The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5. / Thesis (Ph.D.)-University of Natal, Durban, 2002.

The zygotic germplasm of plant species producing desiccation-sensitive seeds can be conserved in the long-term only by cryopreservation. Usually the embryonic axis is excised from the cotyledons and is used as the explant for cryopreservation as it is small and provides a large surface area:volume ratio. However the shoot of the axis of most species studied does not develop after excision, with the result that survival after cryopreservation is often recorded as callus production or simply explant enlargement and/or greening. Thus, besides explant size, factors such as in vitro regeneration techniques, physical injury induced upon excision and developmental status of the seed could compromise the success of cryopreservation. This study investigated the effect of the factors mentioned above, with particular attention to the developmental status of the seeds on explant in vitro development (section 3.1), response to dehydration (section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (section 3.3) of two species: Trichilia dregeana, T. emetica and embryos of a third, Strychnos gerrardii. For all three species, investigations were conducted on the embryonic axes/embryos excised from mature seeds immediately after fruit harvesting and from mature seeds stored under hydrated conditions for different periods (in order to achieve different degrees of development). In addition, preliminary studies were carried out on axes of T. dregeana to assess whether generation of reactive oxygen species (ROS) occurs in response to wounding upon axis excision (section 3.4). Excised embryonic axes of T. dregeana and T. emetica did not develop shoots in vitro unless the explants included attached cotyledonary segments. Following the development associated with short-term storage, however, the excised axes could develop shoots after complete cotyledon excision. The embryos from the (endospermous) seeds of S. gerrardii which included the paper-thin cotyledons, developed normally in vitro, with percentage germination increasing with seed storage time. For all three species, in vitro axis germination was promoted when activated charcoal was included in the germination medium, regardless of the developmental stage of the seeds. When dehydrated to approximately 0.3 g H2O g-1 dry mass (g g-1), embryonic axes from all three species failed to develop shoots even though a minimum of 50% produced roots in all cases. Hence, shoot production was shown to be more sensitive to desiccation than was root production. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not ameliorated with seed storage for T. dregeana and T. emetica, but did decrease for S. gerrardii when seeds were stored for 6 – 8 weeks. The application of certain cryoprotectants did facilitate production of shoots after dehydration by a few axes of both Trichilia spp. For T. dregeana explants, combination of glycerol and sucrose allowed for 10% of the axes to retain the ability for shoot production after dehydration while for T. emetica explants, the combination of DMSO and glycerol (10 - 20% shoot production after dehydration) was best. The efficacy of the cryoprotectants was not influenced by storage period. The provision of cryoprotectants still needs to be tested for S. gerrardii. Survival of subsequent cryopreservation of T. dregeana and S. gerrardii explants was best achieved with rapid cooling in nitrogen slush, with the cooling procedure for T. emetica explants still to be optimized. The highest post-cryopreservation survival of T. dregeana axes was achieved when seeds had been stored for three months, while the seed storage period did not affect post-thaw survival of the axes of T. emetica or S. gerrardii. A small proportion of S. gerrardii explants only, produced shoots after cryopreservation, whereas the surviving embryonic axes of T. dregeana and T. emetica regenerated only as non-embryogenic callus. Although callus production is less desirable than successful seedling establishment, it has the potential for micropropagation if embryogenicity can be induced. Ultrastructural examination of the shoot apical meristem of T. dregeana after a 3-d recovery period, following excision, revealed considerable cellular derangement, although damage of individual organelles could not be resolved microscopically. Preliminary studies on T. dregeana involving a colorimetric assay using epinephrine, confirmed the generation of ROS in response to wounding associated with axis excision. Reactive oxygen species generated appeared to persist over prolonged periods rather than occurring only as a single oxidative burst. Hence, ROS production at the wound site could be the primary factor contributing to lack of shoot development. Axes immersed in the anti-oxidant, ascorbic acid (AsA) immediately after excision, showed lower ROS production and 10% shoot development when cultured in vitro, indicating that the oxidative burst coincident with, and after excision might be counteracted if immediate ROS production can be adequately quenched. Future investigations should aim to identify the specific ROS associated with wounding and optimize an anti-oxidant treatment(s) that will facilitate shoot development. Thus, the successful cryopreservation of the germplasm of the species tested, and others producing recalcitrant seeds, depends on a spectrum of species-specific factors, some still to be elucidated. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2007.

Recalcitrant seeds are those that are shed at high water contents, are actively metabolic throughout development, when they are, and remain, desiccation-sensitive, and may also be chilling sensitive. These properties preclude their conventional storage. Because recalcitrant seeds lose viability rapidly (within a few days to several months depending on the species) the long-term storage of their germplasm is achievable only by cryopreservation [i.e. storage at very low temperatures, generally in or over, liquid nitrogen at -196°C or -150°C, respectively. Generally the seeds are far too large to be cryostored, thus explants - most conveniently, excised zygotic embryonic axes - are used. As the axes of recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing have to be applied in order to avoid lethal ice crystal formation. During the course of this study, cryopreservation protocols were developed for excised zygotic embryonic axes of two different species (Quercus robur L. and Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes of both species, with the use of a 1% sodium hypochlorite solution containing a wetting agent, emerging as the best. For both species, the vigour and viability of axes, assessed by in vitro germination performance, was tested after the implementation of four different rates of desiccation (achieved by a laminar-airflow; silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate (fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur and E. capensis axes after 240 and 20 min, when water contents were reduced to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively. Consequently, fast flash-drying was used in combination with three different freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was lost after slow or intermediate cooling, good survival was obtained for each species after ultra-rapid cooling. In addition to the optimisation of culture conditions, desiccation and freezing rates, the efficacy of different thawing media (distilled water, mannitol, sucrose, full-strength MS medium supplemented with sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The only thawing medium that ensured normal seedling production was the Ca2+Mg2+ solution, in which electrolyte leakage was significantly curtailed. In addition to vigour and viability assessment the responses of the embryos to the various manipulations were monitored by light microscopy and/or transmission electron microscopy. The results of the various manipulations are discussed in terms of the stresses imposed on the excised axes, by each of the procedures. For axes of Q. robu, the outcome of the presently developed successful procedure and two unsuccessful protocols from the published literature are compared and contrasted. It is concluded that while in vitro germination media need to be assessed on a species basis, use of the mildest effective surface-sterilant, in conjunction with the most rapid means to achieve dehydration and cooling/freezing, are likely to underlie generally successful cryopreservation. Additionally, thawing parameters have emerged as being critically important. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

Mitochondria are essential for promoting cell survival and growth through aerobic metabolism and energy production. Mitochondrial function is typically analyzed using mitochondria freshly isolated from tissues and cells because they yield tightly coupled mitochondria, whereas those from frozen tissue can consist of broken mitochondria and membrane fragments. A method, utilizing a well-characterized cryoprotectant such as dimethyl sulfoxide (DMSO), is described. Such mitochondria show preserved structure and function that presents us with a possible strategy to considerably expand the time-frame and the range of biochemical, molecular and metabolic studies that can be performed without the constraints of mitochondrial longevity ex vivo. Mitochondrial dysfunction is implicated in Alzheimer’s disease (AD) mainly through oxidative stress and altered metabolism. Mitochondria are isolated from post-mortem brain samples from selective regions of AD and control patients and, utilizing the cryopreservation strategy, analyzed for respiration and oxidative damage. While we did not observe increases in free radicals, we did observe decreased respiration and increases in oxidative damage markers in AD patients, suggesting a role for oxidative stress in mitochondrial dysfunction. While in the mitochondria, calcium (Ca2+) increases free radical generation by processes not completely understood. A new isoform of nitric oxide synthase (mtNOS) has been isolated and localized to mitochondria; though its existence and physiological role is debated. Nitric oxide synthase (NOS), when activated by Ca2+, produces nitric oxide (NO•) that can interact with ROS producing various reactive nitrogen species (RNS). These highly reactive radical species can damage DNA, proteins and lipids, ultimately resulting in cell death via apoptosis or necrosis. The current research is aimed at understanding the role of Ca2+ and NOS in oxidative stress leading to mitochondrial dysfunction. We observed a significant reduction in mitochondrial respiration with increasing doses of calcium. We also observed NOS enzyme activity and detected NOS protein in the purified mitochondrial fraction. Lastly, we were also able to show that Ca2+ increased the levels of free radicals and changes in oxidative damage markers. These results suggest the presence of NOS in mitochondria that could play a role in Ca2+ induced mitochondrial dysfunction and potentially leading to cell death as relevant to aging and neurodegenerative diseases.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

The cryopreservation of cells and tissues requires the addition of a cryoprotectant in order to prevent cellular damage caused by ice. Unfortunately, common cryoprotectants such as DMSO and glycerol exhibit significant toxicity which makes their use unfeasible for many clinical procedures. Our laboratory is interested in the development of alternative, non-toxic cryoprotectants which possess ice recrystallization inhibition (IRI) activity. Potent IRI activity has recently been discovered in certain small molecules, but the structural features required for this process are unclear. Herein we report the development of a library of O-aryl glycosides in order to probe the importance of electron density and hydrophobic moieties for IRI activity. It was found that the degree of electron density at the anomeric oxygen does not correlate with IRI ability in para-substituted aryl glycosides, nor does changing the position of the aryl substituent impart a predictable effect on activity. However, the addition of hydrophobic alkyl or acyl chains was beneficial for IRI activity; generally, increasing chain length was found to correlate with increasing activity. In some instances, an optimal alkyl chain length was identified, after which continued lengthening results in a loss of potency. We conclude from this study that a certain extent of hydrophobic character is beneficial for the IRI activity of aryl glycosides, and that a balance between hydrophobicity and hydrophilicity is required for optimum IRI ability. It is hoped that these findings will aid future efforts towards the rational design of novel cryoprotectants.

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

L'ús d'esperma criopreservada en la inseminació artificial (IA) d'espècies d'interès productiu permet un major control sanitari i la creació de bancs de germoplasma d'alt valor genètic, entre d'altres avantatges. En el mercat porcí la major part de les inseminacions són encara realitzades amb semen refrigerat degut a l'èxit de l'aplicació de diluents de llarga durada i també a causa de la sensibilitat de l'esperma porcina a la criopreservació. Malgrat que aquesta sensibilitat ve donada per característiques particulars de la fisiologia espermàtica en l'espècie, algunes ejaculacions mantenen els paràmetres de qualitat espermàtica després de la criopreservació (ejaculacions amb bona "congelabilitat", GFEs) enfront d'altres que no sobreviuen al procés (ejaculacions amb mala "congelabilitat", PFEs). El primer objectiu de l'estudi va ser comparar ambdós grups en termes de fertilitat in vivo. El segon objectiu va ser testar l'eficiència de la inseminació postcervical (post-CAI) amb l'esperma criopreservada. El tercer objectiu va ser buscar predictors de la congelabilitat de les ejaculacions, tant en les GFEs com en les PFEs i en tres passos del procés de criopreservació (a 17ºC, a 5ºC i a 240 min postdescongelació). Aquest objectiu es va dur a terme mitjançant l'avaluació de paràmetres convencionals de qualitat espermàtica i a través de l'estudi de la localització i la reactivitat sota el microscopi de tres proteïnes (GLUT3, HSP90AA1 i Cu/ZnSOD) relacionades amb la fisiologia espermàtica i amb possibles rols en la congelabilitat. El quart objectiu va ser quantificar l'expressió de les tres proteïnes per transferència western, tant en espermatozoides d'ejaculacions GFEs com en els d'ejaculacions PFEs i en els tres passos abans esmentats, per tal de determinar el seu potencial com a predictores de la congelabilitat. Pel primer i el segon objectiu, 86 truges van ser inseminades per post-CAI amb 26 ejaculacions de mascles Piétrain dividides en una porció refrigerada a 17ºC (tractament control) i una porció criopreservada, ambdues porcions classificades alhora com a GFEs o PFEs. Els resultats més rellevants van demostrar que les probabilitats d'embaràs eren dues vegades menors en inseminacions amb esperma criopreservada d'ejaculacions PFEs (P < 0.05) que en inseminacions amb esperma criopreservada d'ejaculacions GFEs, fet que indica que les ejaculacions amb percentatges elevats d'espermatozoides mòbils progressius i d'integritat de membrana (per sobre del 40% en les GFEs) són més favorables a provocar embarassos que no pas aquelles ejaculacions amb una pobra funció espermàtica in vitro (PFEs). Ni el nombre de truges que van donar a llum, ni la quantitat de garrins, ni el risc de reflux espermàtic van ser significativament diferents entre les inseminacions amb esperma criopreservada d'ejaculacions GFEs i les inseminacions control amb semen refrigerat, la qual cosa demostra la bona aplicabilitat de la inseminació post-CAI amb l'esperma criopreservada. Finalment, pel tercer i quart objectius van ser criopreservades 29 i 11 ejaculacions de mascles Piétrain, respectivament. Dos paràmetres cinètics espermàtics, la linealitat (LIN) i la rectitud (STR), van mostrar una hiperactivació de la mobilitat superior en les ejaculacions PFEs que en les GFEs després de 30 min a 5ºC durant la criopreservació. A més, la combinació d'ambdós paràmetres va donar una fiabilitat propera al 72% en la predicció de la congelabilitat de les ejaculacions porcines. Tot i que no va ser possible predir la congelabilitat mitjançant l'avaluació de les tres proteïnes al microscopi, els resultats de transferència western van revelar diferències en l'expressió de la HSP90AA1 en l'esperma a 17ºC, molt possiblement relacionades amb la millor supervivència a la criopreservació dels espermatozoides d'ejaculacions GFEs. Aquests resultats suggereixen que la promoció de la criopreservació d'esperma porcina per la seva aplicació en IA passa pel desenvolupament de tests per la predicció de la congelabilitat en semen refrigerat. / The use of frozen-thawed (FT) sperm in artificial insemination (AI) of species with productive interest allows higher sanitary control and the creation of high genetic value sperm banks, among other advantages. In the swine market, most inseminations are still performed with refrigerated semen (FS) because of the successful application of long-term extenders and the sensitivity of boar sperm to cryopreservation. Although this sensitivity is provided by particular features of the sperm physiology in boars, some boar ejaculates maintain the sperm quality parameters after freezing (good freezability ejaculates, GFEs) in front of others that do not survive the process (poor freezability ejaculates, PFEs). The first objective of the study was to compare both groups in terms of field fertility. The second objective was to test the efficiency of the post-cervical AI (post-CAI) with FT sperm. The third objective was to search for predictors of the ejaculate freezability by assessing, both in GFEs and in PFEs and in three steps during the cryopreservation procedure (17ºC, 5ºC and 240 min post-thaw), conventional sperm quality parameters and the location and reactivity, under the microscope, of three proteins related to the sperm physiology with potential roles in freezability (GLUT3, HSP90AA1 and Cu/ZnSOD). The fourth objective was to quantify, through western blotting, the expression of the three proteins, both in GFEs and in PFEs and in the three steps mentioned, to determine their potential as freezability predictors. For the first and second objectives, 86 sows were inseminated by post-CAI with 26 ejaculates from Piétrain boars divided into a 17ºC FS portion (control treatment) and a cryopreserved (FT) sperm portion, the two portions in turn classified into GFEs and PFEs. The most relevant outcomes were that the probabilities of pregnancy resulted two times lower after inseminations with FT-PFEs (P < 0.05) compared to FT-GFEs, which indicates that ejaculates with high post-thaw sperm progressive motility and membrane integrity (over 40% in GFEs) are more likely to result in pregnancies than those with poor in vitro sperm function (PFEs). Neither the number of farrowing sows and the litter size nor the risk of sperm backflow was significantly different in FT-GFEs from that achieved in FS control treatments, which showed the good applicability of post-CAI to boar FT sperm. For the third and fourth objectives, 29 and 11 Piétrain boar ejaculates, respectively, were cryopreserved. Two kinematic indices, the sperm linearity (LIN) and the sperm straightness (STR), revealed higher hyperactivated motility in PFEs than in GFEs when assessed after 30 min at 5ºC during cryopreservation, the combination of the two motility parameters showing around 72% confidence in the freezability prediction of ejaculates. Whereas it was not possible to predict the freezability of the boar ejaculates by assessing the three proteins under microscope, results from western blot showed differences in the HSP90AA1 expression in sperm at 17ºC that are most possibly related to the best cryosurvival of GFEs. This finding aims to promote the cryopreservation of boar sperm intended for use in AI through the development of tests for freezability prediction in FS.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.