الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 157 |  |  |
| | | from | 157 | | |
Abstract
This study was carried out at the International Livestock Management Training Center, Sakha, Kafr El-Sheikh Governorate belonging to the Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture during the period from August 2008 to August 2009.
Three experiments were conducted in this study. The aim of the first experiment was to determine the effect of some factors affecting oocyte vitrification of rabbit does including, three cryoprotectants (EG 40%, DMSO 40% and their combination, EG 20% + DMSO 20%) and three carrier systems [Straws (S), Open pulled straws (OPS) and Sealed close Open pulled straws (SOPS)]. The second experiment aimed to study the effect of vitrification of rabbit oocyte (based on the best results of the 1st experiment) on in vitro maturation rates (IVM) as compared to fresh oocytes (non-vitrified oocytes). As for, the third experiment it aimed to study the effect of in vitro maturation of vitrified rabbit oocytes on in vitro fertilization rates.
A total of 36 New Zealand white (NZW) rabbit does with approximately 5 months of age and 3.25±0.25 kg live body weight (LBW) and 5 NZW bucks with approximately 7.5 months of age and 3.75±0.31 kg LBW were used in this study.
Ovaries were collected immediately after slaughtering and oocytes were harvested. All visible follicles on the ovarian surface with ≥1 mm in diameter were counted. The oocyte yield was recorded and the number of oocyte/ovary was calculated. The recovery rate was determined as percentage of oocytes in proportional to total number of visual follicles present on the ovarian surface of each doe and oocyte categories were evaluated. Only morphologically high quality immature oocytes (COCs) were utilized in this study. Three types of cryodevice were done with all cryoprotectants including conventional straws(S), open pulled straws (OPS) and sealed open pulled straws (SOPS). Oocyte viability was evaluated morphologically and damage oocytes with crack in zona pellucida, split in two halves, change in shape and leakage of contents were recorded. In vitro maturation (IVM) and in vitro fertilization of vitrified oocytes was conducted to obtain maturation rate of vitrified oocytes.
The obtained results could be summarized as follows:
1. Survival rate:
- Total number of follicles including count of vesicular follicles, small follicles < 3 mm, medium follicles 3-10 mm and large follicles >10 mm, was 1296 follicles, averaging 36.0/doe and 18.0/ovary, regardless the ovarian side.
- The total oocyte recovery rate (86.2%) was the highest for compact oocytes (61.96%), followed by denuded and partial denuded (11.65 and 6.48%, respectively), while the lowest rate was obtained for expanded oocytes (6.10%).
- The highest frequency distribution was obtained for compact oocytes (71.89%), followed by denuded (13.52%) and partial denuded (7.52%), while the lowest distribution was recorded for expanded oocytes (7.07%).
- Relative to total number of vitrified oocytes, post-thaw survival rate of oocytes in normal type was significantly (P<0.05) higher in case of using the combination of EG and DMSO than that with each of EG or DMSO alone (72.56 vs. 56.92 and 61.97%, respectively). Regarding the, survival rate of oocytes in abnormal type was significantly (P<0.05) the lowest (144.66%) with the combination, moderate (21.48%) with DMSO and the highest (28.46%) with EG.
- When post-thaw survival rate was expressed relative to total survival oocytes, post-thaw survival rate of oocytes in normal type was significantly (P<0.05) the highest (83.19%) and those in abnormal type was significantly (P<0.05) the lowest (16.81%) using the DMSO+EG combination. Using DMSO alone resulted in moderate survival rates of oocytes in normal (74.26%) and abnormal (25.74%) types. However, EG as a cryoprotectant significantly (P<0.05) showed the lowest survival rate of normal (66.67%) and the highest one as abnormal (33.33%).
- Post-thaw survival rate of normal oocytes relative to total vitrified oocytes was significantly (P<0.05) the highest (68.33%) with SOPS and the lowest (59.43%), while OPS showed moderate values (63.31%), but did not differ significantly than each of straw and SOPS. On the other hand, survival rate of oocytes in abnormal type was significantly (P<0.05) higher with straw than those with OPS and SOPS (27.87 vs. 19.78 and 17.44%, respectively.
-Total survival oocytes, post-thaw survival rate significantly (P<0.05) decreased for oocytes in normal type and increased for those in abnormal types when straw was used as cryodevice as compared to OPS or SOPS. Inspite of these differences, SOPS as cryodevice showed significantly (P<0.05) the highest survival rate of normal oocytes (79.76%) and the lowest one of abnormal oocytes (20.33%), but did not differ significantly from OPS0
- Using SOPS as a cryodevice significantly (P<0.05) increased post-thaw survival rate of total and normal oocytes and decreased abnormal oocytes relative to total vitrified oocytes. Also, post-thaw survival rate of normal oocytes increased and abnormal oocytes relative to total survival oocytes decreased by using SOPS.
- The highest post-thaw survival rate of total and normal oocytes was obtained with two steps procedure and 90 sec exposure time, while, the lowest rates were obtained with one step procedure and 30 sec exposure time to cryoprotectants.
2. Oocyte abnormality:
- Survival rate of abnormal oocytes relative to total vitrified or survival oocytes had an opposite trend to normal oocytes as affected by the interaction between cryosystem and exposure time to cryoprotectants.
- Relative to total number of vitrified oocytes, percentages of abnormality in types of splitting and lacking were significantly (P<0.05) the highest (3.56and 9.88%) with EG and of crack with DMSO (13.38%), while their lowest values (0.75, 8.27 and 4.14%) were obtained with EG + DMSO combination, respectively. However, percentages of abnormality in change type insignificantly showed the same trend
-Total number of survival oocytes, percentages of abnormality in types of splitting and lacking were significantly (P<0.05) the highest (4.17 and 11.57%) with EG and the lowest (0.86 and 4.74%) with EG + DMSO combination, respectively. Also, percentage of crack was significantly (P<0.05) the lowest (9.48%) with the combination, but was the highest (16.03%) with DMSO.
- Total number of abnormal oocytes, percentages of abnormality in types of splitting and lacking were significantly (P<0.05) the highest (4.17 and 11.57%) with EG and the lowest (0.86 and 4.74%) with EG + DMSO combination, respectively. Also, percentage of crack was significantly (P<0.05) the lowest (9.48%) with the combination, but was the highest (16.03%) with DMSO.
- Relative to total number of abnormal oocytes, abnormality in types of crack showed the highest percentages with all cryoprotectants, followed by lacking and change. While, splitting oocytes showed the lowest percentages with all cryoprotectants. However, abnormality in types of crack was significantly (P<0.05) the highest with DMSO (62.3%) and the lowest with EG (37.5%), while their combination showed moderate percentages (56.4%) and did not differ significantly than each cryoprotectant alone.
- Oocytes abnormality in crack type showed the highest percentage relative to survival oocytes with all cryodevices (S, OPS and SOPS) as compared to the other abnormality types, being significantly (P<0.05) the highest with straw (16.90%) and the lowest (9.54%) with SOPS. However, the differences in the other types of abnormality between different cryodevices were not significant.
- Oocytes abnormality in crack type showed the highest percentage relative to abnormal oocytes with all cryodevices (S, OPS and SOPS) as compared to the other abnormality types. However, there was a tendency of higher abnormality in change and crack types with straw, in split type with OPS and in lacking type with SOPS.
Maturation of vitrified rabbit oocytes:
- No significant differences were found in frequency distribution of oocytes at GV, GVB and M I stages between oocytes vitrified by different cryodevices and thawed at 20oC and those in fresh case. However, frequency distribution of oocytes at M II stage was significantly (P<0.05) the highest (69.2%), while that of degenerated (1.1%, was significantly (P<0.05) the lowest for fresh than oocytes vitrified by S, OPS and SOPS.
- Inspite of the observed lower maturation rate of oocytes vitrified by SOPS than fresh oocytes (69.2 vs. 59.3%), the differences were not significant. However, oocyte vitrification by S or OPS showed significantly (P<0.05) lower maturation rate than fresh oocytes (46.6 and 53.2 vs. 69.2%).
- Frequency distribution of degeneration category of fresh oocytes was significantly (P<0.05) lower than those vitrified by all cryodevice types (1.1 vs. 11.6-15.9%).
In vitro fertilization:
- During insemination process, nearly percentages of oocyte loss were observed for fresh and vitrified oocytes (8.5 and 11.4%, respectively). After fertilization, another loss had occurred in inseminated oocytes, being higher in vitrified than in fresh oocytes (42.3 vs. 11.3%) and in vitro fertilization rate was lower in vitrified oocytes than that of fresh oocytes (85.6 vs. 57.7%), reflecting higher embryos at morula and blastocyst stages for fresh (63.9%) than vitrified ones (28.9%).
Based on the foregoing results concerning vitrification media and cryodevice, the current study may conclude the possibility of rabbit embryo production from vitrified oocytes (by combination of cryoprotectants (EG and DMSO) using SOPS) and matured by TCM-199.