Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes (79.60 ± 0.77 μm vs. 101.46 ± 1.07 μm, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found (79.51 ± 2.36 μm vs. 101.46 ± 1.07 μm, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured (1.48 ± 0.42 μm) than in vitro matured for 72 and immature oocytes (1.10 ± 0.16 and 0.43 ± 0.12 μm, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.

In canine, oocytes are ovulated at the GV (germinal vesicle) stage and they have to fulfill maturation phase before reaching metaphase II stage. The efficiency of in vitro maturation is still very low. Therefore, the aim of this study was to investigate the effect of in vitro maturation on nuclear changes of immature canine oocytes recovered from different reproductive stages ovaries and different culture conditions. The oocytes were cultured in TCM-199 with supplement at 5% and for 72 h. The nuclear maturation of canine oocytes was evaluated with Hoechst 33342 stain under fluorescence microscope (Fig. 1). The results of this study detected differences in in vitro maturation rate between oocytes recovered from follicle status and non-follicle status ovaries. However, these differences were not significant as indicated in Table 1 and Fig. 2. In regard to the effect of culture condition with supplements, we did not found significant differences compared with control group (Table 2, Table 3). One of the reasons for this data could be the conditions that ovaries were exposed during slaughtering process or the long distant transportation of the ovaries. Although these data have not shown clearly significant differences results compared with control, furthermore the different reproductive status ovaries was beneficial for maturation of oocytes in vitro and can be a basic part of knowledge to improve in vitro maturation of canine oocytes.

These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03～0.10 mM SN and 0.3～1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a CO2 incubator (5% CO2, 95% air, 38℃). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were 25.9±3.5%, 36.4±3.2%, 33.3±3.5%, 28.8±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03～0.07 mM SN were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were 28.0±4.2%, 36.5± 3.6%, 30.0±3.8%, 19.2±3.5%, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.05 mM SN were 26.0±3.2%, 28.0±3.4%, 38.0±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.5 mM NO were 22.0±3.0%, 30.0±3.8%, 36.0±4.2%, respectively. These result was significantly increased compare to the control.

These study was to investigate the in vitro fertilization and viability of fresh and vitrified oocytes. Also, the developmental capacity of IVF and intracytoplasmic sperm injection (ICSI) oocytes were investigated. Then vitrification was performed with the use of 20% ethylene glycol + 20% DMSO + 0.5 M sucrose + 10% FCS + TCM-199 medium. Vitrification immature oocytes are cultured in vitrification solution for 10 min afterwards transferred to expose at room temperature for 5 min. and transferred to the ice water for 5 min. The oocytes were sealed in a 1.0 mm straw and placed in a LN2 container. Frozen oocytes were rapidly thawed in a water bath at 30～35℃, and then placed in TCM-199 medium containing 0.5 M sucrose for 5 min each, respectively, at 38℃. After being washed for 2～3 times, using fresh medium the oocytes were cultured in TCM-199 medium supplemented with 5% FCS at 38℃ in 5% CO2 and air. The normal morphology of fresh and vitrified-thawed oocytes were 87.1±2.1% and 54.8±2.5%, respectively. The viability rates of fresh and vitrified-thawed oocytes were 70.0±2.2% and 41.9±2.6%, respectively. Viability rates of vitrified-thawed oocytes were lower than that of fresh follicular oocytes (p<0.05). The in vitro maturation rates of fresh and vitrified oocytes were 45.1±3.6% and 28.9±4.4%, respectively. The IVF rates of fresh follicular and vitrified-thawed oocytes were 34.0±2.2% and 20.2±2.6%, respectively. The in vitro maturation and fertilization rates of vitrified-thawed oocytes were lower than those of the fresh follicular oocytes (p<0.05). A total of 350 oocytes were fixed and stained after co-incubation with spermatozoa, of which 88 had identifiable nuclear material. After IVF for 20 hrs, 25.1±3.4% of the oocytes found to have been penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, and 105 oocytes contained identifiable nuclear material. After IVF and ICSI for 20 hrs, 34.3±3.4% and 59.0±2.0% of the oocytes were found to have been penetrated by spermatozoas. The developmental rates upon ICSI were significantly higher than those of the IVF method (p<0.05).

The in vitro maturation rate of vitrified-thawed canine oocytes was 30.8±3.4%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (52.0±2.5%, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation and developmental rates of the vitrified-thawed oocytes were 17.5±2.5% and 8.8±3.4%, respectively. This results were lower than the control group (43.6±3.2% vs 20.0±3.0%). SOD1 gene expression of 1～2 mm of follilce size were higher than those of above 6 mm follicle size. SOD2 gene expression of 1～2 mm of follicle size were significantly higher than those of above 6 mm follicle size (p<0.01). The expression pattern of SOD1, 2 was constantly expressed in both groups but strongly expressed in follicles (1～2 mm) group when compared to the above 6 mm follicles. SOD gene expression between groups the fresh and vitrified oocytes groups were significant differences in rates. However, RGS gene expression between groups the fresh and vitrified oocytes groups were no significant differences in rates.

These study was carried out to investigate the effects of the recovery time, diameter of oocytes on in vitro fertilization or intracytoplasmic sperm injection (ICSI). The in vitro maturation rates to MII stage of oocytes recovered at the inactive, follicular and luteal stages matured for 72 h were 1.4±0.0%, 43.4±3.2% and 10.8±2.7%, respectively. The fertilization rates of in vitro cultured oocytes recovered from ovaries at the in active, follicular and luteal stages were 0.0±0.0%, 15.7±3.4% and 7.6±3.5%, respectively. The in vitro maturation rate of oocytes recovered from ovaries at the follicular stage of the reproductive cycle was significantly higher than those at the inactive and luteal stages (p<0.05). The penetration rate determined that the percentages of oocytes with diameters in the <100 μm, 100 to 100 μm and 110 to 120 μm ranges were 17.5±4.7%, 43.9±4.5%, 21.3±3.4%, respectively. The penetration rate of oocytes with diameters between 100 to 110 μm was significantly higher than that of oocytes whose diameters were 100< μm and 110～120 μm (p<0.05). The penetration rate of oocytes determined that the percentages of ovaries with diameters between 1 to 5 mm and 6 to 10 mm were 32.9±3.2% and 17.5±3.7%, respectively. Thus, the diameters of the ovaries were significantly higher at 1 to 5 mm (p<0.05). A total of 264 oocytes were fixed and stained after co-incubation with sperm, of which 72 had identifiable nuclear material. After in vitro fertilization for 20 hrs, 27.3% of oocytes were penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, of which 38 oocytes contained identifiable nuclear material. After in vitro fertilization and ICSI for 20 hrs, to 27.3% and 67.9% of oocytes were penetrated by spermatozoas. The in vitro fertilization rates by ICSI was significantly higher than that in vitro fertilization method (p<0.05).

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% CO2, 95% air, 38℃. The in vitro maturation rate to MⅡ stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44～72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

In the present studies, we have intended to compare the EDS (20% EG + 20% DMSO + 0.4 M sucrose + 10% FCS) and EDT (20% EG + 20% DMSO + 0.3 M trehalose 10% FCS) methods for vitrification of canine oocytes, in order to improve the vitrification methods. The survival rate of vitrified‐thawed oocytes using the EDS method was 15.1±1.8% (p<0.05), which was lower than that of the control group (66.7±2.5%). About 45～55% of the vitrified‐warmed oocytes showed normal morphology, as assessed by PI staining. However, the ratio of survival rate of oocytes showed lower than that of normal morphology in comparison between EDS method and control group. The survival and developmental rates of vitrified‐warmed oocytes by the EDS and EDT methods were 16.7±1.4% and 11.1±0.8% and 8.3±1.4% and 4.4±1.8%, respectively (p<0.05). The results were significantly lower than the control group (66.7±2.5% and 16.7±3.7%). However, the survival rate of vitrified‐warmed oocytes using EDS method showed higher than that in the ETS group.

The techniques of IVM, IVF and IVC of canine oocytes may provide useful information for gamete salvage programs and the conservation of endangered canidae. This investigation has been made to determine the efficiency of in vitro maturation (IVM) as a basic experiment to study the development of canine oocytes after in vitro fertilization (IVF). The rate of oocytes developing to the MII stage was higher in the hormone treated group (10 IU/ml hCG+eCG, 14.7%, p<0.05) than in the control group (0 IU/ml hCG+eCG, 10.0%). The monospermy and pronuclear rates of canine oocytes were investigated after caffeine treatment on IVF. Canine oocytes were fertilized in the Fert‐TALP medium supplemented with 0, 10, 20 or 30 mM caffeine (Fert I, Fert II, Fert III or Fert IV, respectively). The highest pronuclear formation rate was obtained in the Fert I for 24 h IVF (6.7%, 6/89). Therefore, it is believed that unlike in other mammals, caffeine in canine IVF does not increase the efficiency of fertilization rate, and is not an important factor.