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Embryonic stem (ES)-like cells were derived from mid-blastula stage embryos of a freshwater fish, catla Catla catla, under feeder-free condition and designated as CCES cells. The conditioned media was optimized with 10% foetal bovine serum (FBS), fish embryo extract (FEE) having 100 µg ml(-1) protein concentration, 15 ng ml(-1) basic fibroblast growth factor (bFGF) and basic media containing Leibovitz-15, DMEM with 4·5 g l(-1) glucose and Ham's F12 (LDF) in 2:1:1 ratio using a primary culture of CCES cells. Cells attached to gelatin-coated plates after 24 h of seeding and ES-like colonies were obtained at day 5 onwards. A stable cell culture was obtained after passage 10 and further maintained up to passage 44. These cells were characterized by their typical morphology, high alkaline phosphatase activity, positive expression of cell-surface antigen SSEA-1, transcription factor Oct4, germ cell marker vasa and consistent karyotype up to extended periods. The undifferentiated state was confirmed by their ability to form embryoid bodies and their differentiation potential.

Condensed sperm chromatin is a prerequisite for natural fertilization. Some reports suggested the prevalence of chromatin condensation defects in teratozoospermia cases with head anomalies; conversely, earlier studies exemplified its occurrence in morphologically normal spermatozoa too. The aim of this study was to compare the condensation defects in correlation with head anomalies among different groups of subfertile males and its impact on the rate of fertilization in assisted reproduction procedures. Ultrastructure analysis of spermatozoa through scanning electron microscopy and atomic force microscopy could facilitate an in-depth evaluation of sperm morphology. Nuclear condensation defects (%) in spermatozoa were analyzed in 666 subjects, and its effect on the rate of fertilization was analyzed in 116 IVF and 90 intracytoplasmic sperm injection cases. There was no correlation of condensation defects with head anomalies (%). Student's t-test showed no significant changes in mean values of condensation defects in abnormal semen samples in comparison with the normal group. Condensation defects were observed in normal spermatozoa too, which was negatively associated with the rate of fertilization in IVF (p < 0.01), but intracytoplasmic sperm injection outcome remained unaffected. Ultrastructure study revealed sperm morphological features in height, amplitude, and three-dimensional views in atomic force microscopy images presenting surface topography, roughness property of head, and compact arrangement of mitochondria over axoneme with height profile at nanoscale. In pathological forms, surface roughness and nuclear thickness were marked higher than the normal spermatozoa. Thus, percentage of normal spermatozoa with condensation defects could be a predictive factor for the rate of fertilization in IVF. From diverse shapes of nucleus in AFM imaging, it could be predicted that defective nuclear shaping might be impeding the activity of some proteins/ biological motors, those regulate the proper Golgi spreading over peri-nuclear theca.

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The e¡ects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization e⁄ciency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer-aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with di¡erent extenders and cryoprotective agents (CPAs). Extender-4 with an osmolality 260 mOsmol kg À 1 and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 1C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 1C. Semen frozen at À 16 1C min À 1 with1.4 M DMSO showed 70% fertilization, which was signi¢cantly higher (Po0.05) than other freezing rates. Samples thawed at 35 1C water showed a fertilization rate comparable with that of fresh semen. Computer-assisted semen analysis of fresh and frozen semen after thawing showed variations in di¡erent types of motility in spermatozoa and in their class. There was no signi¢cant di¡erence in motility before or after cryopreservation; however, signi¢cant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender-4 by addition of 1.4 M DMSO to a ¢nal cryopreservation medium (MED 2) cooled at a rate of À 16 1C min À 1 , stored in liquid nitrogen ( À 196 1C) and utilized after thawing at 35 AE 2 1C.

AIM:The aim of the following study is to find out the prevalence of abnormal spermatozoa and associated functional parameters in clinical semen samples of sub-fertile males with the tobacco chewing habit.SETTINGS AND DESIGN:Retrospective study was conducted at infertility unit of a tertiary health care center, in a period of 3 years.MATERIALS AND METHOD:Semen of 642 males were analyzed; of them 194 men (30.2%) were tobacco chewers and they were grouped according to their intensity of chewing (<10 and ≥ 10 packets/day). Counts, motility, vitality, and morphology of sperms were analyzed.RESULTS:In tobacco chewers, 66% of subjects were oligozoospermic, 85% asthenozoospermic and 28% teratozoospermic. Sperm counts (odds ratio [OR] =2.2; 95% confidence interval [CI]: 1.5-3.09), motility (OR = 3.2; 95% CI: 2.05-4.9), and normal morphology (OR = 8.4; 95% CI: 4.9-14.6) were significantly affected (P = 0.001) in tobacco chewers than the non-chewing group. Further, in comparison to the intensity of tobacco chewing, patients with the intensive practice of using ≥10 packets/day had a significant effect on sperm morphology (P = 0.003, OR = 2.7; 95% CI = 1.41-5.08) only. Structural defects in head (P = 0.001) and cytoplasmic residues (P = 0.001) were found to be positively correlated with the intensive chewing, but no significant changes were found in anomalies in mid-piece and tail.CONCLUSION:The adverse impact of tobacco chewing on semen parameters was evident even with mild chewers, but with the intensive chewing practice, phenotypes of sperms, mainly defects in the head and cytoplasmic residue were severely affected.

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This study aimed at developing a suitable cryopreservation protocol for embryonic stem (ES)-like cells of a tiny freshwater ¢sh Leopard danio (Brachydanio frankei). Embryonic stem (ES)-like cells derived from blastomeres of the early blastulae stage of the developing embryo were cultured in vitro in a medium containing Leibowitz-15 supplemented with 10% foetal bovine serum, leopard danio embryo extract, sodium bicarbonate, sodium selenite, basic ¢broblast growth factor, epidermal growth factor and leukaemia inhibitory factor. The ES-like cells showed properties similar to ES cells in other species. They were morphologically small, round to polygonal and present in patches and extensively expressed alkaline phosphatase and stage-speci¢c embryonic antigen. The toxicity and chilling sensitivity of these cells were determined using ethylene glycol (EG), propylene glycol (PG) and glycerol as cryoprotective agents at molar concentrations of 0.6, 1.0, 1.4, 1.8 and 2.0. Among them, 1.8 M EG showed 70% signi¢cant viable ES-like cells (Po0.05). The post-thawed cells retained similar properties of non-cryopreserved ES-like cells with a viability rate of 65%. Similarly, blastomeres cryopreserved following the slow cooling rate with EG and PG yielded a viability of more than 70%.SSEA-1, stage-speci¢c embryonic antigen; ES, embryonic stem; AP, alkaline phosphatase. Aquaculture Research, 2010, 41, 579^589 Cryopreservation of embryonic stem like cells of B. frankei P Routray et al.Figure 9 Embryonic stem (ES) cell colonies under confocal microscopy labelled with 4 0 ,6 0 -diamino-2-phenylidone,dichloride DNA £uorescent stain (DAPI) and immunostained with stage-speci¢c embryonic antigen (SSEA-1). (A) DAPI, (B) SSEA-1 and (C) DAPI/SSEA-1 (merged view). Images are in 2D view. Aquaculture Research, 2010, 41, 579^589 Cryopreservation of embryonic stem like cells of B. frankei P Routray et al.