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In vitro Fertility Assessment of sperms
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The widespread use of AI in livestock breeding, initiated scientists to persistently develop laboratory tests that would accurately predict the fertility of sires.
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The commonly employed semen evaluation tests have been effective in controlling the quality of bovine frozen semen used for A.I. but cannot be relied upon to accurately predict fertility.
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Hence, there is a need for evolving some methods to assess the fertility of spermatozoa in vitro before it is used for AI.
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Zona-free hamster ova penetration bioassay is a recently developed technique that could be successfully employed for in vitro fertility assessment of spermatozoa.
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Zona-free ova of most mammalian species retain very strong or fairly strong species specificity, rejecting entry of spermatozoa of most other species.
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The Golden hamster is exceptional and permits sperm entry of wide variety of other species provided the sperm have completed capacitation.
The hamster egg penetration is a heterologous sperm penetration test. The ova of any species will not allow the other species sperms to enter inside even after the removal of the zona pellucida. But the hamster egg will allow the other species sperms after the zona removal by enzymatic digestion.
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Sperm penetration bioassay (SPB) has been evolved as a reliable test to assess the fertility of spermatozoa.
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Various reports also indicate that there is a positive correlation between bioassay measures and fertility.
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SPB could also be used in screening and selection of donors with high fertility for in vitro fertilization and embryo transfer studies.
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The logistics of SPB is some what cumbersome, because a colony of hamsters is to be maintained, require scheduled hormonal injections, the animal has to be sacrificed and the oocytes are to be recovered at the optimum time.
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Further, the spermatozoa to be tested should also be available at a fixed time.
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To eliminate these problems, the use of cryopreserved hamster oocytes has been attempted.
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In addition, cryopreservation of hamster oocytes has practical advantages such as long-term storage, ready supply and easy transportation of viable oocytes.
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Ready availability of hamster oocytes is essential for infertility clinics and semen banks where maintenance of hamsters is difficult.
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Now, by research in this Semen Bank, it is established that cryopreserved hamster oocytes can also be successfully utilised for SPB.
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SELECTION AND SUPEROVULATION OF HAMSTER AND COLLECTION AND PREPARATION OF OOCYTE
Experimental animals
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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
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PMSG
hCG
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Medium for handling oocytes
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For handling oocytes, the Biggers, Whitten and Whittingham (BWW) medium (Biggers et al. 1971) with minor modifications should be used.
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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.
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The media used for the study, should be prepared as freshmedium, and sterilized by filtration using sterile single use syringe filter unit.
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The triple glass distilled water for the preparation of media should also be autoclaved before use.
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Recovery of the oviduct
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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.
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Then, the hamster should be fixed on a dissection board and under aseptic precautions and the abdomen is opened by midventral incision.
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By using curved scissors, the oviducts are removed after severing the uterine and ovarian ends.
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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
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The petridish containing oviducts are viewed under the stereozoom microscope and observed for the contractions and relaxations of the ampullar portion of the oviducts.
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The cumulus mass are then released by puncturing the ampullar portion with pointed forceps.
Digestion of cumulus mass
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Washing of oocytes
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After digestion, the oocytes are released from cumulus cells.
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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
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The washed oocytes should be evaluated under stereozoom microscope for their morphology.
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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
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Zona pellucida is removed by treating the oocytes with freshly prepared 0.1% trypsin in mBWW medium for 30 to 60 seconds.
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During digestion, the oocytes should be constantly monitored through the stereozoom microscope to avoid excessive digestion.
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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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Participants