MVSc

The study was designed and conducted with the objectives of assessing:
(1) efficiency of two different temperatures, media and duration of storage for
short term preservation of bovine preantral follicles in situ and (2) efficiency of
conventional vitrification (CV) and solid surface vitrification (SSV), for long
term cryopreservation of bovine preantral follicles in situ.
Ovaries of six freshly slaughtered adult crossbred cows were collected
and transported to the laboratory within one hour in normal saline at room
temperature. The ovarian cortex from both ovaries of each animal was
separated, divided further into fifteen pieces. One hour after the slaughter a
cortical piece was selected at random from each animal and fixed in Bouin’s
fluid for histological processing later on (control). Twelve cortical pieces from
each animal were randomly allotted for short term preservation, of which, six
pieces each were placed in vials containing normal saline and remaining in
Dulbecco’s phosphate buffered saline (DPBS). Three vials from normal saline
group and DPBS group were stored at room temperature (25ºC) and the
remaining three vials from each group were stored at refrigeration temperature
(3-5ºC). One cortical piece was taken out, from each media, from both the
storage temperatures, at four, eight and twelve hours of storage and fixed for
histological analysis. The remaining two cortical pieces were further divided
into smaller fragments of approximately 1mm3 size. The fragments from one
piece were vitrified using conventional vitrification (CV) and the rest by solid
surface vitrification (SSV) and stored in liquid nitrogen for ten days. The
fragments were warmed and fixed after the storage period. The quality of
preantral follicles in the fixed ovarian tissues was evaluated based on
morphology in histological sections. The preantral follicles were counted,
classified as primordial, primary and secondary.
Storage at refrigeration temperature preserved the preantral follicles very
much better than the storage at room temperature. The amount of normal
preantral follicles reduced progressively with passage of time. Primordial
follicles were better adapted to preservation than primary and secondary
follicles. The preantral follicles were best preserved at refrigeration temperature
up to four hours of storage. The morphologically normal preantral follicles in
normal saline (55.17 %) and DPBS (56.17 %) at this temperature and storage
period did not show significant difference from that of control (59 %). The
primordial follicles were preserved in both normal saline (61.42 %) and DPBS
(61.15 %) at refrigeration temperature up to four hours, at levels similar to
control (62.63 %). However, only DPBS at refrigeration temperature could
ideally preserve primary (51.13 % vs. 53.06 % in control) and secondary
follicles (42.10 % vs. 48.83 % in control).
The number of morphologically normal preantral follicles in situ was
significantly reduced after both CV (25.5 %) and SSV (37.17 %). But, SSV was
significantly better than CV for preserving the quality of preantral follicles.
According to this study DPBS at refrigeration temperature is ideal for
preserving all the three classes of bovine preantral follicles in situ up to four
hours of storage. Though vitrification of bovine ovarian tissue could not preserve
preantral follicles at ideal levels, SSV was found to be far superior to CV in
preserving bovine preantral follicles in situ.