Cryo letters最新文献

Background: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos.

Objective: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance.

Materials and methods: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified.

Results: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification.

Conclusion: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.

The preservation of the nuclear genome's integrity is paramount for the viability and overall health of cells, tissues, and organisms. DNA, being susceptible to damage under physiological conditions and vulnerable to both endogenous and environmental factors, faces constant threats. To assess DNA damage and repair within individual eukaryotic cells, the comet assay presents itself as a versatile, gel electrophoresis-based, relatively simple, and highly sensitive method. Originally designed to monitor DNA damage and repair within populations of mammalian cells, the comet assay has now found applications across diverse domains, including yeast, protozoa, plants, and invertebrates. This technique has proven invaluable in cryopreservation studies, serving as a valuable adjunct for determining suitable cryopreservation protocols. These protocols encompass choices related to cryoprotectants, sample preparation, as well as storage conditions in terms of time and temperature. In the realm of animal cryopreservation research, the comet assay stands as a gold-standard method for assessing DNA integrity. Nevertheless, when applied in plant-oriented investigations, additional efforts are essential due to the distinct nature of plant cells and associated technical challenges. This review elucidates the fundamental principles underlying the comet assay, discusses its current iterations, and delineates its applications in the cryopreservation of both animal and plant specimens. Moreover, we delve into the primary challenges confronting the comet assay's utility as a monitoring tool in the context of plant sample cryopreservation. https://doi.org/10.54680/fr24110110112.

Background: Increasingly, sheep breeders are using artificial insemination to produce lambs, so finding methods that preserve ram sperm can be useful.

Objective: To determine the protective effects of different concentrations of laminarin on ram sperm motility, viability, abnormalities, membrane, and DNA integrity, superoxide dismutase enzyme (SOD) activity, and malondialdehyde (MDA) production after freeze-thawing.

Materials and methods: The ejaculates of four rams were collected and stored at 35 degree C. Semen samples were diluted with a tris-base extender containing 100, 200, 400, and 800 ug/mL of laminarin and a control extender containing no laminarin, then frozen in liquid nitrogen after 4 h in the refrigerator.

Results: In the treatment of frozen-thawed spermatozoa with 800 ug/mL laminarin, motility, viability, membrane integrity, and DNA integrity were significantly higher than in the control. In spermatozoa that were exposed to 800 ug/mL laminarin after thawing, MDA production was significantly lower than in the control group. The percentage of abnormal spermatozoa in 800 ug/mL laminarin was significantly lower than that in the control.

Conclusion: The addition of 800 ug/mL laminarin to the freezing extender increases motility, viability, SOD activity, and plasma membrane integrity, while reducing abnormality and MDA production in freeze-thawed ram semen. https://doi.org/10.54680/fr24110110812.

Background: Extensive dilution of cattle semen with tris-based extender compromises certain sperm kinetic and functional traits following cryopreservation.

Objective: To study sperm functions of buffalo bulls under high dilution rates.

Materials and methods: Twenty-four ejaculates were harvested twice a week from four buffalo bulls, and diluted to sperm concentrations of 80, 60, 40 and 20 million/mL. Diluted samples were filled in straws, equilibrated at refrigeration temperature for 4 h, and frozen in liquid nitrogen. Frozen sperm samples were thawed for evaluation of kinetic and functional attributes.

Results: Compared to 20 million/mL (million/mL) sperm sample, the total motility, progressive motility and rapid motility were reduced (P < 0.05) in 5 million/mL sample. The proportion of live sperm were significantly (P < 0.05) higher in 10, 15 and 20 million/mL samples than in 5 million/mL sample. The percentage of moribund sperm, dead sperm, and sperm with lipid per oxidation increased significantly (P < 0.05) in 5 million/mL sample.

Conclusion: The reduction of sperm concentrations to < 10 million/mL affects post-thaw Buffalo sperm kinetic and functional attributes.. https://doi.org/10.54680/fr24110110712.

Background: Mico-volume semen freezing is essential and used popularly for fertility preservation of patients suffering cancer or undergoing male reproductive system related surgeries, and for other reasons that may risk fertility potential in ART cycles. However, clinicians and embryologists still face some unresolved technical and theoretical issues about the frozen-thawed efficiency.

Objective: To choose the appropriate freezing method for different volumes of normal semen samples.

Materials and methods: We investigated the frozen-thawed outcomes of semen with different volumes (20 uL, 50 uL, 100 uL, 200 uL, 500 uL and 1 mL) using two freezing methods (FLNV, static liquid nitrogen vapour cooling followed by liquid nitrogen preservation; RFLN, direct rapid freezing in liquid nitrogen) and analyzed the vitality, progressive motility and DNA fragmentation index of thawed sperm.

Results: We found that semen freezing with volumes more than 100 uL had better outcomes than volumes less than or equal to 50 uL after thawing. FLNV presented a higher efficiency for cryopreservation of semen with volumes less than 50 uL.

Conclusion: For smaller (micro) volumes, the FLNV technique is better than the RFLN method. https://doi.org/10.54680/fr24110110412.

Background: When sperm are cryopreserved, reactive oxygen species (ROS) are formed that are detrimental to the sperm.

Objective: To evaluate the effects of oleic acid and trehalose added to ram semen extender on sperm parameters, lipid peroxidation (MDA), and superoxide dismutase (SOD) enzyme levels of spermatozoa following the freeze/thawing processes.

Materials and methods: Ejaculates were collected from four rams and pooled at 35 degree C. Pooled ejaculates were diluted with oleic acid at 0 mM and trehalose at 0 mM (O0 T0) as the control. The Tris-based extender was supplemented with either 0.5 (O0.5) or 1 (O1) mM of oleic acid or 25 (T25) or 50 (T50) mM of trehalose alone, and in combination [0.5 mM oleic acid + 25 mM trehalose (O0.5 T25), 0.5 mM oleic acid + 50 mM trehalose (O0.5 T50), 1 mM oleic acid + 25 mM trehalose (O1 T25) and 1 mM oleic acid + 50 mM trehalose (O1 T50)]. The semen was frozen by the traditional liquid nitrogen vapour method and stored at -196C in the liquid nitrogen tank.

Results: Semen extender containing O1T25 significantly improved the total motility, when compared with other treatment groups (P<0.05), except for O1 T50. O1 T50 had a higher viability rate than any other treatment. The addition of O1 T25 and O1 T50 increased DNA and membrane integrity of spermatozoa post-thawing compared to other treatments (P<0.05). The level of MDA was significantly (P<0.05) lower in extenders supplemented with O1, O0.5 T25, O0.5 T50, O1 T25 and O1T50 compared to the other treatment groups. In addition, SOD levels were higher in groups treated with O1 T25 and O1 T50 than the other treatment groups (P<0.05).

Conclusion: The addition of a combination of oleic acid and trehalose concentrations to Tris-based extender improved the quality of ram semen post-thawing. Doi.org/10.54680/fr23610110712.

Background: Antioxidant present in sperm cells protects them from oxidative damage. However, sperm are more susceptible to peroxidative damages due to the loss of these enzymes during cryopreservation and their survival and fertility may be compromised. Insulin like growth factor-1 (IGF-1) has an antioxidant effect and could maintain sperm motility.

Objective: To improve seminal parameters, mitochondrial membrane potential (MMP), oxidative status and DNA integrity of buck semen after freeze-thawing by fortification of goat semen diluent with various concentrations of IGF-1.

Materials and methods: Fifty ejaculates were collected and were extended with tris- citric acid- fructose diluent with 10% egg yolk and 6% glycerol with sperm concentrations of 1×10⁸ mL^-1. Post-cryopreserved sperm were assessed for motility and a range of other functional parameters.

Results: In post-thaw semen sperm motility, live sperm count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, malondialdehyde (MDA), protein carbonyl content (PCC), TUNEL positive sperm differed significantly (P<0.05) with the various concentrations of IGF-1 used. Sperm functional parameters post-thawing were significantly (P<0.05) better in 250 ng/mL IGF-1. IGF-1 protects against lipid peroxidation by lowering MDA and PCC production, thus reducing the harmful effect of reactive oxygen species. The kidding percentage using the artificial insemination technique was significantly higher ( i.e., 40%) in the group supplemented with 250 ng/mL of IGF-1 than in the non-supplemented group (i.e., 30%).

Conclusion: IGF-1 may be used to improve post-thaw semen quality and fertility as measured by actual kidding rate. Doi.org/10.54680/fr23610110312.