This study was carried out primarily to evaluate the cryo-tolerance of equine semen from four stallions through assessing the spermatozoa motion characteristics with Computer-Assisted Sperm Analysis (CASA). Four stallions were collected during the breeding season (summer). For each ejaculate, fresh and cryopreserved samples were taken for sperm motility characteristics evaluation. Data analysis demonstrated that sperm cells of stallions were significantly affected by (P<0.05) cryodamage. Stallion (A) was cryotolerant, and was classified as a good freezer, whereas stallion (D) was not and classified as a poor freezer regardless of the concentration of sperm. In addition, a concentration of 0.4 x 109 sperm cells/ml had higher percentages of rapid sperm and velocity parameters (P<0.05) compared to 0.8 x 109 sperm/ml. Further research is necessary to identify potential biomarkers for good and poor freezer stallions.

No / Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients. METHODS: We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen¿1q12 region. RESULTS: There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen¿1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy. CONCLUSIONS: Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.

The world is currently facing an amphibian extinction crisis and wild salamanders and newts (Order: Caudata) are the most disadvantaged with 52% of species threatened. Captive breeding programs are been established to act as assurance colonies, but they are overwhelmingly failing due to the lack of environmental cues to stimulate reproduction and other factors. Therefore, assisted reproductive technologies (ART) are being developed to overcome these barriers. This project expands upon the limited existing information on caudate ART through studies in the model species, Ambystoma tigrinum, for application to threatened species. Specific objectives included the characterization of motility longevity in artificially collected sperm samples, investigation of cryoprotective agents and freeze rates in sperm cryopreservation, and application of ART in three at-risk newt species. This study informs needed future advances in caudate ART protocols, especially sperm cryopreservation, and demonstrates their transferability to threatened species across families.

Many factors influence male fertility, and conventional fertility evaluations are not able to reliably identify sub-fertile animals. The overall goal of this work was to explore the expression of phosphatidylserine (PS) on bovine sperm and investigate what factors may impact it, as previous research demonstrated that phosphatidylserine (PS) plays a role in murine fertilization. Despite conventionally being an apoptotic marker, it is present on viable and fertilization-competent murine sperm, however, less is known of the possible role of PS in bovine fertilization. In experiment 1, viable bovine sperm cells expressing PS were identified and PS levels in fresh and frozen semen were compared. Phosphatidylserine levels in frozen samples were significantly less than in fresh samples. We conclude that the cryopreservation process has an impact on PS expression in sperm by altering the proportion of sperm cells which are capable of fertilization. Experiment 2 examined PS levels in bulls with varying fertility levels based on sire conception rate (SCR). There was no difference in PS levels between high and low fertility bulls. There was a significant difference in PS levels of uncapacitated samples and those capacitated for one hour. These results warrant further investigation into the role of phosphatidylserine in bovine fertilization. / Master of Science / Improving the fertility of cattle is incredibly important to meet ever-growing consumer demands for animal protein. Researchers and producers can utilize a variety of reproductive technologies to improve their herds' reproductive efficiency. Phosphatidylserine (PS) is a glycerophospholipid which makes up a part of all cellular plasma membranes. Typically, it is used as a marker for cell death or apoptosis, however, some cells expose it on their surface temporarily while still viable, including sperm. Phosphatidylserine was found to be exposed on sperm from mice that were still viable and able to fertilize oocytes. Following that, the expression of PS in bovine sperm was investigated. Using bulls as a model, fresh semen was collected and analyzed for the level of PS expression then frozen and reanalyzed. We saw that there was a significant decrease in the level of PS expression in sperm that had been previously frozen, possibly due to damage to their membranes during the freezing process. Frozen semen from beef bulls with either high or low fertility was also analyzed. No difference was observed between bulls with varying levels of fertility. Addressing fertility issues in bulls is a complicated and multi-faceted issue which requires the use of many technologies and fertility markers. Further developing the knowledge of PS exposure in bulls and its relation to fertility and fertilization is worthwhile to attempt to improve the reproductive efficiency of cattle herds.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Tankwa goats have been free-ranging in the Tankwa Karoo National Park in the Northern Cape for more than 80 years. A genetic study concluded that these feral goats are a unique genetic resource compared to other goat breeds in South Africa and should be conserved as a distinctive population. A decision taken by the South African National Parks who is the managing authority in the park, was to remove all alien species, which included the Tankwa goats. Several animals were translocated to the Carnarvon Research Station by the Northern Cape Department of Agriculture, Land Reform & Rural Development, where the Tankwa goat population has grown to a few hundred individuals. Currently, sound scientific decisions including the application of a wide range of technologies and approaches are applied to conserve the population, such as an informed understanding of the reproductive biology of these goats. The aim of this study was to define sperm quality in Tankwa goats using various macroscopic and microscopic evaluation techniques.

Sexual promiscuity, whereby females copulate with more than one male, is a quite common phenomenon in socially monogamous birds, and especially in songbirds. This behavior is assumed to influence the evolution of various anatomical traits associated with male ability to outcompete other males in the process of sperm competition. High promiscuity is, in a multi- species comparison, associated with higher relative testis mass, but may also affect sperm phenotypes and other male phenotypes. Sperm morphology is clearly differentiated across avian species and some studies suggest that stabilizing post-copulatory selection on sperm length is responsible for a clear association of between male variation in sperm length and levels of promiscuity. However, the association between other phenotypic traits and promiscuity remains less clear. In this study, I focus on sperm characteristics in relation to the estimated levels of promiscuity in songbirds of tropical and temperate zone climates, and across an altitudinal gradient in the tropics. I found that the coefficient of variation in sperm length, both between males (CVbm) and within males (CVwm), was indeed a good index of promiscuity. I also reveal the size of cloacal protuberance as an anatomical trait intimately associated with the level of sperm...

Artificial insemination (AI) with sex-sorted bottlenose dolphin spermatozoa provides female calves for obtaining more cohesive social groups and optimum genetic management of captive populations. However, distance of animals to the sorting facility represents a limit to the procedure. Although one bottlenose dolphin calf has been born using spermatozoa from frozen-thawed, sorted and recryopreserved spermatozoa, critical evaluation of the steps involved in this process is required to maximize its efficiency for future AIs and expansion of the technology to other species. Two experiments were designed to determine the efficiency of the sorting process and the quality of frozen-thawed bottlenose dolphin spermatozoa during sorting and recryopreservation. In experiment 1, the effect of two washing media (with and without 4 percent egg yolk, v/v) following density gradient centrifugation (DGC) on sperm recovery rate and in vitro characteristics of cryopreserved spermatozoa was examined. In experiment 2, cryopreserved semen was used to compare the effects of two recryopreservation methods (conventional straw freezing and directional freezing) on in vitro sperm characteristics of control (non-sorted) and sorted spermatozoa. Egg yolk supplementation of the washing medium in experiment 1 did not influence (P > 0.05) the sperm recovery rate, however, sperm motility parameters and viability were improved (P < 0.05). For Experiment 2, motility parameters and viability were influenced by stage of sex-sorting process, sperm type (non-sorted and sorted) and freezing method (P < 0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P < 0.05) motility parameters over the 24 h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of nonsorted spermatozoa, as assessed by the SCSA, remained unchanged throughout the process. However, a possible interaction between Hoechst 33342 and acridine orange was observed in sorted samples. After recryopreservation, viability of sorted spermatozoa was higher (P < 0.05) than that of non-sorted spermatozoa across all time points. The percentages of viable spermatozoa determined by light (eosin-nigrosin) and fluorescence microscopy (propidium iodide) techniques were correlated (R^2=0.79, P < 0.001). Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Assessment of sperm viability is an important component for evaluating stallion sperm quality. The flow cytometer is considered the standard in the assessment of sperm plasma membrane integrity (viability); however, this instrument is costly to purchase and use, and it requires an experienced technician to operate it. The growing practice of assisted reproductive technologies (ARTs) in the equine industry has increased the need for an accurate but cost-effective means of determining sperm membrane viability. The NucleoCounter® SP-100TM is reported to be an accurate, easy-to-perform, and an efficient stallion-side test for sperm membrane viability. To evaluate usefulness of the NucleoCounter® SP-100TM for assessing sperm membrane integrity, neat semen was subjected to four treatments with varying seminal plasma volumes and sperm concentrations. Sperm membrane viability was assessed immediately, and at 24 and 48 hours after cooled-storage using three methods: 1) flow cytometer utilizing the fluorescent vital stains SYBR-14/propidium iodide; 2) NucleoCounter® SP-100TM utilizing the fluorescent vital stain propidium iodide; 3) eosin-nigrosin stained air-dried smears of semen. Sperm motion characteristics (total and progressive motility) were assessed using a computer assisted sperm motion analyzer (CASMA) and results were compared to sperm membrane viability to determine the relationship between sperm membrane viability and motion characteristics. Results were compared statistically by: 1) analysis of variance (ANOVA); 2) linear regression analysis; 3) coefficient of variation on untransformed and transformed data (arc sine square root); and 3) the agreement of two instruments, by means of which the difference between measurements of the two instruments were plotted on the y-axis and the average of measurements from the two instruments were plotted on the x-axis. Results obtained with the NucleoCounter® SP-100TM agreed best with the flow cytometer, and least with eosin-nigrosin staining. Coefficients of variation were ≤ 5% for the three methods (transformed data). Sperm motion characteristics and sperm viability were similar among treatments at Time 0. At Times 24 and 48, sperm motion characteristics decreased at a more significant rate compared to viability in the treatments containing ≥ 50% seminal plasma, whereas differences among treatments were only significant at seminal plasma concentrations above 50% when only sperm membrane viability was considered.

Introduction. Sperm parameter analysis is the standard method of male fecundity testing; however, minimal evidence supports associations between individual sperm parameters and reproductive outcomes. Our previous work shows strong associations between sperm mitochondrial DNA copy number (mtDNAcn) and time-to-pregnancy (TTP) in general populations, and between mtDNAcn and fertilization outcomes in clinical populations. Thus it is possible for sperm mtDNA biomarkers to act as summary measures of semen quality. In this study, we developed a sperm quality index (SQI) from semen parameters and compared its ability to measure fecundity to sperm mtDNAcn. Methods. We received 384 semen samples from the Longitudinal Investigation of Fertility in the Environment Study. Sperm mtDNAcn and mtDNA deletions (mtDNAdel) were quantified using a triplex probe-based qPCR method. The SQI was developed by ranking and summing select sperm parameters within the study population, including sperm concentration, sperm count, normal morphology, high DNA stainability, and DNA fragmentation to create a cumulative index. Discrete-time proportional hazards models were used to determine fecundability odds ratios (FOR), indicating associations between mtDNAcn, SQI, and TTP. Receiver operating characteristic (ROC) analyses determined the validity of the SQI and mtDNAcn as predictors of pregnancy within 12 months. Results. The SQI was highly associated with mtDNAcn, both continuously (Spearman Rho: -0.487; p-value: <0.001) and in deciles (ANOVA p-value: <0.001). The SQI (FOR: 1.25; 95% confidence interval (CI): 1.09, 1.43) and mtDNAcn (FOR: 0.754; 95% CI: 0.657, 0.866) performed similarly in discrete-time survival models and indicated a significant decrease and increase in TTP, respectively. MtDNAcn more effectively predicted pregnancy within 12 months (AUC: 0.703; 95% CI: 0.617, 0.789) than the SQI (AUC: 0.642; 95% CI: 0.531, 0.753). With multiple predictors, mtDNAcn outperformed summary models, with addition of the SQI and percent normal morphology minimally increasing model efficacy (AUC: 0.718, 95% CI: 0.617, 0.819). Conclusion. The association between the SQI and mtDNAcn suggest that mtDNAcn may serve as a summary biomarker for overall sperm quality. Neither individual nor summed sperm parameters are useful indicators of couple fecundity and reproductive outcomes compared to mtDNAcn. These results suggest that mtDNAcn has potential for use as a biomarker of fecundity.