Orientador: Frederico Ozanam Papa / Banca: Fernanda da Cruz Landim Alvarenga / Banca: Julio Cesar Ferraz Jacob / Resumo: A colheita de espermatozóides da cauda do epidídimo e sua criopreservação representam um grande avanço biotecnológico na equinocultura, e pode ser a última chance de preservação do material genético de garanhões após sua morte inesperada ou lesões que impossibilitem a cobertura ou colheita de sêmen. Os objetivos dessa pesquisa foram comparar a efetividade de dois diluidores utilizados para a recuperação dos espermatozóides epididimários em relação aos índices de congelabilidade determinados laboratorialmente (Experimento I) e as taxas de concepção proporcionadas por cada metodologia ao utilizar baixa dose inseminante (Experimento II). No Experimento I foram utilizados 58 testículos, o ducto epididimário de um testículo de cada garanhão foi lavado com o diluidor sem pentoxifilina-Botu-Sêmen®1 (Controle) e o ducto epididimário do testículo contralateral, com o diluidor contendo 7,18 mM de pentoxifilina-Botu-Turbo®2 (PTX). No Experimento II, foi utilizado um "pool de espermatozóides" dos testículos de dois garanhões. As amostras foram congeladas e direcionadas às inseminações artificiais em diferentes doses inseminantes, formando os seguintes grupos: 800x106 espermatozóides recuperados com o diluidor sem pentoxifilina (800 Controle), 100x106 espermatozóides recuperados com diluidor sem pentoxifilina (100 Controle) e 100x106 espermatozóides recuperados com o diluidor com pentoxifilina (100 PTX). O índice de concepção para as éguas inseminadas nos grupos 800 Controle, 100 Controle e 100 PTX foi de 68% (11/16), 31,25% (5/16) e 50% (8/16), respectivamente. Frente aos resultados obtidos, conclui-se inseminações artificiais com apenas 100x106 espermatozóides colhidos com o meio diluidor contendo pentoxifilina garantem índices aceitáveis de concepção e maximiza o uso de espermatozóides epididimários congelados / Abstract: The recovery of spermatozoa from epididymal cauda and its cryopreservation represents a great technological advance, since it is the last possibility to preserve genetic material from dead or deceased valuable males. The aims of this study were to compare the effectiveness of two extenders used for recovery of epididymal sperm in relation to its freezability (Experiment I) and conception rates provided by each methodology using a low insemination dose (Experiment II). In Experiment I, 58 testis were used, epididymal cauda of each stallion were separated and flushed with a skim milk-extender either without (Botu-Sêmen®; Control Group) or with 7,18 mM of pentoxifylline (Botu-Turbo®; PTX Group) and then frozen. In Experiment II, a "pool" of epididymal sperm from 2 stallions was used. The samples were frozen and artificial insemination was performed using different insemination doses, comprising the following groups: 800x106 sperm recovered without PTX (800 Control); 100x106 sperm recovered without PTX (100 Control) and 100x106 sperm recovered with PTX (100 PTX). The conception rates were 68% (11/16), 31.25% (5/16) e 50% (8/16) for 800 Control, 100 Control and 100 PTX group, respectively. Based in these results, it was possible to conclude that artificial inseminations with 100x106 sperm recovered with PTX guarantee acceptable conception rates and maximize the use of frozen equine epididymal sperm / Mestre

Philosophiae Doctor - PhD / In the marine environment, penguins have been described as curators and serve a critical role in ecological balance. The African penguin (Spheniscus demersus) has undergone a rapid population decline, mainly due to disturbances in their natural habitat. The African penguin was up-listed from vulnerable to endangered on the IUCN Red List for Threatened Species in 2010 and thus urgent conservation action is required. Integral to long-term conservation action of any species is a basic knowledge of its reproductive biology, which is currently lacking for African penguins. The main aim of this investigation was to evaluate techniques for the collection of semen in African penguin and to determine sperm quality in order to cryopreserve sperm for in vitro fertilization (IVF) purposes of captive and wild populations. Semen was collected once a week during two breeding seasons from two captive African penguins. Ejaculates (n=51) were obtained over two breeding seasons (Jan-Feb and Jun-Oct) and evaluated for semen volume, sperm concentration, sperm vitality, sperm motility and sperm morphology. In addition twelve (six females and six males, n=4 were breeding pairs) captive African penguins were monitored for hormone (estradiol, testosterone, progesterone) levels prior to and after the egg-laying period.

The South West Botanical Province of Western Australia is one of the most floristically rich areas of the world with over, 8,000 species present, the majority of which (70%), are endemic to this region. Coupled with this high level of endemism, many taxa are threatened which makes them vulnerable to habitat alterations, modifications and destruction. Significant habitat alteration in many areas has resulted in 27 species becoming extinct in the South West Botanical Province, while an additional 327 species are classified as rare and endangered. In the context of stemming this loss of biodiversity, research in cryopreservation was undertaken to provide offsite protection and conservation of somatic germplasm.Cryostorage techniques were evaluated in this study to determine the key factors which may affect the ability of somatic tissues of Haemodoraceae species to survive, recover and grow following liquid nitrogen (LN) immersion and storage. Using Anigozanthos viridis as a comparator in most experiments, the base vitrification protocol was established, which involved: (1) preculturing shoot apices on 0.4 M sorbitol for 48 h; (2) incubation in a vitrification cryoprotective solution (PVS2) for 25 min at 0 degrees celsius; (3) LN immersion; (4) recovery to active growth through warming (immersion in a 40 degrees celsius water bath). Using this procedure the highest post-LN survival of shoot apices for A. viridis was 41.4 plus or minus 6.1% Four additional taxa were successfully cryopreserved with this base protocol (Anigozanthos manglesii, A. rufus, Conostylis wonganensis and A. rufus x A. pulcherrimus); a fifth taxon, Macropidia fuliginosa, however, proved unresponsive.To improve on post-LN survival, further research established that four of the six study taxa responded to the following amendments to the basic protocol: (1) longer preculture duration; (2) preculture on ++ / 0.8 M glycerol rather than sorbitol, (3) utilisation of PVS2 solutions with reduced DMSO content; and (4) incorporation of an additional loading phase (2 M glycerol plus 0.4 M sucrose for 20 mins at 0 degrees celsius).Macropidia fuliginosa, a species with poor recovery after LN exposure, was successfully cryostored using somatic embryos. Treatments which resulted in the highest survival (67.3% 5.7 plus or minus %) included preculture with 0.4 M sorbitol, and incubation in PVS2 for 30 min. Further experimentation indicated that preculture for two days on 0.8 M glycerol (replacing 0.4 M sorbitol) was more beneficial for achieving high post-LN survival.Post-LN survival was significantly correlated to the use of polyalcohols when the total number of hydroxyl (-OH) groups (regardless of molarity) present was the same as that found in 0.4 M sorbitol. It was hypothesised that hydroxyl number is more important than molarity in membrane stabilisation, during dehydration and cooling. Post-LN survival was also found to be significantly influenced by stereochemical arrangement of the -OH groups of polyalcohol molecules used in the preculture media. Finally, post-LN survival was also found to be significantly influenced by the size of the molecule, with smaller polyalcohols with more -OH groups on one flank of the carbon chain being superior as cryoprotective agents.The influence of plant growth regulators on post-LN survival and recovery growth was also investigated. The survival of shoot apices was not correlated to cytokinin or auxin treatments administered in culture media prior to cryostorage. However, in the recovery medium, a combination of cytokinin and 0.5 mu M GA(subscript)3 in the medium was found to be the most efficacious for obtaining healthy plantlets.Genetic fidelity was then examined using Amplified Fragment Length Polymorphism (AFLP). Plantlets of one done kept ++ / or maintained under the following conditions: (1) standard tissue culture conditions; (2) cold storage and (3) cryostorage, over a 12 month duration, showed no detectable genetic changes.Further, shoot apex viability evaluated at regular intervals (after 0, 3, 6 and 12 months of LN storage) suggested that medium term storage of samples cryopreservation did not reduce shoot apex viability over this time span.This study has provided a better understanding of the factors influencing post-LN survival and recovery and, as a result, the cryopreservation protocols have been refined. Consequently, the prospects for conserving threatened Haemodoraceae species from Western Australia through cryostorage is now significantly improved.

PhD / The aim of this thesis was to investigate the possibility of integrating frozen-thawed boar semen into reproductive technologies and into commercial production of pigs in Australia. This was to be achieved by establishing a semen freezing and AI regime that was of a standard acceptable to industry, and integrating the resultant frozen-thawed sperm into other reproductive technologies, such as flow cytometric sperm sorting and IVF. Initially, a protocol for freezing and thawing boar semen was established, based on the method described by Westendorf et al. (1975) and attempts were made to modify this protocol to improve the post-thaw sperm quality, as determined by in vitro assessment of motility, acrosome integrity and longevity. First, the egg yolk used in the freezing extenders was investigated, and the chicken yolk was replaced with either duck or quail yolk. It was shown that there was no benefit in substituting yolk from duck or quail for the chicken yolk traditionally used in freezing extender. Second, the effect of seminal plasma addition to the freezing extender, or seminal plasma addition to resuspension medium post-thaw was tested. Incorporating whole seminal plasma into the freezing extender at levels above 50% was found to be detrimental to post-thaw sperm quality. Reducing levels to 20% of the final volume improved acrosome integrity, but adversely affected motility of sperm. However, adding 20% seminal plasma to the resuspension medium used after thawing of boar semen had no significant influence on sperm quality compared with resuspension in medium without seminal plasma. The antioxidant catalase, and the iron chelator desferal added to the freezing extender, did not improve post-thaw sperm quality, nor was any benefit seen with addition of these substrates to the resuspension medium post-thaw. However, the bioactive phospholipid PAF and its regulating enzyme PAF:AH appeared to enhance post-thaw motility and acrosome integrity of sperm, respectively, when added to the semen pre-freezing. Unfortunately, due to the restrictions imposed on rPAF:AH as a research drug, it was not possible to test the in vivo effects at this time. After the in vitro experiments were completed, the in vivo fertility of frozen-thawed sperm was tested using the optimal freezing protocol and a novel technology, enabling non-surgical deep intrauterine insemination of sows. The aim was to establish the lowest possible dose of frozen-thawed sperm that could be used, without compromising fertility. Successful pregnancies were achieved with doses as low as 62.5 x 106 frozen-thawed sperm but the farrowing rates were too low to be practicable on a commercial scale. This is the first report of litters born after insemination of such a low dose of frozen-thawed sperm and using the novel DIU insemination technique. However, it was concluded that a double dose of 250 x 106 frozen-thawed sperm was the minimum dose required for maintaining acceptable fertility. Reduction in sperm numbers to such an extent made it possible to consider non-surgical insemination of sex-sorted, frozen-thawed semen. Previously, pregnancies had been achieved only after surgical insemination of sex-sorted boar sperm, or with DIU insemination of unfrozen sperm, immediately after sex-sorting. The low numbers of sex-sorted sperm available restricted the inseminate dose used here to 50 x106 motile sperm. A litter of 5 piglets was born after a low-dose, DIU insemination of sex-sorted, frozen-thawed sperm. This is the first report of piglets born after insemination with sex-sorted frozen-thawed sperm and non-surgical insemination. The low farrowing rate achieved in this experiment prompted the investigation of integrating sex-sorted, frozen-thawed boar sperm into IVF. Morulae were produced after IVF with sex-sorted, frozen-thawed sperm and successfully transferred using non-surgical techniques. This is the first report of pregnancy achieved with non-surgical transfer of embryos produced after IVF and IVC of IVM oocytes with sex-sorted, frozen-thawed boar sperm. Unfortunately, the pregnancy did not hold, and the embryos were lost prior to Day 32, but PCR of non-transferred embryos confirmed successful pre-selection of sex. Overall, this thesis demonstrated that it is still not economically feasible to incorporate frozen-thawed boar semen into the commercial production of pigs although it has considerable application in breeding programmes. However, the development of novel techniques enabling reduction in sperm dose, and for non-surgical transfer of embryos into recipient sows and incorporation of frozen-thawed semen into these technologies means that progress is being made with the integration of reproductive technologies and frozen-thawed semen into the pig industry.

Preservation of cells and tissues by freezing at temperatures below 70°C has led to new possibilities for the storage of germ cells for fertility preservation. During the freezing process problems might occur, the greatest being ice crystallization which can cause membrane destruction and thus cell death. To minimize this risk, solutions that reduce the freezing point can be added to reduce crystallization and increase survival rates. These solutions are called cryoprotectants. The best method for freezing is still not known.The aim of this study was to analyze the effect of various parameters on the survival rate of human semen frozen with liquid nitrogen. The parameters investigated were thawing method (incubator or water bath) and container choice (straw or ampoule). In addition, two different cryoprotectants were tested.The method used was the instruction for preservation with Sperm CryoProtec™ II from Nidacon. In total 16 samples were collected for the first test and 13 samples for the second test. Sperm concentration and motility was measured.There seem to be no significant differences depending on container choice or thawing method leading to the conclusion that the most cost effective method of storage and thawing may be used. A small but significant difference was found in survival after thawing dependent on cryoprotectant p=0.041. However the study sample was limited and further studies might be of value.

Donor cells for nuclear transfer are usually prepared by the culture of fresh tissue. However, animal carcasses are sometimes frozen without cryoprotectants and if it were possible to obtain live cells from carcasses (tissue) preserved in this manner, it could be very beneficial in nuclear transfer cloning of trophy or extinct animals. This study tested the hypothesis that tissue samples of skin, muscle, and oral mucosa could be cryopreserved without cryoprotection. The tissue samples were taken from euthanized goats and placed into a -20°C freezer for varying lengths of time. The samples were thawed by two different methods. One method was in 37°C water bath and the other was on ice, thawing to room temperature from 1°C to 25°C. The samples were then processed and placed into an incubator to evaluate cell growth. Skin samples frozen for up to 34 days obtained cell growth to confluency and the cells were then cryopreserved with cryoprotectant. The cells were able to tolerate the potentially lethal effects of ice nucleation and dehydration brought about by ice formation and colligative factors. Although this method of cryopreservation has been shown to yield growth of cells that might be useful for nuclear transfer cloning, it is not the recommended method to cryopreserve tissues if cryoprotectants are available or if only short term storage is needed. These procedures would be especially useful when a precious animal dies unexpectedly and cryoprotectant is not available and the sample can not be processed before 10 days.

The influences of loading cholesterol into stallion spermatozoa membranes prior to cold storage or cryopreservation were determined using cholesterol loaded cyclodextrin (CLC) before preservation, followed by the unloading of cholesterol after preservation using methyl beta cyclodextrin (MBCD). Experiment I: dose response trials determining optimal amounts of CLC and MBCD based on percentages of progressively motile spermatozoa (PMS) following preservation. Experiment II: influences of CLC and MBCD on PMS, the percentages of live intact (LI) and live non-intact (LNI) spermatozoa following cold storage. Experiment III: influences of CLC before cryopreservation and MBCD on PMS, LI, and LNI post-thaw. Addition of CLC improved (P<0.05) PMS and LI following preservation when compared to the control. Unloading cholesterol using MBCD does not alter PMS, LI nor LNI. Addition of CLC is beneficial to survival of spermatozoa following preservation and addition of MBCD in small amounts does not negatively influence PMS, LI or LNI.

The developmental potential of mammalian oocytes cryopreserved with procedures similar to those used for embryos has been limited, inasmuch as oocytes differ from embryos in advanced stages of development, both physiologically and morphologically. The objective of this work was to elucidate the precise nature of freeze-thaw damage with the expectation that identification of specific targets will enable devising optimal procedures for cryopreservation of bovine oocytes to prevent specific damage and minimize the loss of developmental capacity. / In the first series of experiments, bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed morphologically and also by in vitro fertilization (IVF) and culture (IVC). Morphological integrity and developmental capacity were greater in S-oocytes than in V-oocytes (P $<$ 0.05). Transfer of four embryos (2 morulae and 2 blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in the birth of twin calves. / In the second series of experiments, oocytes (GV and IVM) were exposed to a cryoprotectant solution (DAP213: 2M DMSO, 1M acetamide, 3M propanediol) for 1.5 or 5 min and viability assessed by IVM-IVF-IVC. Oocytes were also examined by transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound premature cortical granule (CG) release and vesiculation. These changes were less pronounced in oocytes exposed to DAP213 only after IVM. The results suggest that: (1) the extrusion of CG is one of the important cytological events affected by the treatment of oocytes with DAP213; (2) GV oocytes are more sensitive to the cryoprotectant than IVM oocytes. / In the third series of experiments, GV and IVM oocytes were vitrified with DAP213. On rewarming, DAP213 was removed by a one- or three-step dilution procedure and survival assessed by development after IVM-IVF-IVC. Morphology was assessed by TEM study immediately following DAP213 removal. Both assessments indicated that: (1) IVM oocytes are more tolerant to vitrification than are GV oocytes; (2) the three-step dilution is less damaging than the one-step procedure; (3) changes in the zona pellucida (loss of plasticity) of IVM oocytes following vitrification may result from the premature release of cortical granules.

Originally described from the Asian honey bee, Apis cerana, the microsporidian Nosema ceranae is an obligate, intracellular parasite that has recently been discovered infecting the western honey bee, Apis mellifera. More research on the biology of N. ceranae as well as on the impact it may have on A. mellifera is greatly needed. However, conducting studies on N. ceranae is not only dependent on seasonal availability of Nosema spores, but also on reliable methods for determining spore viability. This study presents the results of using cryogenics to provide long term storage of viable N. ceranae spores and a differential staining procedure that details how to use bright field microscopy with the fluorescent viability dye, propidium iodide (PI), and the fluorescent stain, 4', 6-diamidino-2-phenylindole (DAPI) to differentiate viable and nonviable spores. Using these methods, it was found that freezing N. ceranae at -70 °C in 10% glycerol yielded the lowest mean rate of spore mortality after thawing (24.2% ± 2.2).

No description available.