O objetivo deste trabalho foi o de avaliar o efeito da suplementação da dieta de Tilápias (Oreochromis niloticus) utilizando sal comercial composto dos microminerais cobre, manganês e zinco, sobre a histologia e o desenvolvimento das gônadas dos machos. Foram utilizados 1200 machos masculinizados de tilápia, com média de peso de 120g, suplementadas com níveis crescentes (0,00; 0,35; 0,70; 1,05; 1,40 e 1,75 mg/kg) de um produto comercial composto por Cu 13,40 g/kg; Mn 26,70 g/kg e Zn 66,70 g/kg. O experimento foi conduzido em um sistema de recirculação de água, composto por 24 tanques divididos ao meio totalizando 48 unidades experimentais. O delineamento experimental foi inteiramente casualizado, com medidas repetidas no tempo. As biometrias e coletas foram realizadas em 16 e 32 semanas de experimento, para avaliar o desenvolvimento dos peixes. As gônadas coletadas foram preparadas e cortadas para confecção de lamina histológica, após coradas com hematoxilina e eosina, e posteriormente fotografadas, utilizando microscópio com câmera acoplada. A contagem das fotos foi realizada manualmente, e avaliou-se o número de células por mm3, para espermatogônias, espermatócitos, espermátides e espermatozoides. As correlações entre número de espermatozoides, espermatócitos, espermátides e tratamentos foi de -0,30, -0,20 e -0,30, respectivamente, demonstrando que o efeito da suplementação crescente com o sal, é prejudicial para o desenvolvimento das células reprodutivas masculinas. Foi observado que os tratamentos com adição igual ou superior a 0,70 mg/kg do sal comercial causaram danos as gônadas para os parâmetros da gametogênese de número de espermatócitos, espermátides e espermatozoides. Sendo assim, a utilização de sais que contenham combinação de Cu 13,40 g/kg; Mn 26,70 g/kg e Zn 66,70 g/kg reprodutores machos de tilápia, não é recomendada em função dos danos causados nas etapas da gametogênese, reduzindo a produção de espermatozoides. / The objective of this study was to evaluate the effect of supplementing the diet of tilapia (Oreochromis niloticus) using commercial salt compound of micro minerals copper, manganese and zinc, on the histology and gonadal development of males. A total of 1200 masculinized male tilapia were used, with 120 g average weight, supplemented with increasing levels (0.00, 0.35, 0.70, 1.05, 1.40 and 1.75 mg / kg) of a product commercial composed of Cu 13,40 g/kg; Mn 26,70 g/kg e Zn 66,70 g/kg. The trial was conducted in a water recirculation system, consisting of 24 tanks divided in half, totaling 48 experimental units. The experimental design was completely randomized with repeated measures over time. The biometry and samples were taken at 16 and 32 weeks of experiment, to evaluate the fish development. The collected gonads were prepared and sliced for making histological slide, after stained with hematoxylin and eosin, and then photographed using a microscope with attached camera. The count of the photos was performed manually, and was rated the number of cells per mm3 to spermatogonia, spermatocytes, spermatids and sperm cell. The correlations between the number of spermatozoa, spermatocytes, spermatids and treatments was of -0.30, -0.20 and -0.30, respectively, demonstrating that the effect of increasing supplementation of the salt is detrimental to the development of male reproductive cells. It was observed that the addition equal or superior to 0.70 mg / kg of commercial salt caused damage in the gonads for gametogenesis parameters number of spermatocytes, spermatids and sperma cell. Thus, the use of combination of salts containing 13.40 g Cu / kg; Mn 26.70 g / kg Zn and 66.70 g / kg breeding male tilapia is not recommended due to the damage caused in the stages of gametogenesis, reducing the sperm production.

Sperm competition occurs when the sperm of multiple males concurrently occupy a female's reproductive tract and compete for fertilization. Sperm competition may have been a recurrent adaptive problem over human evolutionary history. Women's orgasm may facilitate retention of a particular man's sperm. I therefore hypothesized that men experiencing greater sperm competition risk will be particularly interested in the occurrence of their partner's copulatory orgasm. Men who are more satisfied with and invested in their relationship may experience greater costs in the event of sperm competition and potential cuckoldry. Therefore, these men may be more interested in ensuring their partner's copulatory orgasm. I hypothesized that men's relationship satisfaction and investment would predict interest in their partner's copulatory orgasm and moderate the link between sperm competition risk and interest in partner's copulatory orgasm. Using data secured from 229 men in a committed relationship, I tested and found support for these hypotheses. / by Vincent M. Bates. / Thesis (M.A.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Orientador: Carlos Roberto Daleck / Banca: Paulo Henrique Franceschini / Banca: Maria Denise Lopes / Resumo: A correta orientação do Médico Veterinário, aos proprietários de cães, usados com finalidades reprodutivas, submetidas à quimioterapia com cisplatina, é importante na medida que este agente citostático age nas células em constante divisão, podendo ser citotóxicos para as células germinativas testiculares. O objetivo desse trabalho foi avaliar a qualidade espermática através do espermograma de cães que receberam cisplatina em diferentes momentos de análise espermática. A dose utilizada foi de 70 mg/mø, em intervalos de 21 dias, totalizando 4 infusões. Os cães foram divididos em dois grupos de 4 animais cada, sendo que um dos grupos recebeu a quimioterapia e o protocolo de diurese para proteção renal, já o grupo controle não recebeu a cisplatina, estando sujeito apenas aos fatores ambientais. Os resultados obtidos demonstraram que a cisplatina influenciou na qualidade espermática de cães, pois elevou as patologias maiores e totais acima do aceitável para cães aptos a reprodução. Portanto, infere-se que este citostático possa acarretar alterações morfofuncionais nos túbulos seminíferos e conduto epididimário. / Abstract: The correct veterinary's orientation for male dogs' owners used for reproduction goals, undergone cisplatin administration, is important because of this cistostatic act in cell with frequently proliferation, and could to cause germ cell injury. The objections of this experiment was to analysis the sperm quality through dogs' spermogram that received cisplatin's infusions. The dose used was 70 mg/mø in 21 days periods, with 4 infusion in total. The dogs were divided in 2 groups with 4 animal each one. One of the groups received all the diuresys protocol (to protect the kidney) and the citostatic. And the other control group just didn't receive the cisplatin infusion to know the real action of cisplatin effects without environmental stresses. The results show that cisplatin influence at the sperm quality in the dogs, because it elevated the major and total defects above that would be acceptable for competent dog to reproduct. It could deduct that cisplatin cause phisiologic alteration in the testis and epididymis. / Mestre

A preocupação com a qualidade do sêmen ovino congelado tem sido motivo de muitas pesquisas, principalmente pela dificuldade da transposição cervical durante a inseminação artificial. Contudo, a inseminação intra-cervical é frequentemente usada em ovinos e resulta em redução da fertilidade com o uso do sêmen congelado. Neste sentido, esse estudo foi dividido em dois experimentos. No 1o experimento foi verificado o potencial da ciclodextrina pré-carregada com colesterol como aditivo ao diluidor na proteção da cinética espermática, integridade das membranas plasmática e acrossomal, função mitocondrial, capacitação espermática e produção de espécies reativas ao oxigênio (ROS) em espermatozoides criopreservados ovinos. Cinco ejaculados de seis carneiros (n = 30) foram divididos em três tratamentos: apenas diluidor (CON); diluidor + colesterol incorporado a ciclodextrina (CLC + CHO); e diluidor + ciclodextrina pura (CLCP). Após a diluição (50x106 espermatozóides/mL), o sêmen foi envasado em palhetas, identificado e criopreservado utilizando um sistema automatizado. Duas palhetas da mesma partida de cada tratamento foram descongeladas (a 37°C durante 30 segundos) e analisadas quanto motilidade (CASA), morfologia dos espermatozoides (DIC), integridade do plasma (PI-H342) e acrossomal (FITC-PSA) membranas, potencial mitocondrial (JC-1), produção de radicais livres (CellRox), peroxidação lipídica (BODIPY) e fluidez da membrana celular (merocianina 540). As comparações entre os grupos foram analisadas pelo PROC MIXED do SAS e o efeito de grupo foi detectado pelo teste de Tukey (p<0,05) ou pela estatística não paramétrica de ordem (Kruskal-Wallis), quando era necessário. No 2º experimento os mesmos ejaculados foram divididos por congelabilidade baseados na motilidade total pós-descongelação (MTPD) e na porcentagem de redução na motilidade total (%RMT) quando comparados o sêmen in natura e o sêmen pós-descongelação (somente diluidor), sendo divididos nos grupos alta (MTPD>60% e %RMT<40%), intermediária (60%>MTPD>40% e 60%>RMT>40%) e baixa (MTPD<40% e %RMT>60%) congelabilidade. Foram analisados os tratamentos CON e CLC+CHO dentro de cada animal e de cada grupo quanto a motilidade (CASA), a integridade das membranas plasmática (PI-H342) e acrossomal (FITC-PSA), potencial mitocondrial (JC-1), peroxidação lipídica (BODIPY) e fluidez da membrana celular (merocianina 540). As comparações foram realizadas por análise de variância (ANOVA) em arranjo fatorial 3X3 (Tratamento CLC+CHO, CON e in natura X grupos alta, intermediária e baixa congelabilidade), as comparações entre os grupos foram analisadas pelo PROC MIXED do SAS e o efeito de grupo foi detectado pelo teste de Tukey (p <0,05) ou pela estatística não paramétrica de ordem (Kruskal-Wallis), quando era necessário. No 1o experimento o tratamento CLC+CHO se mostrou mais eficaz em preservar os parâmetros de motilidade, integridade de membranas e potencial mitocondrial quando comparado aos demais tratamentos. O grupo CLCP mostrou queda nos parâmetros de motilidade, integridade de membrana, potencial mitocondrial, mostrando, com isso, uma queda na preservação espermática. No 2o experimento observou-se que o tratamento CLC+CHO mostrou maior grau de preservação para motilidade total e progressiva, células rápidas, preservação de membranas plasmática e acrossomal e potencial mitocondrial quando comparado ao CON. Esse efeito teve maior significância nos grupos de baixa e intermediária congelabilidade, não sendo tão expressivo no grupo de alta. Pode-se concluir com esse estudo que o tratamento com ciclodextrina acrescida de colesterol contribui para uma melhor preservação dos parâmetros espermáticos do sêmen ovino, principalmente em animais apresentando baixa congelabilidade, contudo não apresenta diferença em ejaculados de alta congelabilidade. / Frozen ram semen has been the subject of many researches, mainly due to the difficulty of cervical transposition during artificial insemination. However, intra-cervical insemination in sheep is often used, resulting in reduced fertility with the use of frozen semen. To this end, this study was divided into two experiments. In the first experiment was verified the potential of cyclodextrin loaded with cholesterol as an additive to the extender in the protection of sperm kinetics, integrity of plasmatic and acrosomal membranes, mitochondrial function, sperm capacitation and production of reactive oxygen species (ROS) in cryopreserved sheep sperm. Five ejaculates from six rams (n = 30) were divided into three treatments: only extender (CON); extender + cyclodextrin loaded with cholesterol (CLC + CHO); and extender + pure cyclodextrin (CLCP). After dilution (50x106 spermatozoa/mL), semen was stored in straws, identified and cryopreserved using an automated system. Two straws from each ejaculate and treatment were thawed (at 37° C for 30 seconds) and analyzed for motility (CASA), morphology of spermatozoa (DIC), plasmatic (PI-H342) and acrosomal (FITC-PSA) membrane integrity, mitochondrial potential (JC-1), production of free radicals (CellRox), lipid peroxidation (BODIPY) and cell membrane permeability (Merocyanine 540). Comparisons between groups were analyzed using PROC MIXED of SAS and the effect of group was detected by Tukey (p <0.05) or by the order of nonparametric statistics (Kruskal-Wallis) test, when necessary. In the 2nd experiment the same ejaculates were divided by freezability based on the total post-thaw motility (TPTM) and the percentage of reduction in total motility (%RTM) when compared fresh and post-thaw semen (only extender), and divided into the groups: high (TPTM ≥ 60% and %RTM ≤ 40%), intermediate (60%> TPTM > 40% and 60%> %RTM > 40%) and low (TPTM ≤ 40% and %RTM ≥ 60%) freezability. CON and CLC + CHO treatments were analyzed within each animal and each group for motility (CASA), plasmatic (PI-H342) and acrosomal (FITC-PSA) membrane integrity, mitochondrial potential (JC-1), lipid peroxidation (BODIPY) and cell membrane permeability (Merocyanine 540). Comparisons were performed by analysis of variance (ANOVA) with a factorial arrangement 3x3 (groups CLC+CHO, CON and fresh X groups high, medium and low freezability), comparisons between groups were analyzed using PROC MIXED of SAS and the group effect was detected by the Tukey test (p < 0.05) or no statistical order parametric (Kruskal-Wallis), when necessary. In the first experimente, CLC + CHO treatment was more effective in preserving the parameters of motility, membrane integrity, and mitochondrial potential compared to other treatments. The CLCP group showed a fall in the parameters of motility, membrane integrity, mitochondrial potential, showing thereby a decrease in sperm preservation. In the 2nd experiment, it was observed that the treatment CLC + CHO showed greater preservation for total and progressive motility, rapid cell preservation, plasmatic and acrosomal membranes and mitochondrial potential when compared to CON. This effect was most significant in the groups of low and intermediate freezability, not being as significant in the group of high freezability. We can conclud from this study that treatment with cyclodextrin loaded with cholesterol contributes to better preservation of sperm parameters in ram semen, especially in animals displaying low freezability, however there where no diference in the high freezability group.

Estudos sobre a biologia reprodutiva de primatas não humanos são importantes para o desenvolvimento de biotecnologias da reprodução, visando a conservação das espécies. O objetivo deste estudo foi avaliar métodos de criopreservação do sêmen de Callithrix jacchus e C. penicillata. O sêmen foi colhido pelo método da vibroestimulação peniana, de animais adultos mantidos em cativeiro. Imediatamente após a colheita, foram analisadas as variáveis volume, pH, concentração, motilidades total e progressiva, integridade de membrana plasmática, integridade de acrossomo, atividade citoquímica mitocondrial, fragmentação de DNA e morfologia espermática. As amostras foram então refrigeradas em diluidor TEST gema de ovo sem glicerol; após este período, foi adicionado o glicerol a 4 e 6% de concentração e novas análises foram realizadas. O sêmen foi congelado em vapor de nitrogênio e finalmente em imersão em nitrogênio. As amostras foram descongeladas após um período mínimo de um mês e meio, e novas análises realizadas aos 10, 40 e 80 minutos pós-descongelação. Foi observado que o sêmen fresco de C. jacchus e C.penicillata são semelhantes em muitos aspectos. O diluidor TEST gema de ovo com o glicerol tanto a 4 como a 6% de concentração não foi eficaz para a proteção dos espermatozoides das duas espécies durante a criopreservação. São necessários outros estudos para o desenvolvimento de um protocolo de criopreservação do sêmen das duas espécies. Contudo, este trabalho contém informações que podem nortear futuros estudos sobre a criopreservação do sêmen destas e de outras espécies de primatas neotropicais. / Studies on the reproductive biology of nonhuman primates are important for the development of reproductive biotechnologies, in order to species conservation. The aim of this study was to evaluate methods for sperm cryopreservation from Callithrix jacchus and C. penicillata. Semen was collected by penile vibrostimulation from adult animals kept in captivity. Immediately after collection, the variables analyzed were volume, pH, concentration, total and progressive motility, plasma membrane integrity, acrosome integrity, cytochemical mitochondrial activity, DNA fragmentation and morphology. The samples were then chilled in TEST egg yolk extender without glycerol; after this period, the glycerol was added at concentrations of 4 and 6% and further analyzes were performed. The semen was frozen in nitrogen vapour and finally immersion in nitrogen. After a minimum of one month and a half, the samples were thawed and further analysis performed at 10, 40 and 80 minutes after thawing. Fresh semen from C. jacchus and C. penicillata were found to be similar in many aspects. The extender TEST egg yolk with glycerol as much as 4 to 6% concentration was not effective for the protection of spermatozoa from both species during cryopreservation. Further studies are needed to develop a protocol for cryopreservation of semen from both species. However, this work contains information that can guide future studies on sperm cryopreservation of these and other Neotropical primates species.

ABSTRACT The purpose of this study was to compare the efficiency of three sperm elution methods on sexual assault swabs; factors such as solvent type, solvent volume, sperm concentration, and duration of extraction and elution method were evaluated with respect to observed sperm recovery. Swabs containing dilutions of semen ranging from 1:10 to 1:1,000 and simulated post-coital swabs were extracted via the traditional tube extraction, as well as two direct slide elution techniques, tapping and swirling. For the slide elution techniques, a swab cutting was placed directly onto a microscope slide, a small volume of water or buffer was added, and sperm were eluted by either tapping the sample with a stirring stick or swirling it around the slide with metal forceps. The tube method requires a minimum of one and one half hours for extraction, while the slide elution techniques require only ten seconds for extraction. The average sperm counts from 1:10 dilutions processed with the tapping elution method were statistically higher than the 1:10 dilutions samples processed with tube and swirling methods. Elution by tapping also recovered a significantly higher amount of sperm cells from the 1:1,000 dilution compared to the tube extraction of the same dilution. The tapping elution method consistently resulted in the greatest number of spermatozoa observed, followed by the swirling method and then tube extraction; additionally, incidents of false negatives (no sperm observed) were observed with the tube and swirling methods. Simulated post-coital samples produced similar results to the semen samples; however, vaginal swabs from one donor resulted in an extremely high ratio of exfoliated epithelial cells that obscured the spermatozoa, especially with the direct slide elution methods. The slide elution methods resulted in similar and consistent relative standard deviations between dilutions in samples, while the tube extraction results suggest an increase in variance as the dilution increases. Overall, slide elution methods yielded the most observed sperm cells in a significantly shorter amount of time.

Typically, semen testing involves presumptive and confirmatory tests to determine the region in which a semen stain has been deposited prior to initiating DNA analysis. However, previous research showed that the soluble components of seminal fluid, but not spermatozoa, migrated from their original location on cotton cloth upon exposure to porcine decomposition fluids and rainfall/dew6. This indicates that preliminary testing and detection techniques may result in areas being sampled that will not yield a successful DNA profile. The present study assesses how various amounts of water or blood affect migration patterns of seminal fluid components using traditional serological screening methods as well as DNA analysis. The effects of exposing a semen stain to water over the course of several days are also investigated. The final component of the study evaluates whether the presence of acid phosphatase (AP) Spot reagent had any detrimental effects on subsequent antigen P30 (P30) testing, Kernechtrot Picroindigocarmine (KPIC) sperm staining or DNA analysis. Neat semen was deposited onto swatches from cotton sheets and allowed to dry before being sprayed with 2 mL, 5 mL, or 10 mL of water or blood. The swatches were allowed to dry while lying flat, at 45°, or at 90°. Three of the swatches were sprayed directly with AP Spot reagent to determine any potential interference with subsequent P30 and DNA testing. After the water or blood was dry, the swatches were viewed with an alternate light source (ALS) at 450 nm using orange barrier filter goggles. Three-millimeter fabric punches were collected from each swatch in at least thirteen locations (one from the center of the stain and four at 1 cm, 4 cm, and 7 cm from the perimeter of the stain in multiple directions), and were extracted for two hours prior to testing for the presence of P30. Additional fabric punches were collected from each P30 positive location to be used for DNA analysis. AP testing showed positive results beyond the original semen stain with an average distance of 1-3 cm from the perimeter of the original region of deposition (ORD) for all swatches except those moistened with blood. AP mapping was performed on the swatches moistened with blood and negative results were obtained. Positive P30 results were obtained for all swatches with an average distance of 1-3 cm from the ORD. The angle at which the swatch was positioned influenced the direction(s) that the soluble components migrated; however the amount of water (or blood) the swatch was exposed to had a much greater effect on the distance of migration. Microscopic examination of slides made from the extracts of each fabric punch revealed minimal spermatozoa migration for all swatches; the majority of the samples outside of the ORD showed no spermatozoa, although a few showed a single sperm cell. These findings demonstrate that the soluble components of semen stains that often aid in detection migrated when exposed to moisture, while sperm cells containing genetic material largely remained in their original location. The DNA analysis results confirmed the lack of spermatozoa migration. Full DNA profiles were obtained from within the ORD of the flat and 90° swatches. The samples from outside of the ORD produced either partial profiles (maximum dropout rate of 97%) or no profile. If case circumstances suggest that evidence has been exposed to water, multiple regions should be tested in order to maximize the possibility of identifying semen and obtaining a DNA profile. AP Spot reagent was not found to have detrimental effects on P30 testing, sperm staining or DNA analysis. Therefore, direct application of AP Spot reagent could be used for larger pieces of evidence where the location of a stain is unknown. This would eliminate the careful documentation needed for chemical mapping and the reliance on the transfer of acid phosphatase from one substrate to another.

Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

Consumption of toxic endophyte-infected (E+) tall fescue pastures is known to have a negative impact on bull reproductive performance. Since decreased cleavage rates of embryos fertilized with spermatozoa from bulls grazing E+ tall fescue pastures have been observed in several studies using differing sets of bulls, technicians, pastures, and other methods of inducing tall fescue toxicosis (ergotamine tartrate), it is hypothesized that spermatozoa function from bulls grazing E+ is impaired in ways undetectable by gross semen examination. During a three-month grazing study, 6 Angus bulls were utilized to determine the effects of grazing E+ tall fescue pastures on growth performance and spermatozoa function. Bulls were appointed to graze Kentucky 31 tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum, an ergot alkaloid producing endophyte (n=3) or Jesup tall fescue infected with non-ergot alkaloid producing endophyte (NTE) MaxQTM (n=3). Bulls were grouped by body weight (BW) and scrotal circumference (SC) to graze pastures from April 18-June 26, 2007. Blood samples, BW, SC, semen, and rectal temperatures (RT) were collected every 7 d. Scrotal temperatures (ST) were obtained before semen collection each week in June. Semen was evaluated for gross motility, morphology, and Computer Assisted Semen Analysis (CASA) parameters. Semen from a subset of bulls (n=2 per treatment) was used to assess spermatozoa ability to function utilizing in vitro assays. Growth performance was decreased in E+ bulls compared to bulls grazing NTE tall fescue pastures (P = 0.002). Concentrations of prolactin were reduced in bulls grazing E+ compared to bulls grazing NTE tall fescue pastures (P = 0.055). Motility post-thaw and during a 3-hour stress test were decreased (P = 0.024 and P < 0.0001, respectively), in addition to altered CASA parameters for spermatozoa. Penetration was reduced in oocytes fertilized with spermatozoa from bulls grazing E+ (64.54 ± 3.28%) compared to NTE tall fescue pastures (87.42 ± 1.63%, P < 0.0001) coupled with hastened meiotic completion, and reduced intracellular calcium parameters. These findings indicate impaired spermatozoa function in bulls grazing E+ tall fescue pastures that extends beyond gross semen characteristics, and may provide direction for future studies.

The increasing number of patients undergoing treatment with assisted reproductive techniques (ART) during the past years have led to the need of developing different methods for separation of spermatozoa that can be used for different fertilisation procedures and for freezing. Cryopreservation of spermatozoa includes preparation, freezing, storage and thawing. In this study two different cryomedia (Cryo Protec I and Cryo Protec II) for human spermatozoa were compared. The main outcome was spermsurvival rate for spermatozoa after freezing. Sperm viability was assessed using the Hypo-osmotic swelling test which is based on osmolality. A total of 86 samples of semen were used in this study (Cryo Protec I=38, Cryo Protec II=48). The survival rate between the two cryomedia did not differ much but Cryo Protectant I showed a small increase in survival for the spermatozoa after freezing. The Hypo-osmotic swelling test also showed similar values of viable spermatozoa for the two cryomedia both before and after freezing.