Quality parameters and fertility of ram semen cryopreserved in egg yolk and soybean lecithin supplemented extenders.

The aim of the study was to investigate the effect of soybean lecithin as a substitute for egg yolk in milk and tris based extenders in ram semen cryopreservation. Twenty ejaculates were col- lected from four healthy, mature Wrzosówka rams (2-3 years of age). Each ejaculate was divided into four equal aliquots and diluted with four different extenders: 1) milk extender containing 5% egg yolk, 2) milk extender containing 1.5% soybean lecithin, 3) tris extender containing 20% egg yolk, 4) tris extender containing 1.5% soybean lecithin. Extended semen was loaded into 0.25 ml French straws, cooled and frozen in liquid nitrogen vapor. Total motility, curvilinear velocity, plasma membrane integrity and fertilizing ability of sperm were assessed after thawing. Total mo- tility was lower (p⟨0.05) in tris-soybean lecithin extender when compared to other extenders. Curvilinear velocity was higher (p⟨0.05) for spermatozoa cryopreserved in milk-soybean lecithin extender compared to other extenders tested. For the percentage of live sperm no significant difference was observed between extenders. The lambing rate were higher (not statistically signifi- cant) in ewes inseminated with semen doses frozen in milk-soybean lecithin extender (42.9%) than in the tris-egg yolk extender (16.7%). In conclusion, replacing the egg yolk with soybean lecithin was effective in milk but not in tris extender.

The medium- and long-term outcome of total shoulder arthroplasty for primary glenohumeral osteoarthritis in middle-aged patients.

AIMS: Promising medium-term results from total shoulder arthroplasty (TSA) have been reported for the treatment of primary osteoarthritis in young and middle-aged patients. The aim of this study was to evaluate the long-term functional and radiological outcome of TSA in the middle-aged patient. PATIENTS AND METHODS: The data of all patients from the previous medium-term study were available. At a mean follow-up of 13 years (8 to 17), we reviewed 21 patients (12 men, nine women, 21 shoulders) with a mean age of 55 years (37 to 60). The Constant-Murley score (CS) with its subgroups and subjective satisfaction were measured. Radiological signs of implant loosening were analysed. RESULTS: Two shoulders (two patients) were revised and in two shoulders of two different patients, revision surgery was recommended. The mean CS increased from 23.3 (10 to 45) pre-operatively to 56.5 (26 to 81; p < 0.0001), but with a decrease in CS from 62.8 (38 to 93) to 56.5 (26 to 81) between medium- and long-term follow-up (p = 0.01). Without revision surgery, 18 patients (95%) rated their result as good or very good. The mean radiolucent line score for the glenoid components increased from 1.8 (0 to 6) to 8.2 (2 to 18) between medium- and long-term follow-up (p < 0.001). CONCLUSION: TSA in young and middle-aged patients leads to improvement in clinical function and a relatively high satisfaction rate. However, clinical or radiological glenoid loosening worsens in the long term. Further studies are needed to optimise the treatment options in this patient population. Cite this article: Bone Joint J 2017;99-B:939-43.

Factors influencing quality of anticoagulation control and warfarin dosage in patients after aortic valve replacement within the 3 months of follow up.

Warfarin dosage estimation using the pharmacogenetic algorithms has been shown to improve the quality of anticoagulation control in patients with atrial fibrillation. We sought to assess the genetic, demographic and clinical factors that determine the quality of anticoagulation in patients following aortic valve replacement (AVR). We studied 200 consecutive patients (130 men) aged 63 ± 12.3 years, undergoing AVR, in whom warfarin dose was established using a pharmacogenetic algorithm. The quality of anticoagulation within the first 3 months since surgery was expressed as the time of international normalized ratio (INR) in the therapeutic range (TTR). The median TTR in the entire cohort was 59.6% (interquartile range, 38.7 - 82.7). Ninety-nine (49.5%) patients with TTR ≥ 60% did not differ from those with poor anticoagulation control (TTR < 60%) with regard to demographic and cardiovascular risk factors. Coronary artery disease (n = 84, 42%) and previous stroke (n = 5, 2.5%) predicted higher TTR, while possession of CYP2C9*2 variant allele (n = 49, 25%) was associated with lower TTR (P = 0.01). In turn, VKORC1 c.-1639A, CYP2C9*2 and *3 variants were independently associated with actual warfarin dose (P < 0.0001). In AVR patients better anticoagulation control is observed in patients with coronary artery disease and history of stroke, which might result in part from previous lifestyle modification and therapy. Possession of CYP2C9*2 and/or CYP2C9*3 allele variants is associated with lower TTR values and warfarin dose variations in AVR patients, the latter affected also by VKORC1 c.-1693G>A polymorphism.

Sperm DNA damage-the effect of stress and everyday life factors.

The clinical significance of sperm DNA damage lies in its association with natural conception rates and also might have a serious consequence on developmental outcome of the newborn. The aim of the present study is to determine whether stress and everyday life factors are associated with sperm DNA damage in adult men. The study population consisted of 286 men who attended the infertility clinic for diagnostic purposes and who had normal semen concentration of 20-300 m ml(-1) or with slight oligozoospermia (semen concentration of 15-20 m ml(-1)) (WHO, 1999). Participants were interviewed and provided a semen sample. The sperm chromatin structure assay was assessed using flow cytometry. In the present study, we found evidence for a relationship between sperm DNA damage parameters and everyday life factors. High and medium level of occupational stress and age increase DNA fragmentation index (P=0.03, P=0.004 and P=0.03, respectively). Other lifestyle factors that were positively associated with percentage of immature sperms (high DNA stainability index) included: obesity and cell phone use for more than 10 years (P=0.02 and P=0.04, respectively). Our findings indicate that stress and lifestyle factor may affect sperm DNA damage. Data from the present study showed a significant effect of age, obesity, mobile phone radiation and occupational stress on sperm DNA damage. As DNA fragmentation represents an extremely important parameter indicative of infertility and potential outcome of assisted reproduction treatment, and most of the lifestyle factors are easily modifiable, the information about factors that may affect DNA damage are important.

Occupational, life stress and family functioning: does it affect semen quality?

BACKGROUND AND AIMS: Although psychological stress has been implicated as a cause of idiopathic infertility in both men and women, it has received little scientific attention among males as compared to females. The aim of the study was to examine the association between occupational, life stress, family functioning and semen quality. METHODS AND RESULTS: The study population consisted of 327 men who were attending an infertility clinic for diagnostic purposes. Psychological stress was assessed based on two questionnaires: The Subjective Work Characteristics Questionnaire and the Perceived Stress Scale. The level of satisfaction with family functioning and support was evaluated by means of the APGAR Family Scale. The findings suggest that, on the one hand, exposure to occupational stressors can be negatively associated with semen quality (there was a positive association between stress and the percentage of sperm with DNA damage (p = 0.03) and atypical sperm (p = 0.05)); on the other hand, there was no correlation between the level of life stress and semen quality indicators. Negative associations were found between satisfaction with family functioning and the percentage of motile sperm cells (p = 0.02), VAP (p = 0.05), VSL (p = 0.05) and VCL (p = 0.04). CONCLUSION: The study indicates that occupational stress can affect male semen quality; however, due to limited data on this issue, the obtained results should be confirmed in longitudinal studies.

Assessment of plasma membrane and chromatin structure of sperm from transgenic and non-transgenic boars.

The aim of this study was to determine the apoptotic changes and chromatin damage in non-transgenic and transgenic boars carrying the human alpha1,2-fucosyltransferase gene. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars both crossbreds of Polish Landrace and Large White. Two fluorescence methods were employed to measure apoptosis: an assay to assess the early changes in sperm membrane integrity using fluorophore YO-PRO-1 and an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V. The chromatin damage was assessed based on the sperm chromatin structure assay method. No significant differences in the proportion of all detected subpopulations of spermatozoa were found between TG and NTG boars. Similarly, the analysis of the chromatin structure revealed no statistical differences in the sperm chromatin damage between TG and NTG boars. In conclusion, the presence of the human alpha1,2-fucosyltransferase gene in the genome of TG boars did not cause any spermatogenesis process disturbances leading to the increased production of apoptotic spermatozoa. Moreover, the low level of sperm with damaged chromatin in TG boars confirms the high stability of the spermatogenesis process in the TG boars analyzed.

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

Flow cytometric detection of apoptotic bone marrow cells with fractional DNA content after application of WR-2721, cyclophosphamide, cisplatin, and exposure of mice to gamma rays.

Little is known about the mechanisms of apoptosis triggered in normal cells of the haemopoietic system by the aminothiol WR-2721 (Amifostine), chemotherapeutic drugs, and ionizing radiation; thus, the present study was undertaken to evaluate the effects of WR-2721, cyclophosphamide (CP), cisplatin (CDDP), and 60Co gamma rays on induction of apoptotic DNA degradation in bone marrow cells. Adult male Swiss mice were treated with WR-2721 (400 mg/kg b.wt.), CP (200 mg/kg b.wt.), and CDDP (10 mg/kg b.wt.), and exposed to 6 Gy 60Co gamma rays. Alterations in the number of apoptotic cells with fractional DNA content and also the cell cycle position of the non-apoptotic cells were determined in the bone marrow at 7 and 24 hours after treatment of mice with these agents, using flow cytometric assay of the controlled extraction of low-MW DNA from apoptotic cells. The chemotherapeutic drugs CP and CDDP and 60Co gamma rays triggered apoptosis and affected the cell cycle position of the non-apoptotic cells in the mouse bone marrow. The pretreatment of mice with WR-2721 resulted in the modulatory action of the aminothiol on induction of apoptotic cell death and changes in the cell cycle distribution of the non-apoptotic cells caused by the DNA-damaging agents. The patterns of changes in the frequency of apoptotic cells and the cell cycle position of the non-apoptotic cells, observed in the bone marrow, were dependent on the agent(s) applied and the time interval after application of the drug(s) and exposure of mice to gamma rays. Understanding of the mechanisms responsible for triggering of apoptotic cell death and disturbing of the cell cycle by the DNA-damaging agents, and modulation of the apoptotic and cell cycle pathways by the aminothiol WR-2721, can lead to more effective therapy and chemo- and radio-protection of normal cells.

Effect of rabbit age on sperm chromatin structure.

The aim of this study was to determine the relationship between the age of male rabbits and the sperm chromatin structure. The studies involved the semen of New Zealand White rabbits between 5 and 28 months of age. A flow cytometry and sperm chromatin structure assay (SCSA) method was used to determine chromatin structure. The results of cytometric chromatin structure assay suggested a relatively high stability of sperm chromatin in the rabbit. Between 6 and 16 months of age, the mean percentage of sperm with damaged chromatin was the lowest and ranged from 1.7 to 2.4%. Decreased sperm chromatin stability was found in ejaculates taken from male rabbits less than 5 months and more than 20 months of age.

Sperm chromatin structure assay of bulls qualified for artificial insemination.

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.

Sperm chromatin integrity of bucks transgenic for the WAP bGH gene.

The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene. The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.