published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Surgery / Master / Master of Surgery

The overall aim of this thesis was to determine if day old turkey gonads could be cryopreserved and transplanted into recipient poults. This would allow for grafts to develop and mature normally and potentially produce donor-derived offspring. In addition, the monitoring of folliculogenesis in chickens was studied to determine if ultrasonography would be a useful technique to study this biological process, with the intention of using this method in future studies on ovarian graft development. Three studies were conducted: cell and tissue viability of vitrified day old turkey gonads, transplantation of day old turkey gonads into recipient poults, and monitoring of follicle growth in chickens using ultrasonography. The objective of the first study was to determine if day old turkey gonads were viable after vitrification using a standard protocol or with variation in equilibrium solution (ES) and/or vitrification solution (VS) absorption times. Three different ES time points were tested 10, 15 and 20 min (10ES, 15ES and 20ES) and two different VS time points 2 and 3 min (2VS and 3 VS). The cell and tissue viability was determined by Trypan Blue Assay and light microscopy, respectively. Testicular cell viability was conducted using three vitrification protocols and fresh tissue. All vitrification protocols along with fresh tissue were assessed by light microscopy, to evaluate histological alterations, in ovarian and testicular tissue. Protocols with the highest cell viability and best morphological scores were selected as being the most suitable for cryopreservation. Testicular tissue vitrified using 15ES or 20ES with 3VS had the highest cell viability. Ovarian and testicular tissue vitrified using 15ES with (3VS or 2VS), showed the best morphological scores, out of all the protocols. The second study was broken down into two parts: Part A (Trial 1 & 2) was to determine the most suitable age group for poults pre-surgery to give the highest survivability; Part B (Trial 3) was to determine if previously vitrified day old turkey gonads could develop and mature normally, by retrieving grafts post-surgery, at- different time points. In Trial 1, large white turkeys (LWT) 1, 3, 4 and 7 days of age were used and for Trial 2, LWT’s aged 1, 3, 4, and 5 days of age were used. In Trial 3, bronze turkeys at 1 day of age were used, and graft tissue was used from day old LWT’s previously vitrified (10ES/2VS) or fresh. For all Trials, the survivability at each time point was analyzed, and for the third Trial, the grafts recovered were histologically analysed. From Trials 1 and 2, seven and three day old poults had the highest survivability ratios (3/5 and 6/8) respectively. For Trial 3, day old male poults (96%) had a higher survivability than the females (68%). From Trial 3, transplants were only recovered in females and males up until 4 days and 4 weeks post-surgery respectively, with no fresh tissue grafts recovered. The histological analysis of testicular transplants showed deteriorating structure, with a steady progression away from normal morphology, post-surgery. The third study’s objective was to determine the growth rates and patterns of folliculogenesis in Barred Plymouth Rock (BPR) hens by using ultrasonography. Two ultrasound Trials were performed: the first to determine the optimum time interval between serial ultrasound scans to accurately map follicles, and the second to tackle the main objective of the third part of this thesis. For the first Trial, BPR hens were scanned periodically over 24 hours, follicle diameter and position were recorded and mapped with respect to the ovary and neighbouring follicles. Proportional follicle growth, compared to the first scanning session showed that the 24hr time point had the only significant (P<0.001) proportional follicle growth. It is recommended here that scans occur twice a day (morning and afternoon) to capture a more precise growth rate of follicles. In the second Trial, BPR hens were scanned twice a day, over an 11-day period. Follicle diameters (height and width) were recorded to calculate follicle area. The growth of each follicle’s area was compared to the time before ovulation to determine the overall follicle growth rate. Additionally, it was determined if time (night, day) and type of preovulatory follicle (F1-F5) played a significant role in follicle growth rates. The overall follicle growth rate was best described by a cubic equation (R2=0.907, P<0.001). Follicle growth rates were influenced by both time (P=0.009) and type (P<0.001). With F2 and F3 follicles (P<0.05) having a higher growth rate than F1 and F5 types. In conclusion, modifications to the standard vitrification protocol used on quail gonads were necessary to increase cell viability and lower morphological alterations for turkey gonads left whole. For future work it has been shown that day old turkey poults can survive gonad transplantation. The lack of development of grafts is most likely due to a combination of tissue damage after vitrification-warming procedures and insufficient immunosuppression of the host. This work has paved the way for the poultry industry to be able to cryogenically store turkey gonads and revive lines when required. Additionally it was shown that serial ultrasound scans twice a day provided accurate monitoring of follicle growth in Barred Plymouth Rock hens. For BPR hen’s follicle growth rates and patterns were successfully measured. This gives the industry another tool to better select superior laying hens and to create a more homogeneous laying flock. Future application of ultrasonography on gonad monitoring has the potential to show growth and maturation of grafted tissue before the production of donor-derived offspring, enabling earlier detection of successful transplantation.

No description available.

No description available.

- / -

No description available.

The objective of this study was to investigate whether or not the treatment of nuclear donor cells with inhibitors of protein synthesis and cyclin-dependent kinases (CDKs) affect the development of swine embryos produced by somatic cell nuclear transfer. Host oocytes were derived from pre-pubertal females and matured in vitro for 42-46 h under standard conditions. Nuclear donor cells were fetal fibroblasts maintained in culture for 2 to 6 passages. Oocytes were reconstructed with cells treated for 22-24 hours with cycloheximide (CHX; 10mug/ml), roscovitine (ROS; 25 muM), the combination of CHX + ROS (CR), or untreated cells. Two hours after reconstruction, the oocytes were activated using ionomycin (15muM/5 min) and strontium chloride (10mM/4h), maintained for 6 h in the presence of cytochalasin B (7.5mug/ml) and CHX (10mug/ml), and then cultured in porcine zygote medium (PZM3) for 6 days. The cleavage rate, 63.7% (n=318), 55.2% (n=99), 56.7% (n=107) and 60.6% (n=347), at 48 h post-fusion were not significantly different between embryos derived from ROS, CHX, CR and control cells, respectively. Developmental rate to blastocyst stage was higher for embryos reconstructed with ROS (12.2%) and untreated cells (12.1%) when compared to CHX (5.7%) and CR (4.9%). Blastocysts produced with ROS treated cells had similar number of nuclei compared to embryos reconstructed with untreated donor cells (30.9+/-10.4 vs. 32.2+/-8.0). Phosphorylated H2A.X (gammaH2A.X) was highly expressed in donor cells treated with CR compared to non treated cells, but it was similarly expressed in most of 1-cell stage embryos reconstructed with control or treated cells. Flow cytometry analysis showed that the majority of the fibroblasts were at G 0/G1 phase of the cell cycle at the time of nuclear transfer. It was concluded that the treatment of nuclear donor cells with inhibitors of protein synthesis and CDKs did not improve the in vitro development of somatic cell nuclear transfer embryos in pigs.

No description available.