Cryopreservation of Sperm of Spotted Seatrout (Cynoscion nebulosus)

Cryopreservation of fish sperm has applications in preserving genetic resources from stocks of endangered fishes, replenishing fisheries, reducing the number of males needed in hatchery situations, and allowing repeated spawning of specific males. As part of a larger study on artificial breeding of sciaenid fishes, we developed procedures for collection, handling, refrigerated storage, and cryopreservation of spotted seatrout sperm. Hanks\u27 balanced salt solution (HBSS) was used as an extender for collection and storage of sperm. Sperm motility in relation to graded concentrations of HBSS was used to determine the osmolality at which sperm were activated. Based on these findings, HBSS was prepared at 201 mOsm/kg as an extender for sperm storage. To determine if ions present in HBSS were involved in sperm activation, separate activating solutions were prepared by the addition of NaCl, CaCl2, KCl, Na2HPO4, or MgSO4 to aliquots of a stock glucose solution (185 mOsm/kg). The chemicals were added at the concentration of each found in 1-x HBSS. Only the glucose solution containing 8 g/l NaCl(424 mOsm/kg) produced activation of sperm. We also evaluated four chemicals as cyroprotectants: methanol, glycerol, dimethyl sulfoxide (DMSO), and n,n-dimethyl acetamide. Two freezing rates were evaluated by placing samples at either of two heights within a nitrogen vapor shipping dewar. The highest post-thaw motilities were in 10% DMSO with an average retention of 60% of initial motility at the lower position in the dewar, and 37% at the upper position. A third freezing rate was produced using a computer-controlled freezer programmed for a rate of -45ºC/min, yielding a retention of initial motility of 31%. Our freezing and transport of cryopreserved sperm in shipping dewars demonstrate the utility of this procedure for field applications

Cryopreservation of Sperm of Spotted Seatrout (Cynoscion nebulosus)

Cryopreservation of fish sperm has applications in preserving genetic resources from stocks of endangered fishes, replenishing fisheries, reducing the number of males needed in hatchery situations, and allowing repeated spawning of specific males. As part of a larger study on artificial breeding of sciaenid fishes, we developed procedures for collection, handling, refrigerated storage, and cryopreservation of spotted seatrout sperm. Hanks\u27 balanced salt solution (HBSS) was used as an extender for collection and storage of sperm. Sperm motility in relation to graded concentrations of HBSS was used to determine the osmolality at which sperm were activated. Based on these findings, HBSS was prepared at 201 mOsm/kg as an extender for sperm storage. To determine if ions present in HBSS were involved in sperm activation, separate activating solutions were prepared by the addition of NaCl, CaCl2, KCl, Na2HPO4, or MgSO4 to aliquots of a stock glucose solution (185 mOsm/kg). The chemicals were added at the concentration of each found in 1-x HBSS. Only the glucose solution containing 8 g/l NaCl(424 mOsm/kg) produced activation of sperm. We also evaluated four chemicals as cyroprotectants: methanol, glycerol, dimethyl sulfoxide (DMSO), and n,n-dimethyl acetamide. Two freezing rates were evaluated by placing samples at either of two heights within a nitrogen vapor shipping dewar. The highest post-thaw motilities were in 10% DMSO with an average retention of 60% of initial motility at the lower position in the dewar, and 37% at the upper position. A third freezing rate was produced using a computer-controlled freezer programmed for a rate of -45ºC/min, yielding a retention of initial motility of 31%. Our freezing and transport of cryopreserved sperm in shipping dewars demonstrate the utility of this procedure for field applications

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Synchronous Hermaphroditism and Self-Fertilization in a Captive Shortnose Sturgeon

The shortnose sturgeon Acipenser brevirostrum is a federally listed endangered fish species endemic to the East Coast of North America, where captive fish are cultured for recovery efforts. During routine evaluations of broodfish for spawning potential, a single hermaphroditic shortnose sturgeon was discovered. Motile sperm and approximately 7,600 eggs were manually stripped from the individual simultaneously. Eggs were fertilized with the fish’s own sperm (self-fertilized) and with the sperm of two unrelated males (cross-fertilized). Both self- and cross-fertilization procedures were successful. Viable fry hatched from 9% of the eggs produced by self-fertilization and from 2% of those produced by cross-fertilization. Fry survival through 180 d posthatch was 1% in the self-fertilization treatment and 11% in the cross-fertilization treatment. Endoscopic evaluation of gonads confirmed the presence of ovotestes with discrete regions of active testicular and ovarian tissues. The present account provides empirical evidence including gross anatomical and histological photodocumentation that advances knowledge of this rare condition in a fish species threatened with extinction

The relationship of the cryoprotectants methanol and dimethyl sulfoxide and hyperosmotic extenders on sperm cryopreservation of two North-American sturgeon species

Successful sperm cryopreservation techniques have been developed for Eurasian sturgeon species; however, there is little information available on these techniques for North-American species. In this study, two sets of sperm cryopreservation experiments were carried out on the endangered shortnose sturgeon (Acipenser brevirostrum). In the first set, the cryoprotectants methanol (MeOH) and dimethyl sulfoxide (DMSO) were investigated using three concentrations (5%, 10% and 15%). The highest post-thaw motility was found using 5% DMSO (26F13%) while the use of 5% MeOH resulted in the highest rates for fertilization at the 4-cell stage (40F15%), neurulation (38F13%) and hatching (32F12%). In the second set, the Original Tsvetkova’s extender (OT), Modified Tsvetkova’s extender (MT) and modified Hanks’ balanced salt solution (mHBSS) were investigated in combination with three MeOH concentrations. The highest post-thaw motility (18F10%), fertilization (18F11%) and hatching rates (17F12%) were observed with MT extender used in combination with 5% MeOH. In another set of experiments, the effects of two extenders (MT and mHBSS) and two concentrations of MeOH were investigated for sperm cryopreservation of pallid sturgeon (Scaphyrinchus albus). The highest post-thaw motility (70F10%) was observed using MT and 10% MeOH while MT and 5% MeOH yielded the highest rates of fertilization (88F6%) and hatching (73F14%). In general we conclude that although hyperosmotic conditions of extenders and cryoprotectants result in higher post-thaw motility, they seem to reduce the fertilizing ability of the sperm

Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration

Abstract Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8+ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs