Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described

Long-Term Cold Acclimation Extends Survival Time at 0°C and Modifies the Metabolomic Profiles of the Larvae of the Fruit Fly Drosophila melanogaster

Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately -5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum.We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt(50)) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt(50) (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines.Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article

How insects survive the cold: molecular mechanisms - a review

Insects vary considerably in their ability to survive low temperatures. The tractability of these organisms to experimentation has lead to considerable physiology-based work investigating both the variability between species and the actual mechanisms themselves. This has highlighted a range of strategies including freeze tolerance, freeze avoidance, protective dehydration and rapid cold hardening, which are often associated with the production of specific chemicals such as antifreezes and polyol cryoprotectants. But we are still far from identifying the critical elements behind over-wintering success and how some species can regularly survive temperatures below -20°C. Molecular biology is the most recent tool to be added to the insect physiologist’s armoury. With the public availability of the genome sequence of model insects such as Drosophila and the production of custom-made molecular resources, such as EST libraries and microarrays, we are now in a position to start dissecting the molecular mechanisms behind some of these well-characterised physiological responses. This review aims to provide a state of the art snapshot of the molecular work currently being conducted into insect cold tolerance and the very interesting preliminary results from such studies, which provide great promise for the future