Current methods of identifying semen include preliminary methods such as an alternate light source (ALS) and color tests which test for the presence of acid phosphatase (AP). Confirmatory methods for identifying semen include the microscopic identification of spermatozoa which typically use staining methods such as the Christmas Tree Stain (KPIC) or hematoxylin and eosin stain (H&E). Other methods include immunoassay cards that test for the components of semen such as prostate specific antigen (PSA) and semenogelin (Sg). One common fluorescent staining method used to identify the presence of spermatozoa is SPERM HY-LITERTM which uses an anti-human sperm-specific mouse monoclonal antibody coupled to an AlexaFluor 488 dye. This causes the entire head of the sperm cell to fluoresce when a FITC filter is used. It also utilizes a 4,6-diamidino-2- phenylindole (DAPI) fluorescent dye that stains all cell nuclei non-specifically. This allows the analyst to easily identify whether sperm is present in the sample. A drawback of the antibody labeling procedure is that there are many washing and transfer steps that can lead to sample loss; thus a need for a new, optimized staining method exists. During spermiogenesis, protamines replace histones to further condense the DNA of the sperm nucleus. Humans express two proamines, protamine 1 (P1) and protamine 2 (P2). The protamines contain an arginine-rich core as well as cysteine residues. The high levels of arginine create a net positive charge that allows for stronger binding to DNA. The cysteine residues allow for the formation of inter and intra-protamine disulfide bonds which allow for the chromatin compaction. If the disulfide bonds found in protamines can be reduced to produce free thiols, a thiol-reactive probe can bind and label the protamines in the sperm nucleus. Reduction of the disulfide bonds can be performed by use of reagents such as dithiothreitol (DTT) or tris-(2-carboxyethyl)phosphine (TCEP). From the literature on thiol-reactive probes, it is suggested that TCEP be used as a reducing agent due to its structure. DTT contains thiol groups and when removed from the sample, thiol groups may be oxidized back into disulfide bond. In addition, TCEP is more stable than DTT at a higher pH and higher temperatures. Once disulfide bonds have been reduced, a thiol-reactive probe may be introduced. There are many different types of probes that may be used. Maleimides are commonly used for thiol modification and quantitation. When the compound encounters a thiol group, the thiol group is added across the double bond, yielding a thioether. The reagent used is N-7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM). It absorbs light at 376 nm and emits light at 476 nm, producing a blue fluorescence. The dye is nonfluorescent until it reacts with a thiol group. In this research, both sperm and epithelial cells were added to the slides in order to develop a novel staining procedure. The initial protocol used TCEP to break the disulfide bonds followed by DACM to bind free thiol groups. Sodium dodecyl sulfate (SDS) was then added to the protocol to lyse cells. DTT was tested for use as a reducing agent as well. The purpose of establishing this protocol was to design a procedure for rapid labeling of sperm that does not require antibody labeling. In the antibody labeling procedure, there are many washing and transfer steps. The proposed method may limit the number of procedural steps resulting in less loss of biological material. It can also help to limit the time-consuming methods of the current staining techniques, such as KPIC and H&E.

OLIVEIRA, Rodrigo Vasconcelos de. Proteínas espermáticas e dinâmica da cromatina em ruminantes: relação com a fertilidade em touros e com o uso de castanha de caju na dieta de ovinos. 2013. 119 f. Tese (doutorado em zootecnia)- Universidade Federal do Ceará, Fortaleza-CE, 2013. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-04-22T19:42:24Z No. of bitstreams: 1 2013_tese_rvoliveira.pdf: 1755876 bytes, checksum: 938cdae211bb3c2ecc1a2c8db23536a9 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-05-27T17:54:11Z (GMT) No. of bitstreams: 1 2013_tese_rvoliveira.pdf: 1755876 bytes, checksum: 938cdae211bb3c2ecc1a2c8db23536a9 (MD5) / Made available in DSpace on 2016-05-27T17:54:11Z (GMT). No. of bitstreams: 1 2013_tese_rvoliveira.pdf: 1755876 bytes, checksum: 938cdae211bb3c2ecc1a2c8db23536a9 (MD5) Previous issue date: 2013 / The ruminant fertility is influenced by intrinsic sperm factors, like chromatin or proteins. Considering that the reproductive efficiency is dependent on a balanced and feasible nutrition, the cashew nut meal (CNM) is a low cost byproduct that must be analyzed for possible effects on sperm chromatin and proteins.Study 1:. The objectives of study 1 were to determine failures of chromatin condensation, expression levels and cellular localizations of histones; H3.3, H2B and H4, respectively in spermatozoa from low (LF) vs. high fertility (HF) bulls. The data were analyzed by t test and Pearson correlation (P < 0.05). We demonstrated that aniline blue staining was different within LF (1.73 (0.55, 0.19)) and HF Groups (0.67 (0.17, 0.06) (P < 0.0001), which was also negatively correlated with in vivo bull fertility (r = -0.90; P < 0.0001). Although those histones were consistently immune-detectable and specifically localized in bull sperm, this was not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and conserved among bovine, mouse and human. The H2B variants were more conserved between bovine and human than those of mouse. In conclusion, we showed that H2B, H3.3 and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related with bull fertility. Study 2: The objectives of study 2 were evaluate the effects of 13% of CNM inclusion in the diet of Morada Nova rams on the semen parameters, chromatin integrity and sperm proteins. Twenty rams were distributed in two equal groups: cashew nut group (CNG) and control group (COG) that received 13% and 0% of CNM in the diet for 90 days, respectively. The groups were compared for live weight, scrotal circumference, seminal parameters, chromatin integrity and sperm protein profile at 0, 45 and 90 days of the experiment. The data were evaluated by GLM for repeated measures (P < 0.05). At 90 days, CNG (69.00% (7.38; 2.33)) presented percentage of motile sperm superior than control group (60,00% (9,43; 2,98)) (P<0.05). There was not effect from the diet with CNM on chromatin integrity. But, the percentages of protein expression from ODF1 and H2B were larger in the CNG (P<0.05). The proteins: ODF1, GPX4, FTL and H2B were negatively correlated with sperm chromatin quality. In conclusion, the cashew nut meal did not affect negatively the semen quality. / A fertilidade em ruminantes é influenciada por fatores intrínsecos espermáticos como a cromatina e as proteínas. Considerando que a eficiência reprodutiva do macho depende de uma nutrição balanceada e viável, o farelo de castanha de caju (FCC) é um subproduto de baixo custo que deve ser analisado quanto a possíveis efeitos na integridade cromatínica e proteica dos espermatozoides. Estudo 1: O estudo 1 teve como objetivos determinar: falhas na condensação da cromatina, níveis de expressão e localização celular das histonas: H3.3, H2b e H4B, respectivamente, em espermatozoides de touros de baixa (BF) e alta fertilidade (AF). Os dados foram avaliados pelo teste t e correlação de Pearson (P < 0,05). Os resultados do teste do azul de anilina foram diferentes entre os grupos BF (1.73 (0.55, 0.19)) e AF (0,67 (0,17, 0,06) (P < 0,0001), os quais também foram negativamente correlacionados com a fertilidade in vivo de touros (r = -0,90; P < 0,0001). Apesar das histonas terem sido consistentemente imunodetectadas e localizadas nos espermatozoides, estas não apresentaram diferenças entre os grupos. As proteínas H3.3 e H4 apresentaram 100% de identidade e foram conservadas entre bovinos, murinos e seres humanos. Entretanto, as variantes H2B foram mais conservadas entre touros e humanos do que entre humanos e camundongos. Em conclusão, as proteínas H2B, H3.3 e H4 foram detectáveis em espermatozoides de touros e a condensação da cromatina espermática, alterada pela retenção de histonas, é relacionada com a fertilidade de touros. Estudo 2: O estudo 2 objetivou avaliar os efeitos da inclusão de 13% de FCC na dieta de carneiros Morada Nova sobre as características seminais, integridade de cromatina e perfil das proteínas espermáticas. Vinte carneiros foram divididos em dois grupos: castanha (GCA) e controle (GCO).que receberam na dieta 13% e 0% de FCC durante 90 dias, respectivamente. Os grupos foram comparados quanto ao peso vivo, circunferência escrotal, parâmetros seminais, integridade de cromatina e perfil das proteínas espermáticas aos 0, 45 e 90 dias de experimento. Os dados foram analisados pelo método GLM para medidas repetidas (P < 0,05). Aos 90 dias o GCA (69,00% (7,38; 2,33) apresentou porcentagem de espermatozoides móveis superior ao GCO (60,00% (9,43; 2,98)) (P<0.05). Não houve efeito da dieta contendo FCC sobre a integridade da cromatina. Porém, os percentuais das proteínas ODF1 e H2B foram mais elevados nos carneiros do GCA (P < 0,05). As proteínas: ODF1, GPX4, FTL e H2B foram negativamente correlacionadas com a qualidade da cromatina espermática. Em conclusão, a inclusão de FCC na dieta de carneiros não afetou negativamente a qualidade seminal.

Thesis (DTech (Biomedical Technology))--Cape Peninsula University of Technology, 2009 / Many environmental, physiological, and genetic factors have been shown to impair sperm function through oxidative damage. Oxidative stress (OS) arises as a consequence of excessive reactive oxygen species (ROS) production and/or impaired antioxidant defence mechanisms. The decline in male reproductive health generated considerable public and scientific concerns about the possible role of environmental contaminants. A better understanding of how OS affects sperm function will be beneficial as it might help in the design of new and effective treatment strategies to combat the problem of increasing male subfertility. Studies have suggested that antioxidant nutrients and/or medicines play a protective role in human health. Crude red palm oil (RPO) is known to be the richest natural plant source of antioxidants such as carotenoids, tocopherols and metalloporpheryns. The aims of this study were twofold: (i) To establish an in vivo animal model of OS by exposing rat to organic hydroperoxide such as t-butyl hydroperoxide (tbHP) and cumene hydroperoxide (cHP) through repeated intraperitoneal injections that can be used for studying these effects on testicular tissue, epididymal sperm and sperm function as well as male reproductive parameters in general. (ii) To investigate the effects of a RPO supplemented diet on male reproductive parameters and tissue in animals exposed to OS. In the first part of the study, male Wistar rats aged 10-12 weeks were randomly placed in groups and received standard rat chow (SRC) and water ad lib. Animals were injected intraperitoneally with saline (0.5 ml), t-butyl hydroperoxide (5µM, 10µM, 20µM and 40µM; 0.5 ml) or cumene hydroperoxide cHP (2.5µM, 5µM, 10µM and 20µM; 0.5 ml) over a 60 day period. In the second part, male Wistar rats aged 10-12 weeks were placed randomly in three groups and fed with SRC. Group 1 received no supplement while the food of groups 2 and 3 were supplemented with 2 mL and 4 mL RPO (in 25 gm SRC/day) respectively. Each group was further divided into 3 subgroups and injected intraperitoneally daily with either saline, 10µM cHP or 20µM tbHP respectively. This was done for 5 consecutive days per week over a 60 day period. Sperm concentrations, and motility, lipid peroxidation, superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activities as well as apoptosis were assessed.

M.Sc. (Zoology) / This investigation is divided into two sections. Section A deals with aspects of the spermatology of the tiger fish and section B deals with the cryopreservation of spermatozoa of selected freshwater fish species.

Masters of Science / Gonadotropin Releasing Hormone (GnRH) is a hypothalmic decapeptide, which regulates mammalian gonadotropin secretions by binding to specific, high affinity receptors in the pituitary. Two forms of GnRH (GnRH I and GnRH II) are expressed in the brain of human and some primates. Even though primates have been used extensively in a variety of investigations in relation to the role of GnRH in reproduction, there is no evidence of any research to investigate the direct effect of GnRH on primate sperm. / South Africa

No description available.

This study tested different concentrations of egg yolk in canine freezing extender void of glycerol, a commonly used cryoprotectant, by examining the motility and morphology throughout the freezing process: initial (baseline after extender added), post-cool (after three hours at 5"C) and post-thaw (after freezing.)  Initial values of pH, osmolarity, motility and morphology were obtained for comparison of the samples.  Spermatozoa from six normal dogs as determined by progressive linear motility > 70% and normal morphology > 60% was used. Semen was collected and pooled for five freezing trials.  The concentrations of egg yolk used in the extender were: 0%, 10%, 20%, 30% and 40%. Assessment of each sample was blinded to the treatments until all results were obtained and statistics had been analyzed. Based on this study a 20% egg yolk concentration is slightly superior to a 30% egg yolk concentration when assessing post-thaw motility, morphology and longevity and significantly superior to a 0%, 10% or 40% egg yolk concentration. The study also showed motility and normal post-cool and post-thaw sperm morphology did not always correlate.  Utilization of 0% and 10% concentrations of egg yolk has negative effects on semen quality as measured by the motility and/or morphology.  Results confirm freezing does not affect secondary sperm abnormalities, abnormalities of the tail and distal section of the middle piece, during cooling or freezing.  Primary abnormalities, abnormalities of the head and midpiece, increased in the 0% extender during cooling and all extenders during freezing. The pH of the extenders before the addition of sperm was significantly different. Once sperm was added to the extenders, there was no longer a significant difference in pH.  There was a positive correlation for both motility and normal morphology percentages post-cool and post-thaw for the extenders with similar osmolarity to the semen. / Master of Science

No description available.

Introduction: Numerous past reports have mentioned or have been totally concerned with the significance of spermatozoan agglutination following treatment with blood sera, female genital fluids and other biological and synthetic media. Since semen is a colloid (undissolved particles suspended in a suitable medium such as gas, liquid, solid), spermatozoan agglutination should be at least partially definable by physical chemical measurements and should thereby be predicted and controlled... / Ph. D.

Field evaluation of frozen-thawed stallion semen has been limited to tests such as post-thaw motility and morphology that are not only subjective but also evaluate only a small population of cells. Flow cytometry has provided a quick, repeatable, objective method of evaluating a large number of cells, including spermatozoa. Two experiments were designed to first validate the use of several flow cytometric tests on frozen-thawed stallion semen and then determine a model that may best explain variation in fertility. Comparing samples that were live and freeze-killed validated the flow cytometric tests. In experiment one, six ejaculates were collected from each of three stallions. The semen from each ejaculate was centrifuged and frozen in 0.5 ml polyvinyl chloride straws. Two straws from each ejaculate were thawed and evaluated. Semen was evaluated for post-thaw motility, morphology, mitochondrial activity using Rhodamine 123 (R123), plasma membrane integrity using propidium iodide (PI) and ethidium monoazide (EMA), and chromatin structure using the sperm chromatin structure assay (SCSA). Data was recorded as percentages for all but the SCSA for both experiment one and two. The extent of chromatin denaturation was calculated using the SCSA and the alpha-t population [at = red/(red +green) fluorescence]. From the alpha-t population, statistics were calculated such mean (Xat), standard deviation (SDat), percentage of cells outside (COMPat) the main alpha-t population and the mean green fluorescence (mean green) of the population. Results from experiment one demonstrated that all flow cytometric tests except EMA were able to distinguish between live and freeze-killed samples (p < 0.0001). Also the stallion accounted for most of the variation in samples when compared to ejaculate and straw within an ejaculate. Therefore two straws could be chosen at random from a stallion and evaluated in experiment two. In experiment two, twenty-nine stallions were evaluated using the same tests as experiment one excluding EMA. Fertility data was obtained from the 1998 or 1999 breeding season. Multiple linear regression was used to determine the best-fit model to predict overall pregnancy rate. SCSA and R123-PI assays accounted for the largest amount of variation in fertility (R² = 0.65, p < 0.0004). Within SCSA and the R123/PI assays Xat and PI staining had the highest contribution to this variation in fertility (R² = 0.11, R² = 0.47) respectively. The best-fit model for predicting fertility included the assay combination listed above and the interactions between SDat and mean green staining as well as R123 and mean green staining. Post-thaw motility and morphology did not account for significant variation in fertility (p = 0.22, p = 0.46) respectively. Based on this project post-thaw motility and morphology are poor predictors of fertility in frozen-thawed stallion semen. However, through the addition of SCSA and R123-PI to the routine evaluation of frozen-thawed stallion semen time and money may be saved in advance by identifying those stallions with poor post-thaw fertility. / Master of Science