The consequence of a folate deficiency during organogenesis has been investigated in the rat embryo. In vitro culture of 9.5 day embryos in serum from dietary induced folate deficient rats frequently resulted in abnormal embryos. Many were growth retarded and exhibited a defect in the turning mechanism which inverts the embryo from ventrally to dorsally convex. Affected embryos displayed abnormal twisting or kinking of the neural tube. Gross anaemia was also frequently observed and the protein content of the embryos was markedly less than that of embryos grown in normal rat serum. Supplementation of the deficient serum with folic acid improved growth and greatly reduced the occurrence of deformities. It virtually eliminated the incidence of gross anaemia but only partially restored the protein content to the level observed in embryos cultured in normal rat serum. The effects of the folate deficiency could not be reversed by supplementation with multivitamins or by increasing the volume of culture serum. They were, however, eliminated by supplementing the deficient culture serum with normal rat serum. The effects could also be overcome by in vivo folate supplementation; rats which had previously been so folate deficient that culture in their serum would have resulted in malformed embryos, after restoration to a folate supplemented diet produced serum which supported completely normal embryonic growth. The results indicate that embryos undergoing organogenesis require adequate folate in order for normal growth and differentiation to take place. They also suggest that some of the embryopathic effects of maternal folate deficiency are mediated by secondary effects. These may involve complex growth or metabolic factors which can be corrected in vivo but are not readily reversed in vitro.

Experiments were initiated to determine whether exogenous estradiol-17beta (E2) and oxytocin (OT) can be used to dilate the cervix and improve transcervical embryo transfer (ET) procedures for sheep. However, there was concern that the E2-OT treatment may alter luteal function and that embryo quality would decrease as the superovulatory response to FSH increased. In Exp. 1, 32 ewes were assigned to a 2 x 2 factorial array of treatments. On d 7, ewes received an i.v. injection of either 100 micrograms of E2 in 5 mL of 1:1 ethanol:saline or 5 mL of 1:1 ethanol:saline; 12 h later, ewes received i.v. injection of either 400 USP units of OT or saline. Jugular blood was collected on d 7, 8, 9, 10, 12, 14, 16, and 18. Progesterone concentrations were unaffected by the treatments. Experiment 2 was conducted to determine the dose of pFSH needed to induce approximately six corpora lutea (CL). Ten-day Norgestomet implants inserted between d 8-12 of the estrous cycle were used to synchronize estrus in Hampshire and Hampshire x Dorset ewes (n = 23). Ewes received a total of either 0, 18, 27, or 36 mg of pFSH, which was injected i.m. at -24, -12, 0, 12, 24, and 36 h relative to implant removal. The dose at each respective time was 19.4, 19.4, 16.7, 16.7, 13.9, and 13.9% of the total. Ewes received 400 IU of PMSG i.m. at -24 h. The CL were counted laparoscopically on d 6 (d 0 = estrus). Number of CL increased linearly (P < .01) with dose of pFSH; there were 1.8, 3.6, 6.3, and 11.2 CL/ewe, respectively. Experiment 3 was conducted to determine the effect of the E2-OT treatment, mode of transfer or the interaction of E2-OT treatment x mode of transfer on embryo survival and development. Experiment 3 was conducted over two breeding seasons and across two trials. In the first trial ewes were assigned to one of three randomized treatments. Procedural limitations that were later overcome prevented a true 2 x 2 factorial design; therefore, transcervical transfer without hormonal treatment was excluded in the first trial. In the second trial, ewes were assigned to a 2 x 2 factorial array of treatments. On d 6 of pregnancy, embryos rating a fair or better were transferred into recipients either transcervically or laparoscopically. Recipients were administered either an E2 (d 6) - OT (d 7) treatment or an ethanol:saline-saline treatment following the same protocol as in Exp. 1. Embryos were recovered on d 12 in Trial 1 and d 14 in Trial 2. Embryos were evaluated morphologically for development and ranked on a scale of one to four; one represented no development and four represented development to the morphological stages associated with the day of collection. The treatments did not affect the percentage of embryos recovered after transfer or the percentage of embryos that showed some developed. However, there was an effect of mode of transfer on mean rank of embryo development; embryos transferred laporscopically developed further than embryos transferred transcervically (P < .01). This may have been an artifact of a technician effect between trials. There was an effect of E2-OT treatment on transcervical transfer (P < .01), indicating that it may be detrimental to transfer embryos transcervically without dilating the cervix. In conclusion, the E2-OT treatment did not affect luteal function, and the E2-OT treatment can be used to dilate the cervix and enhance success of transcervical transfer of embryos. A 400 IU priming dose of PMSG and a total dose of 27 mg of pFSH can be used to induce the target number of six CL. / Master of Science

<p> Chick embryos at stages primitive streak to three somites were explanted on the vitelline membrane and cultured by the method of New (1955) or by Gallera's modification of the method (Nicolet and Gallera. 1963). Antisera produced in rabbits against adult chicken brain extract. adult chicken kidney extract. and embryo brain extract were placed on the uppermost side of the embryo preparation. The embryos were recovered after 24 to 36 hours further incubation. Defects of the central nervous system. the posterior trunk. and the extra-embryonic membranes occurred in embryos exposed to adult brain antiserum. Embryos exposed to adult kidney antiserum developed exactly the same kinds of defects. Embryo brain antiserum produced similar abnormalities which included defects of the central nervous system and of the extra-embryonic membranes, and in addition de-. fective somites. However, embryos exposed to gamma globulin solutions containing antibodies against neural-specific antigens and not against common tissue antigens were normal. In control experiments, embryos exposed to saline solution, to normal rabbit serum, and to normal rabbit serum gamma globulins developed normally. </p> <p> Histological examination of ~epresentative antisera treated embryos revealed that there were extensive areas of disorganization and necrosis of neural tissue. In embryos with short trunks, the caudal proliferation centre was necrotic. Embryos exposed to gamma globulins of absorbed adult brain antiserum were histologically normal. </p> <p> The antisera used in these experiments were characterized by double diffusion in agar gel. It was demonstrated by this method that the antisera contained antibodies against common tissue antigens as well as against tissue-specific antigens. It was also shown that certain antigens common to all adult organs were present in the embryo and in the extra-embryonic membranes during the time that the embryos were exposed to the antisera. </p> <p> Embryos which had been exposed to various of the tissue antisera for 8.5, 21.5 or 32 hours were sectioned. The sections were stained with FITC-labelled goat anti-rabbit gamma globulins in order to localize distribution of the antibodies. Fluorescence was located on the ectoderm of the embryos and of their extra-embryonic membranes, in the lumen of the neural tube and in the cavity of the otic vesicles. This demonstrated that, under the conditions of the experiment, the antibodies were available to the embryo. </p> <p> Antisera which affected the embryo and the extra-embryonic membranes were shown to contain antibodies against antigens actually present in the embryos and in the extra-embryonic membranes during the time of exposure. The only antiserum which had no adverse effects on the embryos was the one which contained only antibodies against antigens not demonstrated to be present in the embryo during the time of exposure. </p> / Thesis / Master of Science (MSc)

The mineral nutrient storage reserves in various parts of white spruce (Picea glauca) seeds and somatic embryos were investigated. Somatic embryos are produced in tissue culture from single cells or small groups of cells. These cells are induced to produce a callus, which is then stimulated to produce mature somatic embryos that are desiccated down to moisture content levels comparable to those of a mature dry seed. Morphological comparisons revealed that somatic and zygotic embryos are very similar. Somatic embryos of white spruce; however, were larger, had a flared arrangement of cotyledons, had more prominent suspensor regions, had intercellular spaces in the ground meristem tissue and had ground meristem cells that had divided in several planes of division. Using a wet ashing protocol, anion exchange resins and the molybdenum blue colourimetric reaction, the total levels of P and the amount of P bound to phytic acid were measured. Phytic acid (or phytate) is the major nutrient storage compound in seeds. Although differences were found on a dry weight basis, a single somatic and a single zygotic embryo had similar levels of P. Somatic embryos produced in different batches varied in their levels of phytic acid, but a somatic embryo could have similar levels of phytic acid as a zygotic embryo. The large female gametophyte contained 86% of the total P and 95% of the total phytic acid in a single seed. Electron microscopy and energy dispersive x-ray analysis found phytate-rich globoids in the procambium and ground meristem tissues of both types of embryos. Globoids contained high P, moderate K and Mg, and little if any Ca, Fe and Zn. Globoids were generally larger and more frequent in zygotic embryos. Globoids from the cotyledon procambium of zygotic embryos also contained moderate levels of Fe. Iron-rich particles (possible Fe-phytate deposits) were found in the protoderm, procambium and ground meristem of both types of embryos. These deposits contained high P and Fe, moderate K and Mg, and little if any Ca and Zn. Globoid and Fe-rich particle compositions were similar in both embryo types, but significantly higher Fe:P ratios were found in zygotic embryos. Atomic absorption spectroscopy was used to measure total K, Mg, Ca, Fe and Zn levels. Although differences were found on a dry weight basis, a single white spruce somatic or zygotic embryo contained similar levels of all these elements. The female gametophyte contained high levels of these elements and the seed coat contained considerable Ca. Potassium leakage during imbibition in germination medium revealed that a single somatic and zygotic embryo of white spruce leaked similar levels of K. Pretreatment of somatic embryos in a high relative humidity environment resulted in decreased potassium leakage by 80 and 120 min. of imbibition. The seed coat was found to reduce the amounts of K leaked. Surface cells in dry somatic and zygotic embryos were found to be wrinkled, but cells in zygotic embryos were more shrunken in appearance. During imbibition, cells became more turgid; but after 120 min. of imbibition, surface cells still showed signs of wrinkling. To date, this is the first major report of mineral nutrition in white spruce seeds and is the first comprehensive comparison of mineral nutrients in white spruce seeds and somatic embryos. These results may be useful in producing complete artificial white spruce seeds. / Thesis / Master of Science (MS)

Previous research in bovine embryology has found that embryos cultured individually have limited ability to develop compared to their counterparts cultured in a group of other embryos. This investigation aimed to find if any of three different interventions over two experiments would increase development of individually cultured embryos to that of group cultured embryos. In the first experiment both the addition of serum/serum replacer and a co-culture with bovine granulosa cells were applied to individually cultured embryos in a 3x2 design. None of the interventions was found to be significantly different from the others, and all resulted in significantly lower development than embryos cultured as a group (avg. 4.7 +/- 1.93% individual vs. 21.7 +/- 3.76% group). However, a significant difference was found in the hatching rate between blastocysts cultured in media including cells (71.4 +/- 17.07%) and those cultured without cells (18.1 +/- 11.63%). In the second experiment, embryos were either cultured in standard droplets or microwells made at the bottom of culture droplets either in groups or individually for a 2x2 design. This experiment experienced poor development in all treatments including the group control, and none of the treatments were found to be significantly different from each other. However, the hatching rate of blastocysts cultured in multiple microwells was significantly higher than those cultured individually in droplets. To summarize, none of the treatments increased the development rate, but embryos cultured with granulosa cell co-cultures and in group microwells showed improvements in hatching rates. / Master of Science

This study investigated the development potential of equine embryos in vitro after biopsy and vitrification. Twenty embryos were obtained from Quarter Horse, Thoroughbred, and mix-breed light mares between three and ten years old. The twenty embryos were divided into a biopsy (n=10) and control group (n=10). The biopsy group underwent microaspiration biopsy using a micromanipulator to obtain a small tissue sample from the embryo. Both groups were then vitrified using a commercially available technique originally described by Carnevale (2006) at Colorado State \ University. All 20 embryos were cultured in DMEM/Hams F-12 medium under oil at 37°C in 5% CO2 in air (Hinrichs et al., 1990). Embryos were monitored for expansion and hatching. Embryo development was statistically different between the two groups (p<0.05). The biopsy procedure did result in a much lower development potential in the biopsy group as compared to the control group (20% vs. 80%). However, embryos in the biopsy group did show expansion and hatching therefore the combined procedure did not preclude development potential in vitro. Based on these findings, more research needs to be done to increase the success of the combined procedure and the ultimate viability of the embryos needs to be confirmed with the establishment of successful pregnancies.

No description available.

Zugl.: Düsseldorf, Universiẗat, Diss., 2008.

Reclamation of Canada’s threatened wood bison (Bison bison athabascae) herd is complicated by cattle diseases. As part of an overall goal to conserve bison genetics, five studies were conducted to develop or adapting present reproductive technologies to produce disease-free in vivo-derived wood bison embryos. In Chapter 4, the efficacy of pLH and hCG for inducing ovulation and whether the effect was related to the size of the dominant follicle at the time of treatment was examined in wood bison during the anovulatory season . Ovulation rate with hCG (94%) was nearly two times greater than with pLH (54%), and bison with a growing follicle of ≥10 mm had a greater ovulatory response than those of 8-9 mm. In Chapter 5, the efficacy of pLH and hCG after superstimulation with single or two doses (48 hours apart) of FSH diluted in 0.5% hyaluronan was determined in wood bison. A greater superovulatory ovarian response was found in cows treated with hCG vs. LH during the anovulatory and ovulatory seasons (6.6 vs. 2.8 and 6.3 vs. 3.8 corpora lutea respectively). In addition, dividing the dose of FSH two resulted in greater superovulatory response in wood bison. However, the number of corpora lutea was still lower than expected as compared to cattle using the same two dose method of superovulation (15 corpora lutea; Tribulo et al., 2012). Therefore, in Chapter 6, the effect of the addition of a low dose of eCG at the end of the superstimulation protocol on ovarian response and embryo quality was examined. Although the number of ova/embryos recovered was higher in this study when compared with previous reports in wood bison, no effect of eCG on the number of corpora lutea and embryo quality was found. In Chapters 5 and 6, the effect of exogenous progesterone on embryo quality in wood bison during the anovulatory season was evaluated. We found that progesterone did not improve the number of freezable embryos in either study. In Chapter 7, the effect of lengthening of FSH treatment protocol on superovulatory response and embryo quality during the ovulatory and anovulatory seasons was examined. There was no effect of lengthening the FSH treatment protocol on ovarian response and embryo quality during the anovulatory season. However, embryo quality and ovulation rate were increased by the lengthened treatment protocol during the ovulatory season. Additionally, more freezable embryos (Grades 1 and 2) were obtained during the ovulatory season (1.8 embryos) vs. the anovulatory season (0.3 embryos). Overall, results confirm that superovulation can be performed in wood bison throughout the year, but a higher number of freezable embryos were obtained during the ovulatory season. The final chapter (Chapter 8) focused in the production of disease-free embryos in wood bison. Following superovulation, in vivo-derived wood bison embryos were exposed in vitro to Brucella abortus biovar 1. After incubation, embryos were submitted to the 10-step washing procedures recommended by the IETS to remove the pathogen. When the washing medium contained antibiotics, 100% Brucella-free embryos were obtained. These findings validate the washing procedures for the production of Brucella-free embryos in wood bison.

No description available.