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Ubio vet pregnancy_kit_technical_bulletin


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  1. 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. 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. 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. 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. 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.