Biology subjects

Litvak, M. K.

Publications and source records attributed to Litvak, M. K..

4 recordsLinked to original sources

bioRxiv2025

Optimizing Equilibration Time to Enhance Post-Thaw Viability of Cryopreserved Zebrafish (Danio rerio) Ovarian Fragments

Cryopreservation enables the long-term storage of viable biological material at ultra-low temperatures and forms the foundation for germplasm cryobanks that maintain valuable genetic lines of model organisms such as zebrafish (Danio rerio). However, reliable and reproducible cryopreservation protocols for fish germline stem cells remain difficult to develop, partly because key steps such as equilibration are often overlooked or assigned arbitrarily. Here, we optimized equilibration time for cryopreservation of zebrafish ovarian tissue. Ovarian fragments were equilibrated in 2 M methanol + 0.1 M glucose + 10% egg yolk for varying durations (15-120 minutes) before controlled slow-cooling and storage in liquid nitrogen. Post-thaw viability was assessed using a Trypan Blue exclusion assay. A 60-minute equilibration yielded the highest viability of ovarian cells in Experiment 1 (55.68 {+/-} 1.74%), whereas a 30-minute equilibration yielded the highest viability in Experiment 2 (75.86 {+/-} 2.44%), but was not significantly different from the 60-minute equilibration in Experiment 2 (75.58 {+/-} 2.04%) (p = 0.9983). Equilibration alone accounted for a 48.71% increase in post-thaw viability relative to controls. The framework presented here provides a reproducible method for determining species-specific equilibration optima and supports the development of effective germplasm cryobanks for both model and endangered fish species.

cell biology↗

bioRxiv2024

First attempt to validate multiplex PCR with sex markers SSM4 and ALLWSex2 in long-term stored blood samples of eastern North American shortnose sturgeon (Acipenser brevirostrum).

Sex-specific information is crucial for sturgeon culture, conservation, and fisheries management. However, identifying the sex of sturgeon is difficult, especially for immature individuals. Two recent studies identified two female-specific loci (AllWSex2 and SSM4) that are conserved among many Acipenserid species, but they have not been validated for all species within this family. The objectives of this study were to 1) determine whether SSM4 can be used to sex shortnose sturgeon; 2) develop and test a multiplex PCR technique using both ALLWSex2 and SSM4 for sexing shortnose sturgeon; 3) determine if long-term storage of blood samples can be used to sex shortnose sturgeon; and 4) test the effect of storage temperature on DNA degradation. DNA was extracted from frozen RBC samples from 36 fish which had previously been sexed. A multiplex PCR was set up using three pairs of primers: AllWSex2 and SSM4, as female-specific loci, and mtDNA as an internal control which were all run on a 2 % agarose gel. AllWSex2 and SSM4 allowed for perfect discrimination of sex. While there was DNA degradation, as a result of long-term storage and temperature, the signal was still strong enough after 8 years of cold storage to delineate sex. This suggests that researchers now have the ability to reexamine archived/frozen samples to determine sex of their fish.

ecology↗

bioRxiv2024

Exploring fractal dimensions on ultrasound of gonadal images for sex determination in shortnose sturgeon (Acipenser brevirostrum)

Sturgeon are often viewed as monomorphic species because they often lack external features that allow differentiation between sexes. Current practices for sexing sturgeon rely heavily on surgical invasive procedures to visually examine gonadal tissues. An alternative which is widely used in different species of Acipenserids is the use of ultrasound to sex them. The challenge with ultrasound is that it requires an experienced operator to successfully sex the fish by recognizing relevant patterns in the structures of gonads. The objective of this study is to attempt to lay the groundwork for potential systematization of sexing techniques using a portable ultrasound as a medium. We used texture analysis software based on lacunarity measurements and fractal dimensions to determine whether male gonads, female granular tissue (GT) and female pinheads were significantly different from each other using two different resolutions on the ultrasound machine. Male gonads and GT were significantly different from pinheads in lacunarity measurements (p=0.001) using 10-12Mhz general resolution imaging. Fractal dimensions did not result in any significant differences. Lacunarity has the potential to determine sex based on gray level-co-occurrence matrices and the software is freely available. We have also developed a relative probability table based on the data gathered on lacunarity in this study available as supplementary material for quick reference.

zoology↗

bioRxiv2024

The effect of extended cold storage and the use of extenders on motility and swimming kinematics of the sperm of shortnose sturgeon

Shortnose sturgeon are listed as a species of special concern in Canada and as endangered in the US. Increasing knowledge about this species, particularly in the area of reproductive biology, will better the management of wild populations and aid in the development of assisted reproduction protocols. However, access to wild sperm is limited, so short-term and long-term storage of sperm from sturgeon is crucial for reproductive studies. Here we report on testing and development of a short-term storage protocol for shortnose sturgeon. Milt samples were collected from wild shortnose sturgeon caught in the Wolastoq River. Subsets of semen were mixed with different extenders with or without oxygen; control treatments without extenders were also run. We used computer-assisted sperm analysis (CASA) to determine sperm motility and swimming kinematics for the different treatments. All groups were examined immediately after collection and treatment application, and then 1, 2, and 7 days after storage in a fridge (4{degrees}C) for experiment 1, and days 1, 3, 7, 10, 14, 17, 21, and 24 for experiment 2. The response variables motility, curvilinear velocity (VCL), linearity (LIN), and wobble (WOB) showed an overall decrease over time with differences between extender treatments. While untreated milt maintained some motility up to day 21, the addition of an extender reduced decline in motility and improved longevity up to day 24. Milt treated with the Park and Chapman extender had the slowest motility decline of extenders used, and milt treated with the modified Tsvetkova extender showed less potential for contamination.

ecology↗