Selection, superovulation, collection and preparation of oocyte

SELECTION AND SUPEROVULATION OF HAMSTER AND COLLECTION AND PREPARATION OF OOCYTE

Experimental animals

  • Healthy, sexually matured young female golden hamsters (Mesocricetus auratus) of eight to 12 weeks of age weighing between 60 and 120 g should be used for carrying out this test.

Superovulation and recovery of oocytes

  • The number of oocytes ovulated in the normal cycle is increased by superovulating the hamsters with Pregnant Mares' Serum Gonadotrophin (PMSG) and human Chorionic Gonadotrophin (hCG).

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PMSG

  • 50 I.U. of PMSG (Folligon) should be injected intraperitoneally using a sterile single use syringe for stimulating the follicular growth.

hCG

  • After 78 to 82 hours of PMSG injection, 75 I.U. of hCG (Chorulon) should be administered intraperitoneally using a sterile disposable syringe for superovulation.

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Medium for handling oocytes

  • For handling oocytes, the Biggers, Whitten and Whittingham (BWW) medium (Biggers et al. 1971) with minor modifications should be used.
  • The tonicity of the medium should be adjusted to 290-300 mosmol /kg and the pH of the medium should be adjusted to 7.2 to 7.3.
  • The media used for the study should be prepared afresh and sterilized by filtration using sterile single use syringe filter unit.
  • The triple glass distilled water for the preparation of media should also be autoclaved before use.

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Recovery of the oviduct

  • After 16 to 17 hours of hCG injection, the hamster should be sacrificed by exposing to toxic dose of chloroform anesthesia in a closed chamber.
  • Then, the hamster should be fixed on a dissection board and under aseptic precautions and the abdomen is opened by midventral incision.
  • By using curved scissors, the oviducts are removed after severing the uterine and ovarian ends.
  • The oviducts are then transferred to a drop of handling medium placed in a sterile single use 35 x 10 mm petridish.

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Recovery of cumulus mass

  • The petridish containing oviducts are viewed under the stereozoom microscope and observed for the contractions and relaxations of the ampullar portion of the oviducts.
  • The cumulus mass are then released by puncturing the ampullar portion with pointed forceps.

Digestion of cumulus mass

  • The released cumulus mass is then transferred to a drop of freshly prepared 0.1% solution of hyaluronidase in mBWW medium and allowed for five minutes for the digestion to take place.

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Washing of oocytes

  • After digestion, the oocytes are released from cumulus cells.
  • The freed oocytes are then aspirated and washed three to four times by successive transfer of the oocytes to drops of medium to remove the cell debris.

Evaluation of oocytes

  • The washed oocytes should be evaluated under stereozoom microscope for their morphology.
  • Bright, shiny oocytes with uniform, spherical, regular cytoplasm and an intact zona pellucida are considered as normal.

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Removal of zona pellucida

  • Zona pellucida is removed by treating the oocytes with freshly prepared 0.1% trypsin in mBWW medium for 30 to 60 seconds.
  • During digestion, the oocytes should be constantly monitored through the stereozoom microscope to avoid excessive digestion.
  • After the dissolution of zona, the zona-free oocytes are washed three or four times in drops of fresh handling medium and kept ready for coincubation with capacitated bull spermatozoa.

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Last modified: Monday, 4 June 2012, 11:39 AM