Bovine in vitro produced embryos suffer from poor developmental competence and altered metabolism which hinders their cryotolerance. Overall, the goals of this thesis were to improve oocyte and embryo culture with the antioxidant polydatin and to optimize slow freezing procedures. This thesis was designed as three experiments, and in each experiment, oocytes were aspirated from abattoir ovaries, matured for 23h, fertilized with semen from 1 of 3 bulls, and cultured in synthetic oviductal (SOF) based medium (SCF1) in 38.5 °C in 5% O2, 5% CO2 and 90% N2. Stage 7 blastocysts were stained with Nile Red for lipid content or Mitotracker Red CMX-Rosamine for mitochondrial activity or Cell Rox Green for reactive oxygen species. Ten images per embryo were acquired using confocal microscopy at 5-µM step size and detected fluorescence by IMAGE PRO software. Embryos were also slow frozen in ethylene glycol and analyzed for post-thaw re-expansion after 24 and 48 hours. Experiment one was a 2x2x2 factorial design to test two culture media (SCF1 and a conventional SOF media), the use of a sucrose dehydration prior to slow freezing (2 min in 0 or 0.6 sucrose), and the length of equilibration in ethylene glycol prior to slow freezing (10 or 20 min). We determined that embryos cultured in SCF1 had increased blastocyst rate, mitochondrial activity, and cryotolerance and decreased lipid accumulation (pin vitro.

Cryopreservation of in vivo derived Jersey bovine embryos have resulted in a 10% lower pregnancy rate compared to other dairy breeds. Poor embryo survival after cryopreservation has been partially attributed to the high lipid content of Jersey embryos. In vitro-produced (IVP) bovine embryos have darker cytoplasm than their in vivo-derived counterparts because of higher lipid accumulation. High lipid accumulation is associated with impaired embryo quality. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. L-carnitine is required for transport of fatty acids from the intermembrane space of the mitochondria into the mitochondrial matrix to support the process of β-oxidation, and enhances ATP production. We hypothesized that the lipid content of in vivo-produced and IVP Jersey embryos is higher than respective Holstein embryos and that forskolin + L-carnitine would reduce lipid content of IVP embryos and vitrification with embryo collapse would improve the cryosurvival of Jersey IVP embryos. The objectives of this experiment were (1) to analyze lipid content of in vivo and IVP Jersey and Holstein cattle embryos, (2) to evaluate the effect of forskolin + L-carnitine added to IVP culture media, and (3) evaluate Jersey IVP survival rates after three cryopreservation procedures. The factorial experimental design for objectives one and two used two breeds (Holstein and Jersey) and three embryo production methods (in vivo, IVP, and IVP + forsk/L-C). In vivo produced embryos (n = 27 blastocysts) were collected from superstimulated donors by routine procedures 7.5 days after AI. IVP embryos (n = 259 blastocysts) were produced by standard procedures; briefly, oocytes were aspirated from 2- to 8-mm follicles from slaughterhouse ovaries and matured for 24 h in SMM medium (BoviPro, MOFA Global, Verona, WI, USA). Matured oocytes were fertilized using semen from two different bulls for each breed, and embryos were cultured in BBH7 medium (BoviPro, MOFA Global) alone or with the addition of 1.5mM L-carnitine during maturation and embryo culture with forskolin (10 µM) added at Day 5 of culture at 38.5°C in 5% O2, 5% CO2, and 90% N2. The lipid content of embryos was quantified by staining Day 7 blastocysts with 1 μg mL–1 Nile red dye (580–596 nm), after which a digital photograph of the equatorial part of the embryo was taken at 40×, and fluorescence intensity (FI) was measured with Image Pro software. Data was analyzed by ANOVA, and means were compared using Tukey’s HSD. For the third objective, grade 1 Jersey IVP blastocysts (n=356) were divided into six treatments using a 2x3 factorial design comparing intact (IB) vs collapsed blastocoele (CB) and three cryopreservation methods: slow freezing (SF) vs vitrification using open pulled straws (OPS) or cryotop (CT). Slow freezing embryos were equilibrated in 0.7 M glycerol and 0.1 M galactose in holding media for 10 min, held for 10 min at -6°C, seeded after 5 min, decreased to -32 °C at 0.5 °C /min, held at -32°C for 5 min, and finally plunged into liquid nitrogen. Vitrified embryos were equilibrated in 1.5 M ethylene glycol (EG) for 5 min, exposed to 7 M EG + 0.6 M galactose for 30 s while loaded into OPS or placed onto CT, then immediately plunged into liquid nitrogen. SF embryos were thawed in air for 10 s and placed in a water bath at 37°C for 45 s. Vitrified embryos were warmed directly into holding medium at 37°C supplemented with 1.0 M, 0.5 M and 0.25 M galactose for 3 minutes each. Subsequently, embryos were cultured in BBH7 and re-expansion rates were assessed at 24 and 48h post warming and data was evaluated by GLIMX. For objective 1, Jersey and Holstein IVP embryos had higher lipid content than Holstein in vivo-produced embryos (P < 0.05), but were not different than Jersey in vivo-derived embryos (P > 0.1). Forskolin + L-carnitine lowered the lipid content (P < 0.05) of both IVP Jersey and Holstein embryos and was not different (P > 0.1) than in vivo-produced embryos. For experiment 2, re-expansion rates were higher for CT, than OPS, and SF (85 vs. 66 vs. 72% ± 0.4, respectively; p<0.05). Main effect means for re-expansion were higher for CB than IB (79 vs 68% ± 0.3; p<0.05). In conclusion, IVP embryos have higher lipid accumulation over Holstein in vivo embryos. Addition of forskolin and L-carnitine to embryo culture media has the potential to lower embryo lipid accumulation and possibly improve embryo viability and cryotolerance of IVP embryos. The CT method and collapsing the blastocoele prior to cryopreservation resulted in higher blastocyst survival rate. Further studies including transfer of embryos to recipients are necessary to corroborate these results.

Several cell lineages derived from the mesectoderm occupy and contact axons in the midline of the developing Drosophila CNS. Which of these midline cell lineages contribute to commissural axon morphogenesis? In the absence of the midline cells as in mutant embryos of the single-minded gene, the longitudinal axons collapse at the midline and commissural axons are absent. Despite the similarity in axon tract phenotype, the midline cells in slit mutant embryos survive but are displaced. Correct cytoarchitecture of the midline cells is therefore dependent on the activity of Sli protein which is in turn necessary for commissure formation. In mutant embryos displaying a fused commissure phenotype (rhomboid and Star), the anterior and middle midline glia cells failed to migrate and died by apoptosis after commissure development. In these mutants the number of cells in midline neuronal lineages was reduced before defects in midline glia were apparent. In wildtype embryos approximately 50% of cells in three midline glia lineages died by apoptosis after commissure separation as shown by ultrastructural and enhancer trap analysis. Midline glia lineages died by apoptosis as shown morphologically and by their survival in embryos deficient in the cell death gene reaper. Quantitative analysis revealed variable survival of cells in the anterior, middle and posterior midline glial lineages during embryogenesis suggesting heterogeneity among these cells. The presence of extra anterior, middle and posterior midline glial lineages relative to wildtype numbers in reaper mutant embryos suggested that cell death regulates either midline glial proliferation or cell fate determination during wildtype embryogenesis. Alterations in axon-glia contact correlated with changes in midline glia survival. What happens to apoptotic cells in the Drosophila embryonic central nervous system? A variety of glia in the nervous system were capable of phagocytic activity including midline glia, longitudinal tract glia, nerve root glia and subperineurial glia, revealed by electron microscopy. However, the majority of apoptotic cells in the central nervous system were engulfed by subperineurial glia. In the absence of phagocytic haemocytes in embryos mutant for the Bicaudal-d gene, most apoptotic cells were retained in subperineurial glia at the outer edges of the central nervous system. Apoptotic cells were expelled from the central nervous system of Bicaudal-d mutant embryos suggesting that phagocytic haemocytes participate in the removal of apoptotic cells from the central nervous system but are not essential for this process. / Thesis / Doctor of Philosophy (PhD)

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No description available.

Most embryonal mortality occurs during early embryonic development. Two experiments were designed to study aspects of early embryonic development. Experiment 1 was to determine if early ovine embryos were capable of metabolizing arachidonic acid. Cyclic ewes were estrous synchronized with 6⍺-methyl-17β-hydroxy progesterone acetate (MPA) pessaries, superovulated with follicle stimulating hormone (FSH) and bred artificially. Embryos were collected on d 4, 8, 10, 12 or 14 of pregnancy and incubated with 1 μCi of [¹⁴C] arachidonic acid in an atmosphere of 5% CO₂, 45% O₂ and 50% N at 37°C for 24 h. Embryos from all days of pregnancy metabolized arachidonic acid to a number of compounds. Embryos produced primarily an unidentified polar compound, 6-keto-prostaglandin F₁⍺ (6-keto-PGF₁⍺), prostaglandin F₂⍺ (PGF₂⍺), prostaglandin E₂ (PGE₂), 13,14-dihydro-15-keto prostaglandin F₂⍺ (PGFM), prostaglandin B₂ (PGB₂) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Experiment 2 was to determine whether prostaglandins have a role in embryo hatching from the zona pellucida. Ewes were superovulated and bred artificially, and embryos were collected on d 7 of pregnancy. Embryos were incubated with ethanol (control), indomethacin, PGE₂ or indomethacin and PGE₂ in an atmosphere of 5% CO₂ and 95% air at 37°C for 24 h. Indomethacin appeared to decrease embryo hatching rate (indomethacin, 34.5% vs control, 46.4%). Prostaglandin E₂ appeared to increase embryo hatching rate (PGE₂, 60.0% vs. control, 46.4%). However, hatching rates for indomethacin and PGE₂ treatment groups were not different from control (P > .05). When compared to any group with indomethacin treatment, PGE₂ increased (P < .05) embryo hatching rate. The results of this study indicated that early ovine embryos can convert arachidonic acid to various compounds in vitro. Although not conclusive, indomethacin may decrease and PGE₂ may increase embryo hatching rate. Therefore, embryo-produced prostaglandins may be involved in hatching of sheep embryos from the zona pellucida. / Master of Science

The objective was to study the enzymatic production of triiodothyronine (T₃) in the intestine of chickens during perinatal intestinal maturation in two lines of chickens selected for high (HW) or low (LW) body weight at eight weeks of age. Valid assay conditions (proportionality of enzyme activity with enzyme concentration and assay time) were established and the intestinal 5'-deiodinase (5'D) activity was characterized for comparison with other tissues. The characterization studies showed that intestinal 5'D is like the Type I 5'D in liver of birds and mammals previously studied. Specific activity of adult intestinal 5'D is significantly higher in the HW than in the LW line. In both lines intestinal 5'D increases significantly between embryos that have not pipped into the air cell (NP) and embryos that have pipped into the air cell (AC) and 5'D activity peaks in embryos that have pipped through the shell (TS). In contrast to the line differences in adults, LW embryos have much higher 5'D specific activity than HW embryos until 1d posthatch. Plasma thyroxine (T₄) and T₃ also increased between consecutive stages and peaked in embryos pipped through the shell, then decreased abruptly at 1d posthatch. Both plasma hormones were higher at each perinatal stage in the LW line than in the HW line and the LW line hatched earlier than HW. Intestinal alkaline phosphatase (a marker of differentiation) showed a significant increase in activity at each of the stages of development in both lines. Alkaline phosphatase activity was significantly higher in the LW line than the HW line at the NP, AC and TS stages but not at 1d posthatch. Previous work in other laboratories indicates that T₃ plays a role in triggering intestinal differentiation and maturation of intestinal function for posthatching life. The results of this study indicate that T₃ for this signal originates at least partially from 5'deiodination of T₄ within the intestinal tissues as well as from T₃ available in the plasma. / Master of Science

In adult vertebrates, cardiovascular regulation is accomplished by numerous systems with neural, hormonal and local components responsible for the majority of regulation. These regulatory components work in concert to maintain the essential function of blood perfusion to adult tissues. Given the essential nature of this function it is therefore surprising that the development of cardiovascular regulation during gestation is poorly understood. The majority of what is known is based on a single vertebrate model, the fetal lamb. The fetal lamb has been used in multiple studies due to the clear clinical applications and has been pivotal in understanding the onset of regulation in developing vertebrates. However, study on the fetal lamb is limited to the latter 40% of gestation and has the added complication of an in-utero developmental strategy. Therefore the primary focus of this dissertation was to characterize basic cardiovascular regulation in the chicken embryo to provided the needed information for it's use an alternative to the fetal lamb. Developing chicken embryos rely on both alpha and beta adrenergic tones to maintain normal heart rate and arterial blood pressure during incubation. However, on day 21, just prior to hatch, these animals lose both tones on arterial pressure suggesting the onset of adult regulation. Cholinergic tone, however, was absent throughout chicken development indicating that it must mature during the neonatal life. Adult cardiovascular reflexes become apparent late in chicken development with a clear baroreflex specifically operating initially on day. However, an adult response to changes in ambient gas tension was absent during incubation suggesting embryos possess unique regulatory systems that are absent in adult chickens. This mechanism is comprised entirely of adrenergic systems with no cholinergic action during change in ambient gas tension. Similar developmental patterns were determined in embryos of the desert tortoise suggesting fundamental differences between in-utero and ex-utero developing vertebrates.

A delicate balance of steroid and gonadotrophic hormones is essential for intrafollicular oocyte maturation and successful fertilization and embryonic development. Previous studies have demonstrated that a superovulatory dose of pregnant mare serum gonadotrophin (PMSG) has excessive gonadotrophic activity and alters intrafol1icular steroid hormone levels. In a series of four experiments, the morphology of oocytes and embryos retrieved from immature rats, treated with either a low or high dose of PMSG, and mature, cycling rats was compared to determine whether a superovulatory dose of PMSG has an adverse effect on oocyte maturation and subsequent fertilization and embryonic development in immature rats. Morphological criteria for the assessment of intraoviductal oocyte aging were established in the first experiment. During intraoviductal aging, progressive morphological changes directed by the intrinsic developmental program of the oocyte were observed. Further alterations in morphology were attributed to abnormalities of cytoskeletal function. In the second experiment, no difference in morphology was observed between oocytes retrieved from immature rats treated with either 4 or 40 IU PMSG. When compared with mature rats, changes attributable to cytoskeletal instability were observed in aged oocytes from immature rats treated with both doses of PMSG. This was concluded to be a manifestation of altered intrafollicular oocyte maturation as a result of the administration of exogenous gonadotrophin. In the third and fourth experiments, delayed fertilization and a significant reduction in fertilization rate were observed in superovulated, immature rats. The major cause of fertilization failure was determined to be intraoviductal oocyte aging. A significant increase in abnormal embryos was observed as a result of parthenogenetic activation of the aged oocytes. Abnormal, fertilized embryos retrieved from the superovulated group were concluded to be the manifestation of delayed fertilization. In conclusion, the major effect of a superovulatory dose of PMSG on oocyte fertilizability and embryonic development was intraoviductal oocyte aging and delayed fertilization. Changes attributed to altered intrafol1icular maturation were manifested during oocyte aging in immature rats treated with either the low or high dose of PMSG. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

To examine if a rhythm can be entrained in either heart rate or oxygen consumption in late stage embryos (days 17-19.5) with light as a zeitgeber, chicken embryos were incubated in complete darkness (D:D) and 12:12 light:dark cycle (L:D). Light had no impact on oxygen consumption (390 µL O2∙min-1∙egg-1) but increased heart rate for non-internally pipped embryos (260 to 270 beats∙min-1 during light cycle). Oxygen consumption increased independent of pipping while heart rate increased (255 to 265 beats∙min-1) in D:D embryos due to pipping. A light-induced rhythm or effect occurred in heart rate but not oxygen consumption, suggesting heart rate and oxygen consumption may be uncoupled.