Acta vet. scand. 1986, 27, 50!>-519. Prom the Department of Animal Reproduction aind ,the Departmen,t of Anatomy, Royal Veterinary anid Agricultural University, Copen­ hagen, Denmark. CHRONOLOGICAL CHANGES OF BOVINE FOLLICULAR OOCYTE MATURATION IN VITRO* By K. P. Xu, T. Greve, S. Smith and P. Hyttel XU, K. P., T. GREVE, S. SMITH and P. HYTTEL: Chronological changes of bovine follicular oocyte maturation in vitro. Acta. ve.t. sicand. 1·986, 27, 5-0!>-519. - Chronological changes of bovine follicular cumulus-oocyte-complexesi were studii.ed after in vitro maturation oveir a period of 48 h. Accordintg to thei!r thickness and compactness of cumulus investments they were classified into 4 groups and cultured in enrfohed Ham's F-1-0 medium with or without human chorionic gonadot·rophin (hCG) and estradiolbenzoate (EB) for 0, 6., 12., 18, 21, 24, 27, 30 and 48 h. Rep1resentative samples were .taken at each time interval for evalua-tion of nuclear maturation s·tages, ooplasm quality and size of the perivitelline space (PVS). The Tesults showed that oocyte nuclear breakdown (ONBD) required 6 to 12 h culture., and the peak of the first polar abstriction. occurred at 24 h. The culture period required for ONBD and abstniction of the first )?Olar body were related to the thickness and compactness of cumulus: m.vestrnents with and approximately 6 h delay in hea.vily compacted complexes. Ooplasm quaJ.ity e.v;aluation failed to show a clear trend, but the PVS increased in. size from 0 h to 3-0 h and then .retracted again from 3-0 to 48 h. The overall maturation rate in the presence of hCG and EB was 79.1 %, and a substanUal proportion (68.8 % ) of niud'e or partially covered oocytes reached metaphase II stage. In the presence of hCG and EB no block at either metaphase I or at anaphase-telophaoo I was ob­ served. In the absence of hCG and EB the percentage of oocytes ing metaphase II was much lower (48.6 % ) in comparison. with oocytes matured -in the presence of the$e hormones ( 79.1 % ) . I.t was concluded a ve"Y. high p•roportion of oocytes. could be matured in vitro and that .the cumulus investments and addUion of certain hormones affected the maturation rate. nuclear matura.tioill; ho.rmones; cumulus; cattle. • This work was supported by grants from DanJs.h Veterinary and Agricultural Research Council and 506 K. P. Xu et al. MQst of mammalian o.ocytes including those of cattle mature in vitro when they are released from the follicular environments and placed in suitable culture media (Edwards 1965). The in vitro maturation events and the biochemical components involved in mei:osis are poo-rly understood despite considerable research efforts in recent year.s (Jagiello et al. 1974, Sato et al. 1978, Fulka et al. 1981, Siiss & Wiithrich 1985, Motlik & Fulka 1986, Eppig & Schroeder 1986). The only reliable criteria for normal oocyte maturation are fertiilization in vitro and subsequent development of the fertilized egg into Live offspring (Moor et al. 1981, Hensleigh & Hunter 1985, Leibfried-Rutledge et al. 1985). However, the developmental capacity of most in vitro matured oocytes is low (Ball et al.1984) and only recently has a pregnancy been established following in vitro fertilization of in vitro matured oocytes (Critser et al. 1986). Even in vivo matured oocytes have low survival rates following iin vitro fertilization (Greve et al. 1984, Lambert et al. 1986). There is still a need for more detailed studies of the r-equire­ ments for and the conditions that allow proper in vitro matura­ tion of bovine oocytes, and the aims of the present study are: ( 1) to establish a detailed chronology of the nuclear maturaition of bovine oocytes cultured in vitro for different periods of time, and (2) to examine if certain factors such as hormones could influence this nuclear maturation process. MATERIALS AND METHODS Oocyte collection Ovaries were obtained from cows within 30 min after slaugh­ ter at a local abattoir. Follreu'lar contents f.rom small visible antral ·follicles (approx. 1- 6 mm in diameter) were aspirated and placed in a conical centrifuge tube containing 4 ml Hepes buffered Ham's F-10 (F-10), supplemented with 20 % heat treated foetal calf serum (FCS), 0.4 mmol/il gl.utamine, and 50 I.U. penicillin and 50 µg dilhydrostreptomycin per ml. The tubes were kept at 30-38°C. After 30 to 40 min the sedimented oocytes with their cumulus investments were transferred to fresh medium of the same composition. To establish a zero hour control group follicular contents were stored without medium at 0°C unti1l evaluation and fixation, Bovine follicular oocyte maturation 507 Oocyte evaluation and selection W.ithin 2 to 3 h of collection the oocytes weiie brought to the laboratory, washed 3 times in fresh medium and evaluated under a stereomicroscope. In Exp. I (a and b) pools of oocytes were assigned into 4 groups based upon their gross morphology and the integrity of the cumUJ1us ceJils. The features and descdptions of different groups are shown on Plate 1, Figs. 1 to. 5. No attempt was made to cfa.ssiify the ooplasm qualJi.ty of groups I, II and III due to the thick layers of cumulus cells that made proper evalua­ tion impossible. Oocytes with shrunk, vacuolated ooplasm or with other signs of degeneratiion wer·e excluded from group IV. In Exp. II only heal.thy looking oocytes with more than 2- 3 layers of cumulus were included. Oocyte culture The onset of culture time was defined as the mean time of the oocyte collection period. The medium used for oocyte culture wais identical to the F -10 used for the collection with the excep­ tion, that it was bicarbonate buffered. In Exp. lb no further supplementation was done while in Exp. Ia and Exp. II 2 l.U. hCG (Physex, Loo, Denmark) and 1 µg estradiolbenzoate (Ovex, Leo, Denmark) were added per ml. Culture was performed in 4 well multidishes, at 38°C/5 % C02 in humidified air for 0, 6, 12, 18, 24, 30 and 48 h (Exp. Ia and Exp. lb), and 21, 24 and 27 h (Exp. II). For each group, 10 to 15 oocytes were placed in 1.0 ml medium which was filtered through a 0.22 µm milipore £ilter and equilibrated in 5 % co2 for at least 3 h prior to cultivation. Oocyte evaluation before fixation After various periods of culture, oocytes were sampled and the cumulus cells were removed with trypsin (original Trypsin­ EDTA solution, GIBCO U.K.) along with repeated pipetting. The denuded oocytes were mounted on grease free slides and ex­ amined under a stereomicroscope. Oocytes were classified ac­ carding to the size of the PVS as: absent, smaH, mediiium and large. Based upon the character of the ooplasm, three oocyte types could be defined. Those with finely granulated and evenly distributed organelles were regarded as type I, an.d those that were mare coarsely granulated with unevenly distributed orga­ nelles and some vacuolation were designated as type II. Large 508 K. P. Xu et al. vacuoles and shrunken ooplasm were indicative of degeneration and these oocytes were ol.assified as type III. Following this stereomicroscopic evaluation oocytes were fixed ·in methanol: acetic acid ( 3: 1) for at least 48 h, and then stained with 1 % aceto-orcein for the final examination. Oocyte evaluation after fixation After fixation and staining, oocyte nuclear maturation was evaluated under interference phase contrast microscope (320 x) to define the meiotic stages as follows. 0 o c y t e n u c l e u s s t a g ·e I ( 0 N I) : Oocytes had a distinct nuclear enve'lope, chromatin was only around the nu­ oleolus, whOOh may not always be present ,and/or in the apperur­ ence of a few orcein-positive zones. 0 o c y t es nucleus stage I I (0 NI I): The nuclear en­ velope was s.Ulll present but less distinct, the chromatin was distributed in separate, well stained clumps or the fodividual fillamentous bivalents were distingufahable. D i a k i n e s s i s (D) : The nuclear envelope was no longer vi·sible, and the bivalents remained within the area of the oocyte nucleus. Late di akin es i.s (L D): The oocyte nucleus area had totaHy disappeared and the bi;valents were further condensed. Met a phase I (MI): The chromosomes were maximailLy condensed, and sometimes present as a whole clump from an equatorial view. Anaphase-Telophase I (A-TI): The chromosomes were under division or segregation, and the membrane of the fir.st pola·r body might be present but the spindle was still not detached. M et a p h a s e I I (M I I) : The contracted metaphase chromosomes and the first polar body were both present. If none of the above mentioned nuclear features were identif­ ied in the preparation, the oocytes were as Non-Defined (ND). K. P. Xu, T. Greve, S. Smith and P. Hyttel: Chronological Changes of Bovine Follicular Oocyte Maturation in Vitro. P 1 ate I . Morphologival appearance of cumulus-oocyte-complexes before and after culture. Figure I. (Group I): Oocytes with tight, completely compacted cumulus invest­ ments (70 x). Figure 2. (Group II): Oocytes with complete but not tightly compacted cumulus investments (70 x). Figure 3. (Group Ill): Oocytes with only a few layers of cumulus cells (70 x). Figure 4. (Group IV): Oocytes with only partial or none cumulus investments . Figur e 5.