The main objective of the present study was to characterize buffalo preantral ovarian follicles. Parts of ovarian cortex, collected from postpubertal buffalo females that were having estrous cycles at regular intervals, were selected under stereomicroscopy and processed for optic and transmission electron microscopy. Primordial follicles were characterized as an oocyte encircled by one layer of flattened cells. The buffalo primordial follicle has a mean diameter of 35 microm and the oocyte diameter is 24.9 microm. The oocyte nucleus is relatively large and eccentric; and in the cytoplasm a large amount of mitochondria, vesicles and endoplasmic reticulum cistern, mainly of the smooth type is observed. The primordial follicles cells are rich in plasma membrane invaginations, which are observed within the cell and between the cell and the oocyte. The primary follicles (mean diameter of 41.8 microm) consist of an oocyte, with a medium diameter of 26.9 microm, surrounded by one layer of cubical granulosa cells. At this follicular stage, the beginning of zona pellucida deposition can also be seen in areas between the oocyte and follicular cells. The secondary follicles, which are surrounded by more than one layer of cubical cells, have a diameter of 53.3 microm, and the oocyte has a mean diameter of 29.4 microm. The ultrastructural analysis showed a large amount of coalescent vesicles, more evident in the oocyte periphery. The zona pellucida (ZP) is thicker at this stage and contains a large quantity of glycoproteins. In general, the ultrastructure of buffalo preantral follicles was similar to that of other mammalian species, but some differences were observed, which indicate species specific characteristics. The main differences observed were cytoplasmic vesicles quantity, mitochondria shape and inner content, ZP deposition and granulosa cell-oocyte junctions. In conclusion, the morphological differences described in this paper, could be responsible for some functional differences observed in Bubalus bubalis in vitro embryo production and follicular dynamics, when compared with Bos taurus or Bos indicus species.

The main objectives of the present study were to determine the ultrastructural modifications occurring in the oocyte during late folliculogenesis and to estimate pre-antral follicle population in buffalo. Half the collected ovaries were fixed and prepared for optic microscopy; the antral follicles from the other ovaries were measured and individually punctured. The cumulus-oocyte complexes (COCs) were processed for transmission electron microscopy. The number of pre-antral follicles in buffalo ovaries was estimated at 19 819 structures. Cumulus-oocyte complexes derived from 1-mm antral follicle had an eccentrical nucleus and compact corona radiata, ooplasm vilosities were fully embedded in zona pellucida (ZP) and a well-defined junction could be observed. Mitochondria were predominantly round and well distributed in ooplasm, as were small lipid vacuoles. In COCs derived from 2-mm antral follicles, the initial formation of perivitelline space was observed. The nucleus was peripherally located and the number of pleomorphic mitochondria increased. Cortical granules were clustered at oocyte periphery and lipid vacuoles increased in number and size. In COCs derived from 6-mm antral follicles, the organelles were located mainly in the perinuclear region. Golgi complexes and smooth endoplasmic reticulum (SER) were more developed. Mitochondria migrated to the cortical region and lipid vacuoles migrated to the medullar region. In COCs derived from 10-mm antral follicles, the lipid vacuoles coalesced and occupied the medullar region of the oocyte, together with a well-developed SER. Mitochondria were pleomorphic and located at the oocyte periphery. In conclusion, the morphological differences described in this paper could be responsible for some functional differences observed in in vitro embryo production and follicular dynamics for buffalo, when compared with cattle.

The objective of the present study was to describe ultrastructural changes in the nucleus and cytoplasmic organelles during in vitro maturation (IVM) of buffalo cumulus-oocyte complexes (COCs). The structures were collected by ovum pick-up (OPU). Some COCs, removed from maturation medium at 0, 6, 12, 18 and 24 h, were processed for transmission electron microscopy. The average number of COCs collected by OPU/animal/session was 6.4, and 44% of them were viable. Immature oocytes had a peripherally located nucleus, Golgi complex and mitochondrial clusters, as well as a large number of coalescent lipid vacuoles. After 6 h of IVM, the oocyte nucleus morphology changed from round to a flatter shape, and the granulosa cells (GC) lost most of their contact with zona pellucida (ZP). At 12 h the first polar body was extruded and the aspect of lipid droplet changed to dark, probably denoting lipid oxidation. Cortical granules were clearly visible at 18 h of maturation, always located along the oocyte periphery. At 24 h of IVM the number of cortical granules increased. Ultrastructure studies revealed that: (1) immature oocytes have a high lipid content; (2) the perivitelline space (PS) increases during IVM; (3) Golgi complexes and mitochondrial clusters migrate to oocyte periphery during IVM; (4) 6 h of IVM are enough to lose contact between GC and ZP; (5) the oocyte lipid droplets' appearance changes between 6 and 12 h of IVM.