The purpose of this study was to evaluate the effects of three different diluents used during the cryopreservation process on the motility and vitality of sperm cells. The three diluters used in this study were: A) trice-serum-egg yolk-glycerin, B) serum-egg yolk-glycerin, and C) Dulbecco’s serum-egg-yolk- glycerin. Diluters were tested in proportions of 64-15-15-6% (N1), 54-20-20-6% (N2), and 44-35-15-6% (N3). Llama semen was collected at the Mejoramiento Genético y Diagnóstico Clínico Del Servicio Agropecuario (SEDAG) in the Los Andes Province of the Department of La Paz. The procedure took place at the Unidad Académica Campesina de Tiahuanaco by a direct optimized bulbourethral collection method with an artificial vagina.

For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.

Includes bibliographical references (leaves 232-257). / xv, 257 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to determine if the compatible solutes (proline, glycine betaine, and trehalose), the epididymal compounds (taurine, hypotaurine and inositol) or the antioxidants (carnosine and ascorbic acid) in tris-citrate based diluents could improve the post-thaw survival and/or fertility of ram spermatazoa. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1996?

Thesis (MScMedSc (Obstetrics and Gynaecology))--Stellenbosch University, 2008. / BACKGROUND: The cryopreservation and transplantation of ovarian tissue have been shown to restore ovarian function temporarily and may also preserve the fertility of young female cancer patients until after their sterilizing cancer treatment. Since tissue samples are large and morphologically complex, the cryopreservation methodology is difficult to optimize and standardise. Ovarian tissue cryopreservation is therefore, still in its experimental stages and is not a routine option offered to cancer patients. OBJECTIVES: Our main aim was to initiate, develop and implement a practical ovarian tissue cryopreservation and re-transplantation protocol which would restore ovarian function, and possibly fertility, in young female cancer patients undergoing sterilizing cancer therapies in South Africa. The objective of this study was to improve the slow cryopreservation protocol for human ovarian tissue. The ultrastructural effects after cryopreservation with two well-known cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propylene glycol (PROH), on early human follicles were investigated and compared to identify and the better cryoprotectant. MATERIALS AND METHODS: A single group experimental study design was used. The participants consisted cancer patients of the Gynaecological Oncology Unit of Tygerberg Hospital who entered on a basis of voluntary informed consent. Ovarian tissue was obtained by laparoscopic oophorectomy. After dissection of the ovary(ies), some fresh cortical tissue was sent for metastatic analysis and a few strips taken as fresh control. Remaining dissected ovarian cortical tissue sections of each patient were equally divided into the two cryoprotectant groups. Five resulting groups could be compared: i) fresh tissue (control group); tissue equilibrated in ii) DMSO; or iii) PROH and tissue equilibrated and cryopreserved in iv) DMSO or v) PROH. Five tissue samples per patient were therefore fixed for standard histological haematoxylin and eosin (HE) staining and transmission electron microscopy (TEM). Tissue samples showing early follicles on HE slides were sent for TEM processing. Ultrastructural studies on micrographs of primordial and primary follicles were assessed according to a scoring system which gave an indication of follicular health. Appropriate statistical tests were applied to analyse the mean scores where P≤0.05 was considered as statistically significant. RESULTS: No significant overall cryopreservation treatment effect was evident in any of the follicular ultrastructures evaluated. This result indicated that the cryopreservation protocol used did not induce significant damage to the cortex tissue compared to the fresh control group. Comparison of the effect of PROH and DMSO on the follicular ultrastructures showed that PROH tend to induce more extensive damage, especially after cryopreservation. Correlation studies showed significant positive relationships between the majority of the evaluated ultrastructures, especially between the oocyte and granulosa cell layer and basal membrane. The stromal cells and extracellular matrix did not correlate well with other structures. Correlations indicated that the granulosa cells, oocyte and basal lamina are affected similarly and that the damage in one of these structures may be representative of the damage in the other structures. CONCLUSION: The main aim of the study was achieved since results showed that no significant damage was induced by the cryopreservation protocols. Ovarian tissue cryopreserved in this study has shown to restore endocrine function temporarily after heterotopic autotransplantation in menopausal patients. From the electron microscopy evaluations, DMSO showed better cryopreservation results. The DMSO cryopreservation protocol was also more time efficient and has shown the most successful outcomes in the literature. The stromal tissue seemed to be affected differently by cryopreservation compared to the primordial follicle ultrastructures. Younger patients are needed for future studies since a larger initial follicular reserve may allow for larger follicle sample sizes.

Ph.D. (Zoology) / This project involves a detailed study of three South African antelope species, springbok, impala and blesbok. The study investigates the origins of sperm in terms of testicular histology and subsequently the major storage organ, the cauda epididymis. Sperm of these species were characterized in terms of their quality (morphology, motility, vitality characteristics among others and their physiology: when exposed to different media and cryopreservation protocols. Finally sperm fertilization biology of the three species and evaluation of fertilization and developmental success when using homologous and heterologous oocytes (relative comparison) were assessed. Cauda epididymal spermatozoa was recovered post-mortem from the testes of culled springbok (n =38); impala (n =26) and blesbok (n =42) during winter months in South Africa and cryopreserved in a Tris-fructose-citric acid extender (Biladyl) supplemented with 20% egg yolk and 7% glycerol under field conditions. Longevity of sperm was assessed in Tris and Citrate extenders and modified Tyrode lactate in vitro fertilization (IVF) media. Oocytes were collected from the ovaries of domestic cows (n =165), springbok (n = 72) and blesbok (n = 42) and matured in domestic cattle M199 maturation media supplemented with 10% FCS, 10IJg/mi LH, 10IJg/mi FSH and antibiotics. Heterologous (zona intact and zona free) and homologous fertilization was carried using a domestic cattle IVF protocol. Results were analysed using SPSS version 18.0 (Statcon, South Africa). Interspecies comparisons were made using parametric tests: paired t-test for the freezing effect, one-way analysis of variance (ANOVA), Mixed between-within subjects ANOVA for longevity, Non Parametric test for motility characteristics and least squares ANOVA for...

In an effort to minimize the harmful effects of intracellular ice formation (IIF) during cryopreservation of confluent tissues, computer simulations based on Monte Carlo methods were performed to predict the probability of IIF in confluent monolayers during various freezing procedures. To overcome the prohibitive computational costs of such simulations for large tissues, the well-known Johnson-Mehl-Avrami (JMA) model of crystallization kinetics was implemented as a continuum approximation of IIF in tissues. This model, which describes nucleation, growth, and impingement of crystals in a supercooled melt, is analogous to the process of intracellular ice formation and propagation in biological tissues. Based on the work of Weinberg and Kapral (1989), the JMA model was modified to account for finite-size effects, and was shown to predict accurately the results of freezing simulations in 1-D tissue constructs, for various propagation rates and tissue sizes. An initial analysis of IIF kinetics in 2-D tissues is also presented. The probability of IIF in 2-D liver tissue was measured experimentally during freezing of HepG2 cells cultured in monolayers, and compared to Monte Carlo simulations and predictions of the continuum model. The Avrami coefficient and exponent for IIF in HepG2 tissue were estimated to be k = 0.19 and n = 0.45.

O Instituto de Biologia Molecular do Paraná (IBMP) produz kits de diagnóstico para o Sistema Único de Saúde (SUS), que consistem em testes moleculares, realizados por reação em cadeia da polimerase (polymerase chain reaction - PCR). Essa reação ocorre pela atividade da enzima Taq DNA Polimerase (Taq). O IBMP fabrica a Taq a partir do cultivo de células bacterianas, em conformidade às Boas Práticas de Fabricação (BPF) e pretende produzi-la a partir de um banco de células provindas de um mesmo clone visando o aumento da homogeneidade e reprodutibilidade do processo produtivo. O objetivo do presente trabalho é estabelecer um banco de células de trabalho (BCT) e avaliar sua estabilidade para a produção da enzima Taq, partindo de um banco de células mestre (BCM) estabelecido em Bio-Manguinhos. O estabelecimento do BCT consiste em cultivar colônias do BCM, caracterizá-las, avaliar desempenho, e eleger células adequadas para compor o BCT. O estudo da estabilidade contempla testes para comprovar que as células retêm a capacidade de produzir a Taq ao longo do tempo (i.e., viabilidade celular, estabilidade plasmídica, cinética de crescimento, indução da expressão, extração e dosagem do DNA plasmidial, análise de restrição e avaliação plasmidial). O critério decisivo para a seleção de um dos clones para compor o BCT foi o resultado da cinética de crescimento. A estabilidade foi estudada em 10 avaliações realizadas mês a mês. Nelas, a viabilidade celular foi superior a 1,0 x 106 UFC/mL, mostrando que as células permanecem com capacidade de realizar seu metabolismo e reprodução. O crescimento até a fase exponencial se deu entre 6 e 7 horas de cultivo, com taxa específica de crescimento superior a 0,4 min-1 . Os cultivos acrescidos de 1 mM de IPTG expressaram a enzima Taq DNA Polimerase, revelando a qualificação das células para o fim proposto. A estabilidade plasmídica foi superior a 90%, indicando que o plasmídeo pBioMTaq se mantém estável e com boa capacidade de replicação. A concentração plasmidial média foi de 338 ng/μL, e em todos os meses foi maior que 100 ng/μL. Na análise de restrição os plasmídeos foram corretamente clivados e na avaliação plasmidial houve adequada amplificação do fragmento de 500 pb, demonstrando que o plasmídeo se mantém íntegro e estável, com sequências compatíveis com sua construção. O BCT foi testado como substrato para um processo fabril típico do IBMP de produção da Taq DNA polimerase, se apresentando satisfatório em todas as etapas em que foi avaliado. Esses resultados indicam que o banco estabelecido se manteve estável ao longo de 10 meses e está apto a ser utilizado como protótipo para um BCT em conformidade às BPF. / The Molecular Biology Institute of Paraná (Instituto de Biologia Molecular do Paraná - IBMP) produces diagnostic kits for the Brazilian Unified National Health System (Sistema Único de Saúde - SUS), that consist in molecular tests, carried out by polymerase chain reaction (PCR). This reaction is performed by the enzyme Taq DNA Polymerase (Taq). The IBMP manufactures Taq from bacterial cell culture in accordance with Good Manufacturing Practices (GMP) and intends to produce it from a cell bank using cells from the same clone in order to increase the homogeneity and reproducibility of the production process. The objective of the present work is to establish a working cell bank (WCB) and to evaluate its stability for the production of the Taq enzyme, starting from a master cell bank (MCB) established in BioManguinhos. The WCB establishment consists of cultivating MCB colonies, characterizing them, evaluating performance, and electing proper cells to compose the WCT. The stability study contemplates tests to prove that cells retain the ability to produce Taq over time (i.e., cell viability, plasmid stability, growth kinetics, expression induction, plasmid DNA extraction and dosage, restriction analysis and plasmid evaluation). The decisive discretion for the selection of one of the clones to compose BCT was the result of growth kinetics. Stability was studied in 10 month-to-month evaluations. In them, cell viability was higher than 1,0 x 106 CFU/mL, showing that the cells remain capable of performing their metabolism and reproduction. Growth to the exponential phase occurred between 6 and 7 hours of culture, with a specific growth rate greater than 0.4 min-1 . The cultures with 1 mM IPTG expressed Taq DNA polymerase enzyme, revealing the qualification of the cells to the intended purpose. The plasmid stability was greater than 90%, indicating that the plasmid pBioMTaq remains stable and has good replication ability. The mean plasmid concentration was 338 ng/μL, and in all months it was greater than 100 ng/μL. In the restriction analysis the plasmids were correctly cleaved and in the plasmid evaluation there was adequate amplification of the 500 bp fragment, demonstrating that the plasmid remains intact and stable, with compatible sequences with its construction. The WCB was tested as substrate for a typical IBMP production process of Taq DNA polymerase, presenting satisfactory in all the stages in which it was evaluated. These results indicate that the established bank remained stable over 10 months and is apt to be used as a prototype for a WCB in compliance with GMP.

O Instituto de Biologia Molecular do Paraná (IBMP) produz kits de diagnóstico para o Sistema Único de Saúde (SUS), que consistem em testes moleculares, realizados por reação em cadeia da polimerase (polymerase chain reaction - PCR). Essa reação ocorre pela atividade da enzima Taq DNA Polimerase (Taq). O IBMP fabrica a Taq a partir do cultivo de células bacterianas, em conformidade às Boas Práticas de Fabricação (BPF) e pretende produzi-la a partir de um banco de células provindas de um mesmo clone visando o aumento da homogeneidade e reprodutibilidade do processo produtivo. O objetivo do presente trabalho é estabelecer um banco de células de trabalho (BCT) e avaliar sua estabilidade para a produção da enzima Taq, partindo de um banco de células mestre (BCM) estabelecido em Bio-Manguinhos. O estabelecimento do BCT consiste em cultivar colônias do BCM, caracterizá-las, avaliar desempenho, e eleger células adequadas para compor o BCT. O estudo da estabilidade contempla testes para comprovar que as células retêm a capacidade de produzir a Taq ao longo do tempo (i.e., viabilidade celular, estabilidade plasmídica, cinética de crescimento, indução da expressão, extração e dosagem do DNA plasmidial, análise de restrição e avaliação plasmidial). O critério decisivo para a seleção de um dos clones para compor o BCT foi o resultado da cinética de crescimento. A estabilidade foi estudada em 10 avaliações realizadas mês a mês. Nelas, a viabilidade celular foi superior a 1,0 x 106 UFC/mL, mostrando que as células permanecem com capacidade de realizar seu metabolismo e reprodução. O crescimento até a fase exponencial se deu entre 6 e 7 horas de cultivo, com taxa específica de crescimento superior a 0,4 min-1 . Os cultivos acrescidos de 1 mM de IPTG expressaram a enzima Taq DNA Polimerase, revelando a qualificação das células para o fim proposto. A estabilidade plasmídica foi superior a 90%, indicando que o plasmídeo pBioMTaq se mantém estável e com boa capacidade de replicação. A concentração plasmidial média foi de 338 ng/μL, e em todos os meses foi maior que 100 ng/μL. Na análise de restrição os plasmídeos foram corretamente clivados e na avaliação plasmidial houve adequada amplificação do fragmento de 500 pb, demonstrando que o plasmídeo se mantém íntegro e estável, com sequências compatíveis com sua construção. O BCT foi testado como substrato para um processo fabril típico do IBMP de produção da Taq DNA polimerase, se apresentando satisfatório em todas as etapas em que foi avaliado. Esses resultados indicam que o banco estabelecido se manteve estável ao longo de 10 meses e está apto a ser utilizado como protótipo para um BCT em conformidade às BPF. / The Molecular Biology Institute of Paraná (Instituto de Biologia Molecular do Paraná - IBMP) produces diagnostic kits for the Brazilian Unified National Health System (Sistema Único de Saúde - SUS), that consist in molecular tests, carried out by polymerase chain reaction (PCR). This reaction is performed by the enzyme Taq DNA Polymerase (Taq). The IBMP manufactures Taq from bacterial cell culture in accordance with Good Manufacturing Practices (GMP) and intends to produce it from a cell bank using cells from the same clone in order to increase the homogeneity and reproducibility of the production process. The objective of the present work is to establish a working cell bank (WCB) and to evaluate its stability for the production of the Taq enzyme, starting from a master cell bank (MCB) established in BioManguinhos. The WCB establishment consists of cultivating MCB colonies, characterizing them, evaluating performance, and electing proper cells to compose the WCT. The stability study contemplates tests to prove that cells retain the ability to produce Taq over time (i.e., cell viability, plasmid stability, growth kinetics, expression induction, plasmid DNA extraction and dosage, restriction analysis and plasmid evaluation). The decisive discretion for the selection of one of the clones to compose BCT was the result of growth kinetics. Stability was studied in 10 month-to-month evaluations. In them, cell viability was higher than 1,0 x 106 CFU/mL, showing that the cells remain capable of performing their metabolism and reproduction. Growth to the exponential phase occurred between 6 and 7 hours of culture, with a specific growth rate greater than 0.4 min-1 . The cultures with 1 mM IPTG expressed Taq DNA polymerase enzyme, revealing the qualification of the cells to the intended purpose. The plasmid stability was greater than 90%, indicating that the plasmid pBioMTaq remains stable and has good replication ability. The mean plasmid concentration was 338 ng/μL, and in all months it was greater than 100 ng/μL. In the restriction analysis the plasmids were correctly cleaved and in the plasmid evaluation there was adequate amplification of the 500 bp fragment, demonstrating that the plasmid remains intact and stable, with compatible sequences with its construction. The WCB was tested as substrate for a typical IBMP production process of Taq DNA polymerase, presenting satisfactory in all the stages in which it was evaluated. These results indicate that the established bank remained stable over 10 months and is apt to be used as a prototype for a WCB in compliance with GMP.

The semen cryopreservation in cryotubes reduces the time needed for filling, freezing and thawing of the samples, while optimizing the procedures for artificial fertilization. However, no study has yet been performed with tambaqui semen in this container. The aim of this study was to evaluate the influence of cryotubes (1.6 and 4.5 mL) and thawing time (60ºC/70s and 60ºC/90 s) on the quality and fertility of tambaqui cryopreserved semen. For that, semen samples were diluted in freezing solution (1:9) composed with 75% glucose 290 mOsm , 10% methylglycol and 5% egg yolk, and frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). The semen samples was thawed at 60ºC in bath water during 70s or 90s and the semen quality was evaluated (Total motility - MT; Progressive motility - MP; Curvilinear velocity - VCL; Straight-line velocity - VSL and Average path velocity - VAP). In this study was also determined the time viability of spermatozoa thawed, maintained under refrigeration at 5°C, and assessed over 24 hours. Besides the kinetic parameters were evaluated sperm morphology and membrane integrity of spermatozoa. The fertilizing capacity of semen was evaluated from the samples thawed in the best time. All parameters of sperm kinetic showed higher values when the semen samples were thawed in 90 s compared to 70 s, independently of cryotube type. No significant differences were observed in sperm kinetic parameters after thawing between samples frozen in 1.6 ml and 4.5 mL cryotubes, except for total motility that was higher in 1.6 mL (47 ± 14% ) compared with 4.5 mL cryotubes (40 ± 11%), independently of thawing time. After activation, the spermatozoa significantly reduced the values of kinetic parameters within 37 seconds, except the MT which remained constant during this period. Relying on the sperm parameters evaluated (VCL, VSL, VAP and membrane integrity for both cryotubes and MT, MP and morphology only for 1.6 mL cryotube) the frozen semen maintained the quality during 3h after thawing. The fertilization rate obtained with fresh semen (74±6%) was higher than cryopreserved semen (1.6 mL - 45±9% and 4.5 mL - 41±12%). The two cryotubes did not differ in this parameter. A high correlation (p <0.05) was observed between fertility and sperm kinetics parameters (MT - 89%, MP - 86%; VCL - 79%; VSL - APV and 69% - 78%). It is concluded that 1.6 and 4.5 mL cryotubes can be used in the cryopreservation of tambaqui semen being recommended to be thawed at 60°C for 90s and their use in fertilization procedures within 3 hours after thawing since kept at 5°C. / A criopreservação de sêmen em criotubos reduz o tempo necessário para o envase, congelamento e descongelamento das amostras, além de otimizar os procedimentos de fertilização artificial. No entanto, nenhum estudo ainda foi realizado com o sêmen de tambaqui neste recipiente. Assim, o objetivo do presente trabalho foi avaliar a influência do tipo de criotubo (1,6 e 4,5 mL) e do tempo de descongelamento (60ºC/70s e 60ºC/90s) sobre a qualidade e fertilidade do sêmen de tambaqui criopreservado. Para isso, amostras de sêmen foram diluídas em solução de congelamento (1:9 v/v) composta por 75% de glicose 290 mOsm, 10% de metilglicol e 5% de gema de ovo, sendo envasadas em criotubos de 1,6 e 4,5 mL, congeladas em vapor de nitrogênio líquido no botijão dry-shipper (-175ºC) e armazenadas em botijão criogênico a -196°C. Para avaliação do tempo de descongelamento do sêmen, os criotubos foram imersas em água a 60°C durante 70 s ou 90 s e a qualidade espermática imediatamente avaliada (Motilidade total - MT; Motilidade progressiva - MP; Velocidade curvilinear - VCL; Velocidade em linha reta - VSL e Velocidade média da trajetória - VAP). Neste estudo foi determinado também o tempo de viabilidade dos espermatozoides descongelados, mantidos sob refrigeração a 5°C e avaliados durante 24 horas. Além dos parâmetros de cinética espermática foram avaliadas a morfologia e a integridade da membrana plasmática dos espermatozoides. A capacidade de fertilização do sêmen foi avaliada a partir das amostras descongeladas no melhor tempo. Todos os parâmetros de cinética espermática apresentaram valores superiores quando as amostras de sêmen foram descongeladas por 90s em relação ao tempo de 70s, independentemente do tipo de criotubo. Não foram observadas diferenças significativas nos parâmetros de cinética espermática pós-descongelamento entre as amostras congeladas nos criotubos de 1,6 e 4,5 mL, com exceção da MT que foi superior nos criotubos de 1,6 mL (47±14%) em comparação com os criotubos de 4,5 mL (40±11%), independentemente do tempo de descongelamento. Após ativação, os espermatozoides reduziram significativamente os valores dos parâmetros de cinética dentro de 37 segundos, exceto a MT que se manteve constante neste período. Baseando-se na maior parte dos parâmetros espermáticos avaliados (VCL, VSL, VAP e Integridade da membrana plásmatica para ambos os criotubos e MT, MP e Morfologia espermática somente para o criotubo de 1,6 mL) o sêmen congelado manteve sua qualidade durante 3h após o descongelamento. A taxa de fertilização obtida com o sêmen in natura (74±6%) foi superior ao sêmen criopreservado (1,6 mL - 45±9% e 4,5 mL - 41±12%). Os dois criotubos não diferiram entre si neste parâmetro. Uma alta correlação significativa (p<0,05) foi observada entre a fertilização e a cinética espermática (MT - 89%; MP - 86%; VCL - 79%; VSL - 69% e VAP - 78%). Conclui-se que os criotubos de 1,6 e 4,5 mL podem ser utilizados na criopreservação do sêmen de tambaqui, sendo recomendado seu descongelamento a 60°C por 90s e seu uso em procedimentos de fertilização dentro de 3 horas após o descongelamento desde que mantido a 5°C.

M.Sc. / Climate change, loss of habitat and over-exploitation of natural resources as well as the introduction of invasive alien species through human activities are resulting in an ever increasing risk of extinction of many plant and animal species. There are two major approaches to conserving threatened and endangered species. Firstly the large scale preservation of natural habitat and ecological processes, thereby protecting the species inhabiting the habitat. The second approach involves the ex-situ breeding of rare and endangered species. It is estimated that in the next 200 years approximately 800 mammalian species will require the assistance of breeding programs to ensure long term genetic viability. Biological Resource Banks (BRB) can potentially contribute to this challenge by providing a source of genes that can be used to counter the effects of external selection pressures, genetic drift and inbreeding depression in small or fragmented populations. These banks commonly contain biological materials such as cryopreserved sperm, embryos and cell cultures mainly as genetic and research resources. . Biological resource banks can potentially use these cryopreserved gametes together with assisted reproductive technologies (ART), such as artificial insemination (AI), in vitro fertilisation (IVF), embryo transfer (ET), intracytoplasmic sperm injection (ICSI) and nuclear transfer (NT) to maintain genetic heterogeneity in ex-situ and wild populations. Ascertaining the appropriate protocols for developing the ARTs necessary for non-domestic species is one of the major challenges faced by reproductive physiologists. Typically, there is very little available information about the processing of semen, the effects of diluents, concentration and type of cryoprotectants and freeze-thaw methods for sperm samples of non-domestic species. Procedures proven to be highly effective in humans and laboratory or domestic species, are frequently adopted and modified for use in related wildlife species. It is thus necessary to gain knowledge of the reproductive physiology of wildlife species in order to define effective protocols for the cryopreservation of biomaterials which assists in the conservation of South Africa‘s diverse wildlife species. Sperm quality assessment is a useful tool for assessing the reproductive health of free-ranging populations as well as for selecting individuals for future assisted reproduction programs.