The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results.

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.

Comprehensive functional analysis reveals that acrosome integrity and viability are key variables distinguishing artificial insemination bulls of varying fertility.

In vitro methods of assessing bull semen quality in artificial insemination (AI) centers are unable to consistently detect individuals of lower fertility, and attempts to reliably predict bull fertility are still ongoing. This highlights the need to identify robust biomarkers that can be readily measured in a practical setting and used to improve current predictions of bull fertility. In this study, we comprehensively analyzed a range of functional, morphological, and intracellular attributes in cryopreserved spermatozoa from a selected cohort of Holstein Friesian AI bulls classified as having either high or low fertility (n = 10 of each fertility phenotype; difference of 11.4% in adjusted pregnancy rate between groups). Here, spermatozoa were assessed for motility and kinematic parameters, morphology, acrosome integrity, plasma membrane lipid packing, viability (or membrane integrity), superoxide production, and DNA integrity. In addition, spermatozoa were used for in vitro fertilization to evaluate their capacity for fertilization and successful embryo development. The information collected from these assessments was then used to phenotypically profile the 2 groups of bulls of divergent fertility status as well as to develop a model to predict bull fertility. According to the results, acrosome integrity and viability were the only sperm attributes that were significantly different between high- and low-fertility bulls. Interestingly, although spermatozoa from low-fertility bulls, on average, had reduced viability and acrosome integrity, this response varied considerably from bull to bull. Principal component analysis revealed a sperm phenotypic profile that represented a high proportion of ejaculates from low-fertility bulls. This was constructed based on the collective influence of several sperm attributes, including the presence of cytoplasmic droplets and superoxide production. Finally, using the combined results as a basis for modeling, we developed a linear model that was able to explain 47% of the variation in bull field fertility in addition to a logistic predictive model that had a 90% chance of distinguishing between fertility groups. Taken together, we conclude that viability and acrosome integrity could serve as fertility biomarkers in the field and, when used alongside other sperm attributes, may be useful in detecting low-fertility bulls. However, the variable nature of low-fertility bulls suggests that additional, in-depth characterization of spermatozoa at a molecular level is required to further understand the etiology of low fertility in dairy bulls.

Genetic parameters of semen quality traits and genetic correlations with service sire nonreturn rate in Nordic Holstein bulls.

Despite the importance of the quality of semen used in artificial insemination to the reproductive success of dairy herds, few studies have estimated the extent of genetic variability in semen quality traits. Even fewer studies have quantified the correlation between semen quality traits and male fertility. In this study, records of 100,058 ejaculates collected from 2,885 Nordic Holstein bulls were used to estimate genetic parameters for semen quality traits, including pre- and postcryopreservation semen concentration, sperm motility and viability, ejaculate volume, and number of doses per ejaculate. Additionally, summary data on nonreturn rate (NRR) obtained from insemination of some of the bulls (n = 2,142) to cows in different parities (heifers and parities 1-3 or more) were used to estimate correlations between the semen quality traits and service sire NRR. In the study, low to moderate heritability (0.06-0.45) was estimated for semen quality traits, indicating the possibility of improving these traits through selective breeding. The study also showed moderate to high genetic and phenotypic correlations between service sire NRR and some of the semen quality traits, including sperm viability pre- and postcryopreservation, motility postcryopreservation, and sperm concentration precryopreservation, indicating the predictive values of these traits for service sire NRR. The positive moderate to high genetic correlations between semen quality and service sire NRR traits also indicated that selection for semen quality traits might be favorable for improving service sire NRR.

Biological aspects of altered bone remodeling in multiple myeloma and possibilities of pharmacological intervention.

Multiple myeloma is a fatal B cell neoplasm often resulting in focal and in some cases more diffuse destruction of bone. The bone destruction is a result of increased activity of bone resorbing cells--multinucleated osteoclasts emerging through of multiple fusions. In multiple myeloma, clonally expanding cancer cells provide a stimulatory signal for osteoclast recruitment, differentiation and excessive bone resorption. The stimulatory actions of myeloma cells are believed to be mediated via the production of cytokines and local factors or by modulating bone microenvironment in order to stimulate osteoclastic bone resorption. However, our recent study revealed potentially a novel and more intimate contribution of myeloma cells to the bone destruction. Our analysis of the bone biopsies from myeloma patients showed fully integrated malignant nuclei inside osteoclasts, which were transcriptionally active. As a result, about 30% of the osteoclasts in the bone marrow biopsies from myeloma patients were in fact osteoclast-myeloma cell hybrids. As the functional relevance of this novel cell type remained uncertain, the aim of my PhD study became to 1) strengthen the evidence of the existence of hybrid cells, 2) elucidate the functional differences between hybrid cells and non-hybrid OCs and 3) relate these findings to the pathogenesis of osteolytic disease in multiple myeloma. To this end, I developed a culture model of osteoclast-myeloma cell fusion between (pre)osteoclasts already committed to fuse and myeloma cells selected for adherence. The model was applied for testing of the bone resorptive properties of hybrid cells identified by labelling with green fluorescence. When comparing the highly fluorescent and non-fluorescent OCs on bone slices, it seemed that the frequency of highly fluorescent osteoclasts actively resorbing bone was increased as compared with non-fluorescent osteoclasts. This was assessed in two independent ways. Furthermore, these fluorescent osteoclasts appear to resorb deeper compared to non-fluorescent osteoclasts. The preliminary data that need to be confirmed suggest that formation of hybrid cells by fusion of myeloma cells with osteoclasts may result in reprogramming of the osteoclasts and contribute to the more aggressive bone resorption by osteoclasts as it is typically seen in myeloma patients. Another aspect of multiple myeloma and associated bone disease is the unmet need for novel and more efficient therapeutic regiments. Resveratrol (trans-3, 4', 5-trihydroxystilbene; RSV) is a natural compound shown to target the key players of myeloma bone disease: bone resorbing osteoclasts, bone forming osteoblasts and myeloma cells. Our in vitro study on RSV showed that it possessed this ideal triad of properties appearing and thus might be of interest as a potential drug for the treatment of multiple myeloma. RSV suppresses the growth and survival of myeloma cells, inhibits osteoclasts and stimulates the formation of osteoblasts. However, the need for high concentrations combined with low biological availability after oral administration and risk of important side effects stimulated a search for RSV derivates with the same spectrum of actions but safer and with better bioavailability. As the other task of my PhD, I screened structurally modified RSV analogues in cultures of myeloma cells, osteoblasts and osteoclasts. Compared to resveratrol, some analogues showed an up to 5,000-times increased potency to inhibit osteoclast differentiation and could still promote osteoblast maturation but they did not antagonize myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but effect on bone loss could not be detected. During my PhD, I also participated in the studies of the effect of the proteasome inhibitor - bortezomib on osteoclasts conducted at the department. Based on its potent activity in multiple myeloma, bortezomib was accepted as a front-line treatment of myeloma patients by EMEA for the European Union. In our study we assessed the effect of bortezomib on osteoclasts in cultures under the conditions that mimic the pulse-treatment regime used for myeloma patients. The pulse administration of bortezomib significantly inhibited OC activity and, moreover, significantly but transiently reduced levels of two bone resorption markers measured in serum of treated myeloma patients. In MM the clonal expansion of malignant plasma cells results in the unbalanced bone remodelling, therefore it is essential to understand the molecular mechanisms governing the actions of osteoclasts and osteoblasts. During my PhD, I was involved in the investigations of mesenchymal stem cells over-expressing delta like protein - 1(Dlk-1) previously shown to inhibit the differentiation of mesenchymal stem cells (MSC) into osteoblasts. In results, the over-expression of Dlk-1 evoked pro-inflammatory phenotype in MSC suggesting the involvement of Dlk-1 in the immune response.