Cryopreservation alters buffalo sperm kinematics and mitochondrial parameters, acrosome and intra-cellular calcium status.

BACKGROUND: Little is known about the effects of different seasons on the cryopreservation success of buffalo sperm in terms of kinematics and sperm functional parameters. OBJECTIVE: To study the effect of three seasons (winter, comfort and summer) and cryopreservation on sperm kinematics and functional properties in buffalo bulls. MATERIALS AND METHODS: Semen ejaculates (n = 90) collected during three seasons i.e. winter (n = 30), comfort (n = 30), summer (n = 30) were evaluated for sperm kinematics and functional properties. RESULTS: Sperm kinematics with respect to total (TM), progressive (PM) and rapid motility (RM) was higher (P < 0.05) in fresh sperm compared to sperm that had been frozen-thawed. Similarly, all kinematic parameters [viz. average path velocity (VAP), straight linear velocity (VSL), curvilinear velocity (VCL), beats cross frequency (BCF), lateral head displacement (ALH), linearity (LIN) and straightness (STR)] were higher (P < 0.01) at the fresh stage. With respect to season, frozen-thawed semen TM (57.67 ± 115 %), PM (50.2 ± 1.15 %) and RM (51.6 ± 1.19 %) were higher (P < 0.01) when using sperm collected during winter. The stage of cryopreservation (i.e., equilibration and freeze-thawing) also showed significant effects (P < 0.01) on mitochondrial superoxide positive status (MSPS), mitochondrial membrane potential (MMP), acrosome status and intra-cellular calcium status. CONCLUSION: The season of sperm collection and cryopreservation have significant effects on buffalo bull sperm kinematics and functional properties. Doi.org/10.54680/fr24410110612.

Assessing bone density on MRI: comparison between routine MRI sequences and DEXA scans.

PURPOSE: Magnetic Resonance Imaging (MRI) is frequently utilised to aid in the comprehensive assessment of back pain, while dual-energy x-ray absorptiometry (DEXA) is the gold standard test for the assessment of bone density. Assessing bone density on MRI could reduce costs and avoid exposing patients to ionising radiation. The aim of this paper is to investigate whether the relative signal intensity of vertebral bodies compared to other structures can detect osteoporosis on MRI. METHODS: 100 patients that had undergone both a lumbar spine MRI and a DEXA scan were identified. The T1 and T2 signal intensity of L1-L4 vertebral bodies (VB), cerebro-spinal fluid (CSF), and psoas muscle were measured within a 1-cm2 region of interest (ROI), and the signal intensity ratios were calculated. The ratios were stratified as normal, osteopenic, or osteoporotic based on DEXA T-scores. RESULTS: The T1 VB /T1 CSF ratio was significantly higher in the osteoporotic group than the normal and osteopenic groups (p < 0.001). The T1 VB /T1 CSF ratio had excellent discrimination (AUC = 0.841) for the presence of osteoporosis. The Pearson correlation coefficient between the DEXA T-score and the T1 VB/T1 CSF ratio was -0.474 (p < 0.001). The intra-observer (ICC = 0.910, 95% CI = 0.757-0.966) and inter-observer reliability (ICC = 0.927, 95% CI = 0.824-0.970) were excellent. In our cohort, a T1 VB / T1 CSF ratio of greater than 4 is 66.7% sensitive but 90.0% specific for the presence of osteoporosis. CONCLUSION: A high T1 VB/T1 CSF ratio suggests osteoporosis on MRI. Prospective validation is needed to confirm these findings.

Genetic analysis for semen quality traits in buffalo bulls.

This study was attempted to estimate the genetic parameters of semen quality traits in buffalo bulls. The study data consisted of 10975 ejaculates from 45 Murrah buffalo bulls (aged 24-72 months) used for breeding program during year 2010 to 2020. Semen quality traits (ejaculate volume, concentration of sperm, mass activity, initial and post-thaw motility, number of sperms per ejaculate, motile sperm number and discard rates) were studied. It was observed that average ejaculate volume was 2.82 ± 1.45 mL with mean concentration of 1040.12 ± 523.26 million/mL. Higher heritability was observed for number of sperms per ejaculate, number of motile sperm and sperm concentration. Significant phenotypic correlation was obtained between volume and number of sperms per ejaculate as well as volume and number of motile sperms. Likewise, significant phenotypic correlation was evident between sperm concentration with sperm number per ejaculate. Highest phenotypic correlation was obtained between sperm count per ejaculate and motile sperm count. Estimated genetic trends showed significant change in volume and motile sperm number. In conclusion, this study ascertains that genetic parameters of semen traits can be considered during the selection of buffalo bulls in breeding program.

The impact of blood conservation techniques on transfusion requirements for posterior adolescent idiopathic scoliosis corrections: do we need a routine cross-match for the operation?

PURPOSE: Various strategies are utilised to reduce blood loss and allogenic blood transfusion for posterior instrumented correction of Adolescent Idiopathic Scoliosis (AIS). The aim of this study was to evaluate post-operative blood transfusion requirements to determine whether routine cross matching of blood is essential. METHODS: This is a prospective case series of 84 patients who underwent posterior correction of AIS between September 2016 and March 2018. We reviewed demographic, operative, radiological data and transfusion requirements. Results of transfusion requirements in 44 patients who underwent Ponte osteotomies (F:M = 36:8; mean age 14.8 years) were compared with 40 patients (F:M = 9:31; mean age 14.4 years) who did not and provided the control group. A transfusion trigger of 80 mg/dl with clinical caveats was utilised. Cross matching and procurement costs of allogenic blood/unit were ascertained. RESULTS: Five patients required postoperative blood transfusion on days 2 or 3. Anaesthetic time (p = 0.0003) and preoperative Cobb angle (p = 0.0166) were significant variables between both groups and post-operative Hb (p = 0.0084) and number of levels fused (p = 0.0312) being significant in patients requiring transfusion. Unutilised units on the day of the operation incurred £30,030 (£380/patient or £154/unit) in operational costs. CONCLUSION: Our audit demonstrates that transfusion on the day of the operation was not required. We recommend that routine crossmatching is not essential for primary posterior correction for AIS with blood conservation techniques. Blood grouping with availability of urgent blood is sufficient at the onset of operation. This has financial implications and cost savings. LEVELS OF EVIDENCE: III.

Effects of different concentration and combinations of cryoprotectants on sperm quality, functional integrity in three Indian horse breeds.

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4-6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.

[Corneal cell therapy-an overview]. / Korneale Zelltherapie ­ Eine Übersicht.

In recent years, the cultivation and expansion of primary corneal cells has made significant progress. The transplantation of cultured limbal epithelial cells represents a successful and established treatment of the ocular surface. Cultivated corneal endothelial cells are undergoing a clinical trial in Japan. Stromal keratocytes can now be expanded in vitro. A wide range of stem cell sources is being tested in vitro and animal models for their possible application in corneal cell therapy. This article gives an overview of recent advancements and prevailing limitations for the use of different cell sources in the therapy of corneal disease.

Risk factors for contact lens-related microbial keratitis in Singapore.

PURPOSE: To investigate independent risk factors for contact lens-related microbial keratitis in Singapore and estimate their impact on disease load. METHODS: Cases were contact lens wearers presenting to Singapore National Eye Centre with microbial keratitis between 2008 and 2010. Community contact lens wearers were recruited as controls. All wearers completed a previously validated questionnaire describing contact lens wear history, hygiene and compliance habits, and demographics. Risk factors significant in univariate analysis (P<0.2) were evaluated in a multivariate model. RESULTS: In all, 58 cases of microbial keratitis and 152 contemporaneous controls were identified. When controlling for other variables, Chinese had a 7 × lower risk compared with other races (95% CI: 2.3-21.3, P=0.001). Those aged between 25 and 44 years were at 3 × increased risk compared with younger wearers (95% CI: 1.1-9.6, P=0.04). Occasional overnight contact lens wear (less often than one night per week) was associated with a 4 × higher risk (95% CI: 1.2-15.4, P=0.03) compared with daily use. Not washing hands before handling was associated with a 13 × increased risk (95% CI: 1.9-84.8, P=0.008). Use of multipurpose solution A carried a 16 × higher risk compared with hydrogen peroxide (95% CI: 1.5-174.0, P=0.02). The combined PAR% for modifiable risk factors (occasional overnight wear, not washing of hands, and MPS A) was 82%. CONCLUSIONS: Consistent with previous findings, independent risk factors for contact lens-related microbial keratitis include poor hand hygiene, occasional overnight wear, and type of lens care solution. Prolonged overnight or extended contact lens use was infrequent in this population.

Dynamic change in natural killer cell type in the human ocular mucosa in situ as means of immune evasion by adenovirus infection.

The most severe form of virus-induced inflammation at the ocular surface is epidemic keratoconjunctivitis (EKC), often caused by group D human adenoviruses (HAdVs). We investigated the dynamics and mechanisms of changes in natural killer (NK) cell types in the human ocular mucosal surface in situ over the course of infection. In the acute phase of infection, the mature CD56(dim)NK cells that comprise a major subpopulation in the normal human conjunctiva are replaced by CD56(bright)NK cells recruited to the ocular surface by chemokines produced by the infected epithelium, and NKG2A-expressing CD56(dim) and CD56(bright) NK cells become the major subpopulations in severe inflammation. These NK cells attracted to the mucosal surface are however incapable of mounting a strong antiviral response because of upregulation of the inhibitory ligand human leukocyte antigen-E (HLA-E) on infected epithelium. Furthermore, group D HAdVs downregulate ligands for activating NK cell receptors, thus rendering even the mature NKG2A(-)NK cells unresponsive, an immune-escape mechanism distinct from other adenoviruses. Our findings imply that the EKC-causing group D HAdVs utilize these multiple pathways to inhibit antiviral NK cell responses in the initial stages of the infection.

A Comparison of Different Operating Systems for Femtosecond Lasers in Cataract Surgery.

The introduction of femtosecond lasers is potentially a major shift in the way we approach cataract surgery. The development of increasingly sophisticated intraocular lenses (IOLs), coupled with heightened patient expectation of high quality postsurgical visual outcomes, has generated the need for a more precise, highly reproducible and standardized method to carry out cataract operations. As femtosecond laser-assisted cataract surgery (FLACS) becomes more commonplace in surgical centers, further evaluation of the potential risks and benefits needs to be established, particularly in the medium/long term effects. Healthcare administrators will also have to weigh and balance out the financial costs of these lasers relative to the advantages they put forth. In this review, we provide an operational overview of three of five femtosecond laser platforms that are currently commercially available: the Catalys (USA), the Victus (USA), and the LDV Z8 (Switzerland).

Comparison of intra-ocular pressure changes with liquid or flat applanation interfaces in a femtosecond laser platform.

Cataract surgery is the most common surgical procedure and femtosecond laser assisted cataract surgery (FLACS) has gained increased popularity. FLACS requires the application of a suction device to stabilize the laser head and focus the laser beam accurately. This may cause a significant escalation in intra-ocular pressure (IOP), which poses potential risks for patients undergoing cataract surgery. In this study we aimed to assess the effect of the Ziemer LDV Z8 femtosecond cataract machine on IOP. We demonstrated through a porcine model that IOP was significantly higher with a flat interface but could be abrogated by reducing surgical compression and vacuum. Pressure was lower with a liquid interface, and further altering angulation of the laser arm could reduce the IOP to 36 mmHg. A pilot series in patients showed comparable pressure rises with the porcine model (30 mmHg). These strategies may improve the safety profile in patients vulnerable to high pressure when employing FLACS with the Ziemer LDV Z8.

A mouse model of lamellar intrastromal femtosecond laser keratotomy: ultra-structural, inflammatory, and wound healing responses.

PURPOSE: The availability of knockout mouse species provide a highly versatile platform for critically examining the corneal wound healing response. We aimed to develop and characterize the wound healing response in a mouse model of intrastromal femtosecond laser (FSL) keratotomy. METHODS: An intrastromal lamellar dissection using a Visumax FSL was performed on 16 wild type mice (C57BL6) . The energy level was optimized at 150nJ. The FSL was programmed to perform a lamellar dissection at 50 µM depth without sidecut. The flap was not lifted. Fellow eyes were used as controls. Slit lamp photography and confocal microscopy were performed immediately before the mice were sacrificed 4 h, 1, 3, and 7 days post surgery. Corneas were harvested for immunocytochemistry, transmission electron microscopy (TEM) and light microscopy (LM). RESULTS: Confocal microscopy showed an absence of keratocytes in the area immediately surrounding the dissection plane. The dissection plane and individual FSL plasma cavitation bubbles were clearly evident on TEM. There was evidence of Keratocyte cell death along the laser resection plane on TEM. LM revealed the dissection plane at a 20 µM depth, although not all epithelial cell layers were intact. Staining for monocytes using antibodies for CD11b (cluster of differentiation 11b) showed early migration at the peripheries at 4 h that increased at 24 h and became more central in treated corneas (p<0.001). Apoptotic cells were evident on TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay in the immediate ablation zone and were significantly raised at 4 and 24 h (p<0.001). Ki67 (Kiel 67 protein) positive proliferating keratocytes are evident at 3 days and increased significantly by 7 days (p<0.001). Minimal fibroblast (cluster of differentiation 90, CD90) transformation was seen at 1 week. No myofibroblasts were detected. DISCUSSION: We have demonstrated that FSL lamellar cuts can be effectively performed on mice and that this model exhibits typical signs of the corneal wound healing response. This model could provide a ubiquitous platform in which to study corneal wound healing responses in both wild type and knockout mice species. The ability to create such a lamellar pocket may be utilizzd for intrastromal drug delivery.

The variation in transparency of amniotic membrane used in ocular surface regeneration.

BACKGROUND/AIMS: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. METHODS: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. RESULTS: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. CONCLUSION: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

Effect of dispase denudation on amniotic membrane.

PURPOSE: To describe the cellular components, biochemical composition, and membrane surface characteristics of denuded human amniotic membrane (DHAM) treated with Dispase II. METHODS: DHAM was incubated with Dispase II (1.2 U/ml) for 30 min, 60 min, or 120 min. This was followed by gentle scraping to remove any remaining epithelial cells using a cell scraper. Histology, immunohistochemistry for extracellular matrix molecules and growth factors, and transmission (TEM) and scanning electron microscopy (SEM) were performed to assess the effects of increasing durations of incubation on DHAM structure. RESULTS: Dispase II treatment was associated with the digestion of several ECM molecules, particularly those in the basement membrane including collagen VI, fibronectin, and laminin. FGF-2 and PDGF-B expression were unaffected by Dispase II, but TGF-alpha, TGF-beta1, TGF-beta 2R, PDGF-A, VEGF, and EGFR expression were all reduced by Dispase II incubation. TEM confirmed the disruption of DHAM ultrastructure with increasing duration of Dispase II incubation, beginning with disruption of the basal lamina and progressing to loosening of the stromal collagen network as well. CONCLUSIONS: The use of Dispase II in the preparation of DHAM causes significant changes to the ultrastructure of the membrane, particularly the BM. Prolonged incubation with dispase may cause significant disruption in DHAM structure which may affect cell growth in cultured explants.

Surgical management and genetic analysis of a Chinese family with the S171P mutation in the UBIAD1 gene, the gene for Schnyder corneal dystrophy.

BACKGROUND: To describe the underlying molecular genetic basis, surgical management and phenotypic variation of Schnyder corneal dystrophy (SCD) identified in a four-generation Chinese family. METHODS: This is an interventional case series of 13 members from a non-consanguineous Chinese family. All patients underwent complete ophthalmological examination and slit-lamp photography. Subsequent corneal transplantations were performed (n = 3). Blood samples were taken for DNA extraction and subsequent genetic analysis. RESULTS: Genotyping indicated linkage to the locus at chromosome 1p36. Screening of the UBIAD1 gene identified a highly conserved mutation, Ser171Pro. Phenotypic variation in this large pedigree is similar to that seen in Caucasian patients. Surgical management of patients with anterior lamellar keratoplasty and deep anterior lamellar keratoplasty showed good visual outcomes. CONCLUSIONS: The S171P mutation is described for the first time in a Chinese family. This is the largest non-Caucasian pedigree described with SCD. Visual rehabilitation may be performed successfully with lamellar surgical procedures as opposed to full-thickness corneal grafts.

Surgical interventions for age-related cataract.

BACKGROUND: Cataract accounts for 50% of blindness globally and remains the leading cause of visual impairment in all regions of the world, despite improvements in surgical outcomes (WHO 2005). This number is expected to rise due to an aging population and increase in life expectancy. Although cataracts are not preventable, their surgical treatment is one of the most cost-effective interventions in healthcare. OBJECTIVES: To compare the effects of different surgical interventions for age-related cataract. SEARCH STRATEGY: We searched CENTRAL, MEDLINE, EMBASE up to July 2006, NRR Issue 3 2005, the reference lists of identified trials and we contacted investigators and experts in the field for details of published and unpublished trials. SELECTION CRITERIA: We included randomised controlled trials (RCTS). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and discrepancies were resolved by discussion. Where appropriate, risk ratios, odds ratios and weighted mean differences were summarised after assessing heterogeneity between the studies. MAIN RESULTS: We identified 17 trials that randomised a total of 9627 people. Phacoemulsification gave a better visual outcome than extracapsular surgery but similar average cost per procedure in Europe but not in poorer countries. Extracapsular surgery with posterior chamber lens implant and ICCE with or without an anterior chamber intraocular lens (IOL) implant gave acceptable visual outcomes but extracapsular surgery had less complications. Manual small incision surgery provides better visual outcome than ECCE but slightly inferior unaided visual acuity compared to phacoemulsification. AUTHORS' CONCLUSIONS: This review provides evidence from seven RCTs that phacoemulsification gives a better outcome than ECCE with sutures. We also found evidence that ECCE with a posterior chamber lens implant provides better visual outcome than ICCE with aphakic glasses. The long term effect of posterior capsular opacification (PCO) needs to be assessed in larger populations. The data also suggests that ICCE with an anterior chamber lens implant is an effective alternative to ICCE with aphakic glasses, with similar safety. Phacoemulsification provides the best visual outcomes but will only be accessible to the poorer countries if the cost of phacoemulsification and foldable IOLs decrease. Manual small incision cataract surgery provides early visual rehabilitation and comparable visual outcome to PHACO. It has better visual outcomes than ECCE and can be used in any clinic that is currently carrying out ECCE with IOL. Further research from developing regions are needed to compare the cost and longer term outcomes of these procedures e.g. PCO and corneal endothelial cell damage.

Protein C levels in patients with Legg-Calve-Perthes disease: is it a true deficiency?

A hyper-coagulable state due to protein C deficiency has been postulated to be the cause of avascular necrosis of the capital femoral physes in Legg-Calve-Perthes disease (LCPD). In order to test this hypothesis, plasma protein C levels were analyzed from 51 unselected cases of LCPD. These were compared with a control group. Our findings showed that the levels were less than the mean for age in 38 (74.5%) of the cases, though were within the normal range. We conclude that clinical thrombosis could be triggered off in these susceptible individuals by prothrombotic insults such as passive smoking, ultimately leading to LCPD.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials.

AIM: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors' aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. METHODS: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. RESULTS: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ss(1) integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. CONCLUSIONS: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.