   of   ovarian   cysts   is   important   in   selection   of   endocrine   therapy.   The   cyst   can   be   differentiated   by   performing   the   test   on   two   samples   taken   7   days   apart   –   a   dark   blue   spot   in   both  tests  indicate  a  follicular  cyst  and  a  clear  white  card  in  both  tests  indicate  a  luteal  cyst.   3. Evaluation   of   endocrine   therapy:     To   evaluate   treatment   of   follicular   cysts   using   GnRH,   ubio   quickVET   rapid   pregnancy   test   can   be   used   to   evaluate   to   progesterone   levels   10   days   after   administration  of  GnRH.  A  light  blue  spot  or  a  clear  white  card  indicates  response  to  treatment.   Similarly,   the   test   can   be   used   to   evaluate   response   to   treatment   of   luteal   cysts   using   Prostaglandin   compounds   as   unobserved   estrus   following   prostaglandin   administration   is   a   common  occurrence.  Response  to  prostaglandin  is  dependent  upon  the  presence  of  a  functional   corpus  luteum.  Therefore,  milk  progesterone  testing  prior  to  and  3  days  after  administration  of   prostaglandin   can   accurately   assess   if   administration   is   justified   (high   concentration   prior   to   administration)  and  if  luteolysis  has  occurred  (low  concentration  on  day  3).   4. Evaluation  of  fitment  for  embryo  transfer:  ubio  quickVET  rapid  pregnancy  test  for  cattle  can  be   used   in   embryo   transfer   programs   to   check   if   there   is   adequate   progesterone   level   in   the   recipient   to   support   the   embryo.   The   recipient   should   show   a   light   blue   test   result   before   an   embryo  is  transferred.   INTERPRETATION  OF  THE  RESULT   The  control  well  is  usually  colorless  or  a  very  pale  blue.  A  test  well  that  is  considerably  bluer  than  the  control   well   indicates   a   non-­‐pregnant   animal   with   low   progesterone   level.   A   test   well   lighter   than   or   of   the   same   shade   as   the   control   well   indicates   a   pregnant   animal   with   higher   progesterone   concentration,   subject   to   caveats.   Slightly   bluer   tinge   in   the   test   well   when   compared   to   the   control   well   indicates   non-­‐pregnant   result   during  approaching  or  past  heat  period  –  in  this  case,  the  test  timing  and  heat  calculation  must  be  checked,   and  the  test  should  be  repeated  if  necessary.                                                                  Not  Pregnant                Not  Pregnant                      Pregnant                            Pregnant  
  • 5. TEST  PROCEDURE                         1.  Collect  milk  sample  from  cow  in  a  clean   vessel  after  discarding  milk  from  the  first  few   KIT  COMPONENTS                   pulls.  For  pregnancy  test,  the  test  should  be   -­‐  One  Aluminium  pouch  containing     done  between  19th  to  23rd  day  after   the  test  cassette.   insemination.   -­‐  Three  droppers  for  transferring                         milk  and  reagents.   2.  Keep  the  test  pack  at  26-­‐30ºC  for  1-­‐2  hours   -­‐  One  transparent  vial  marked  ‘1’   to  bring  it  to  room  temperature.  Remove  the   containing  reagent  1.                         test  cassette  from  Aluminium  pouch  and  keep   -­‐  One  dropper  bottle  marked  ‘2’   on  a  flat  surface.  There  are  two  wells  on  the   containing  wash  buffer  (reagent   card,  marked  C  and  T.   2).   3.  Add  three  (3)  drops  of  milk  to  both  the  wells   -­‐  One  opaque  vial  marked  ‘3’                   (C  and  T)  using  a  dropper  supplied  with  the   containing  reagent  3   test.  Dispose  the  dropper  immediately.     4.  Keep  the  cassette  on  a  flat  surface  for  5   PRECAUTIONS  &   minutes.  Periodically  tap  the  side  of  the   WARNINGS                cassette  for  proper  mixing.  Take  care  not  to   -­‐  Store  in  fridge  at  4-­‐8ºC,  do  not   splash  the  milk  in  the  wells.   freeze   5.  Using  a  fresh  dropper,  Add  three  (3)  drops   -­‐  Do  not  reuse  test  kit.   of  reagent  into  both  the  wells  (C  and  T)  from   -­‐  Use  a  separate  dropper  for  each   the  bottle  marked  Reagent  1.  Let  milk  and   reagent.                     reagent  mix  by  gently  tapping  the  cassette.   -­‐  Do  not  mix  components  from   6.  Keep  the  cassette  on  a  flat  surface  for  20   different  kits.   minutes.  Gently  tap  the  cassette  at  4-­‐minute   intervals.   -­‐  Do  not  touch  the  test  wells.             7.  Tip  the  cassette  and  empty  the  wells  into  a   sink.     8.  Fill  the  wells  with  the  wash  buffer  in  the     dropper  bottle  marked  ‘Reagent  2’,  tap  gently             and  empty  the  wells  into  the  sink.  Repeat  this   washing  5-­‐6  times.  Shake  off  any  remaining     solution  from  the  wells.     9.  Add  5  drops  of  Reagent  3  in  the  opaque  vial   to  both  the  empty  wells.  Keep  the  test  on  a           flat  surface.  The  result  appears  after  8   minutes,  and  can  be  read  till  15  minutes.