High levels of dietary protein may be detrimental to reproductive performance in cattle. The objective of Exp. 1 was to determine the effects of differences in dietary protein on the production and quality of bovine embryos collected from superovulated donors. Angus cows were randomly assigned to receive one of three experimental diets: a daily ration of 5.7 kg poultry litter, 2.0 kg hay, 3.1 kg corn, and 0.5 kg peanut hulls (LITTER; n = 15); a daily ration of 6.2 kg peanut hulls, 2.2 kg soybean meal, 2.0 kg hay, 0.5 kg corn, and 0.4 kg dicalcium phosphate (SBM; n = 15); or a daily ration of 6.2 kg peanut hulls, 2.0 kg hay, and 3.1 kg corn (CON; n = 19). Diets differed in the amount of total, soluble and degradable protein, but were comparable in energy. After 30 d on the diets, all cows were treated to induce superovulation (28.8 mg FSH/cow, Folltropin) and synchronize estrus. After the detection of estrus each cow was inseminated with semen from one of four Holstein bulls. Embryos were collected 7 d after estrus and evaluated for quality (according to the International Embryo Transfer Society (IETS) standards) and stage of development. Prior to treatment to induce superovulation, blood samples were collected 6 h after feeding. Samples were analyzed to assess dietary effects on plasma urea nitrogen (PUN). Mean levels of PUN were higher (P < 0.01) in cows fed the LITTER or SBM diet (16.3 mg/dL, LITTER; 21.8 mg/dL, SBM; 9.7 mg/dL, CON) than in cows fed the CON diet. Additionally, concentration of PUN was higher in cows fed SBM than in those fed LITTER (P < 0.01). An average of 9.2 transferable embryos (Grade 1, 2 and 3) was collected from each cow and there were no significant differences in the number of transferable embryos collected among groups (9.2, LITTER; 9.3, SBM; 9.1, CON). The number of degenerate embryos or unfertilized ova did not differ among dietary groups. High-protein diets elevated PUN, but did not affect the number or quality of embryos collected from superovulated donors. Cryopreservation of bovine embryos is an important aspect of a successful embryo transfer program. The objective of Exp. 2 was to evaluate the post-thaw viability of bovine embryos collected in Exp. 1 in an in vitro culture system after the embryos had been held at room temperature or refrigerated for 2 to 12 h prior to freezing. Upon embryo recovery, each embryo was randomly assigned to be placed in holding media for 2, 6 or 12 h prior to freezing. During this interval, one-half of the embryos were maintained in a refrigerated environment (5 °C), while the remaining half of the embryos were held at room temperature (20.5 to 22 °C) until freezing. Immediately prior to freezing, embryos were removed from the holding media, transferred to a well containing ethylene glycol (10%) in ovum culture media and loaded individually into a 0.25-mL plastic straw. Straws were then placed in a freezer unit (-6 °C) and seeded to induce ice crystal formation through all columns of the straw. The temperature of the freezer was then decreased 0.6 °C/min to -32 °C, and straws were loaded into canes and plunged into a liquid nitrogen tank (-196 °C). After storage, each straw was exposed to a 5-s air thaw and placed in a water bath at 35 °C for 20 s. Each embryo was then washed to remove excess ethylene glycol prior to in vitro culture. Embryos were individually cultured in Ham's F-10 media supplemented with 4% fetal bovine serum for 72 h. Embryos were evaluated at 24 h intervals throughout the culture period and assigned a stage of development and quality grade score (according to IETS standards). The percentage of embryos that developed to the expanded blastocyst stage and hatched from the zona pellucida was greater for embryos held 2 or 6 h prior to freezing (P < 0.05) than for embryos held for 12 h after collection before being frozen (62.9, 52.0 and 31.1%, respectively). The percentage of embryos that degenerated during in vitro culture was lower for embryos held 2 or 6 h prior to freezing (20.4 and 26.6%; P < 0.05) than for embryos held for 12 h before freezing (50.8%). Furthermore, embryo quality grade was more desirable for embryos held for 2 or 6 h (1.5 and 1.7; P < 0.05) than for those held for 12 h before freezing (2.1). The semen used to inseminate donors and the diet fed to donors for 4 wk prior to embryo collection did not influence the proportion of embryos that hatched or degenerated during the 72 h of in vitro culture. Additionally, holding embryos in a refrigerated environment from the time of collection until freezing did not enhance embryonic development during post-thaw culture. Thus, embryonic viability may be impaired when embryos are held longer than 6 h following embryo recovery before being frozen; however, the storage temperature during the interval from collection to freezing does not influence embryonic development post-thaw. / Master of Science

Landolphia kirkii is scrambling shrub forming an integral part of the flora along the coastal areas of north-eastern South Africa. The non-sustainable harvesting of fruit as food source, by monkeys and rural communities and the highly recalcitrant nature of their seeds threatens the continuation of the species. In addition, the ability of the plants to produce high quality rubber makes its long-term conservation highly desirable. Previously, attempts have been made to cryopreserve germplasm of L. kirkii, but no survival had been recorded at cryogenic temperatures of below -140ºC. The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates, thawing and imbibition treatments on survival of embryonic axes excised with cotyledons completely removed, as well as with 3 mm portion of each cotyledon attached, from fresh, mature, recalcitrant seeds of L. kirkii. Survival was assessed by the ability for both root and shoot development in in vitro culture, the tetrazolium test and electrolyte leakage readings. At seed shedding, embryonic axes were at the high mean water content of 2.24 g gˉ¹ (dry mass basis). All axes (with and without attached cotyledonary segments) withstood rapid (flash) drying to a water content of c. 0.28 g gˉ¹; however, the use of chemical cryoprotectants, singly or in combination, before flash-drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g gˉ¹, with no explants showing shoot production after exposure to -196ºC and -210ºC. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology regardless of the thawing temperature employed. Furthermore, analysis of the lipid composition within embryos of L. kirkii revealed negligible amounts of capric and lauric acids, suggested to be the medium-chained saturated fatty acids responsible for triacylglycerol crystallisation when lipid-rich seeds are subjected to cryogenic temperatures. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent thawing and rehydration. Rapid rehydration of embryonic axes of L. kirkii by direct immersion in a calcium-magnesium solution at 25ºC for 30 min (as apposed to slow rehydration on moistened filter paper or with rehydration in water) was associated with highest survival post-dehydration. Cooling at 1ºC minˉ¹ and 2ºC minˉ¹ facilitated survival of 70 and 75% respectively of axes with attached cotyledonary segments at 0.28 g gˉ¹ after exposure to - 70ºC. Viability retention of 40 and 45% were recorded when embryonic axes with attached cotyledonary segments were cooled at 14 and 15ºC minˉ¹ to temperatures below -180ºC. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

The effects of two types of cryopreservation, conventional freezing and vitrification, on the in vitro and in vivo function of a pancreatic substitute were investigated. Conventional freezing uses low concentrations of cryoprotective agents (CPAs), slow cooling and rapid warming and allows ice formation. Vitrification requires high concentrations of CPAs coupled with rapid cooling and warming to achieve a vitreous, or ice-free, state. A previously published mathematical model describing the mass transfer of CPAs through the alginate matrix of the substitute and the cell membrane was expanded to incorporate heat transfer as well as CPA cytotoxicity. Our results indicate that temperature of exposure is the most critical parameter for the proper design of CPA addition and removal protocols. The use of a mathematical model is critical to ensure CPA equilibration and minimize CPA exposure. Properly designed CPA addition and removal protocols were used for vitrification. The effects of cryopreservation on the biomaterial and the cellular function of a pancreatic substitute consisting of murine insulinomas encapsulated in calcium alginate/poly-L-lysine/alginate beads were assessed. In vitro results indicate that both vitrification and conventionally frozen perform comparably to fresh. However, in vivo studies reveal that vitrified beads perform worse than both conventionally frozen and fresh beads. With adjustments, it may be possible to improve the performance of the vitrified beads. Nevertheless, for this pancreatic substitute, conventional freezing is the better method and allows successful cryopreservation.

Key to the success of natural and engineered tissues becoming clinically available until needed is their long-term storage at low temperatures. This can be implemented by means of freezing or vitrification. To this end, vitrification offers an attractive approach for tissue banking by forming an amorphous glass both intra- and extracellularly and thereby avoiding the harmful effects of ice formation. Generally, high concentrations of cryoprotectants (CPAs) are used in conjunction with high cooling and warming rates to achieve this. However, hurdles associated with applying this technique include the ability to adequately deliver and remove CPAs due to cellular osmotic and cytotoxic effects as well as achieving adequate cooling and warming rates throughout the tissue to avoid ice formation. The aim of this work was to account for these factors in designing cryopreservation protocols for native and engineered tissues that had intrinsically different characteristics, including tissue size and extracellular matrix properties. The tissues investigated were two types of three-dimensional, cell encapsulated systems consisting of murine insulinomas and murine embryonic stem cells, and native articular cartilage. A mathematical 3-D CPA transport model was developed to predict cell volume excursions and intracellular CPA equilibration and applied to cryopreserve an engineered tissue. This thesis established a systematic methodology to design cryopreservation protocols using experimental measurements and a mathematical model for tissues.

Long-term storage of tissue by cryopreservation is necessary for the efficient mass production of tissue engineered products, and for reducing the urgency and cost of organ transplantation procedures. The goal of this work was to investigate the physical processes thought result in damage during tissue cryopreservation towards development of tissue cryopreservation strategies. Although mathematical models of cell dehydration and intracellular ice formation (IIF) have been successfully used to optimize cryopreservation procedures for cell suspensions, it is not currently possible to use this approach with tissue because of the lack of tissue-specific permeability parameters for predicting cell dehydration during tissue freezing, and because of the increased complexity of the IIF process in tissue. We have measured the membrane permeability properties of tissue comprising a cell monolayer using a fluorescence quenching technique, and compared the results to the corresponding cell suspensions, revealing significant differences in the membrane transport kinetics between monolayers and suspensions. These data enabled the prediction cell dehydration during freezing of cell monolayers. Whereas the mechanisms of IIF are relatively well understood in cell suspensions, tissue is susceptible to new IIF mechanisms. In particular, cell-cell interactions have been shown to increase the IIF probability by enabling the propagation of ice between neighboring cells. We investigated the effect of cell-cell interactions on IIF using genetically modified cells expressing different levels of intercellular junction proteins. A new IIF mechanism was observed in these cells associated with penetration of extracellular ice into the cell-cell interface, and the incidence of this IIF mechanism was reduced in cells expressing the tight junction protein occludin. In addition, we investigated the effect of the cytoplasm supercooling and viscosity on the kinetics of IIF in tissue. We found that increasing the viscosity or decreasing the supercooling significantly decreased the kinetics of IIF, suggesting that IIF protocols for tissue can be optimized by modulating the cytoplasm supercooling and viscosity. Together, these data represent an important step towards developing cryopreservation strategies for tissue.

Thesis (M.Sc. (Plant Science))--Mahidol University, 2006. / LICL has E-Thesis 0018 ; please contact computer services.

Thesis (PhD)--University of Mastericht, the Netherlands, 2007. / Includes bibliographical references.

D.Sc. (Zoology) / The spermatogenesis of two freshwater species from South Africa, Barbus marequensis and B. polylepis, and three fresh water species from Israel, B. canis, B. longiceps and Capoeta damascina, was studied. A histological comparison of the process of spermatogenesis was undertaken. The breeding cycle of B. marequensis, B. polylepis, B. canis, B. longiceps and C. damascina was similar to the breeding cycle of B. aeneus and four phases occured within the cycle. The four distinctive phases are post spawning phase, rest phase, pre-spawning phase and the spawning phase (Vlok, 1986) . During the post spawning phase a decline in sperm development is observed and possible lisosomal activity is responsible for the resorption of sperm cells not shed during the spawning phase. The presence of collagen structure provides a distinctive character to the tissue of the testis. The resting phase is characterised by the absence of the lobular structure and the testis is dominated by the collagen tissue. The testis is small and unobtrusive in the abdominal cavities of both species. At the onset of the pre-spawning phase, the testis is filled with spermatogonia. The lobular structure becomes more prominent and the interstitial tissue can be distinguished. Later during the phase, the synchronised development of sperm cells in the cysts of the lobules can be observed, whilst sperm cells in adjacent lobules are in different stages of development. During the spawning phase the testis of all species studied contain mature sperm . cells in the lumens of the lobules.

Cryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.

In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.