Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage.

Introduction: Freeze-drying techniques give alternative preservation mammalian spermatozoa without liquid nitrogen. However, most of the work has been conducted in the laboratory mouse, while little information has been gathered on large animals that could also benefit from this kind of storage. Methods: This work adapted a technique known as vacuum-drying encapsulation (VDE), originally developed for nucleic acid conservation in anhydrous state, to ram spermatozoa, and compared it to canonical lyophilization (FD), testing long-term storage at room temperature (RT) and 4°C. Results and discussion: The results demonstrated better structural stability, namely lipid composition and DNA integrity, in VDE spermatozoa than FD ones, with outcomes at RT storage comparable to 4°C. Likewise, in VDE the embryonic development was higher than in FD samples (12.8% vs. 8.7%, p < 0.001, respectively). Our findings indicated that in large mammals, it is important to consider dehydration-related changes in sperm polyunsaturated fatty acids coupled with DNA alterations, given their crucial role in embryonic development.

Controlled spermatozoa-oocyte interaction improves embryo quality in sheep.

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction.

While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used. In order to verify the hypothesis that centriole dysfunction might be responsible for low ICSI outcomes in sheep, we have investigated micro-tubular dynamics, markedly aster nucleation, in fertilized sheep zygotes by ICSI with frozen/thawed (FT) and FD spermatozoa; In Vitro Fertilized (IVF) sheep oocytes were used as control. The spermatozoa aster nucleation was assessed at different time points following ICSI and IVF by immune-detection of α-tubulin. Pronuclear stage, syngamy and embryo development were assessed. No difference was noticed in the timing of aster nucleation and microtubule elongation in ICSI-FT derived embryos with control IVF ones, while a delay was recorded in ICSI-FD ones. The proportion of 2-pronuclear stage zygotes was similar in ICSI-FT and ICSI-FD (47% and 53%, respectively), both much lower comparing the IVF ones (73%). Likewise, syngamy was observed in a minority of both ICSI groups (28.5% vs 12.5% in ICSI-FT/FD respectively) comparing to IVF controls (50%), with a high number of zygotes blocked at the 2-pronuclear stage (71.5% vs 87.5% respectively). While no significant differences were noticed in the cleavage rate between ICSI-FD, ICSI-FT and IVF groups (31%, 34% and 44%) respectively, development to blastocyst stage was markedly compromised in both ICSI groups, especially with FD spermatozoa (10% in ICIS-FD and 19% in ICSI-FT vs 33% in IVF (P < 0.005, ICSI-FD vs IVF and P < 0.05, IVF vs ICSI-FT, respectively). Hence, here we have demonstrated that the reduced cleavage, and the ensuing impaired development to blastocysts stage of ICSI derived sheep embryos is not related to centriole dysfunction, as suggested by other authors. The major recorded problem is the lack of syngamy in ICSI derived zygotes, an issue that should be addressed in further studies to improve ICSI procedure in sheep embryos.

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature.

Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to - 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.

Dry biobanking as a conservation tool in the Anthropocene.

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity - humans. Since we are the cause, we also hold the key to making the change. Any change, however, will take time, and for some species this could be too long. While working on possible solutions, we also have the responsibility to buy time for those species on the verge of extinction. Genome resource banks, in the form of cryobanks, where samples are maintained under liquid nitrogen, are already in existence but they come with a host of drawbacks. Biomimicry - innovation inspired by Nature, has been a huge source for ideas. Searching methods that Nature utilizes to preserve biological systems for extended periods of time, we realize that drying rather than freezing is the method of choice. We thus argue here in favor of preserving at least part of the samples from critically endangered species in dry biobanks, a much safer, cost-effective, biobanking approach.

Freeze-dried spermatozoa: An alternative biobanking option for endangered species.

In addition to the iconic wild species, such as the pandas and Siberian tigers, an ever-increasing number of domestic species are also threatened with extinction. Biobanking of spermatozoa could preserve genetic heritages of extinct species, and maintain biodiversity of existing species. Because lyophilized spermatozoa retain fertilizing capacity, the aim was to assess whether freeze-dried spermatozoa are an alternative option to save endangered sheep breeds. To achieve this objective, semen was collected from an Italian endangered sheep breed (Pagliarola), and a biobank of cryopreserved and freeze-dried spermatozoa was established, and evaluated using IVF (for frozen spermatozoa) and ICSI procedures (for frozen and freeze-dried spermatozoa). As expected, the fertilizing capacity of cryopreserved Pagliarola's spermatozoa was comparable to commercial semen stocks. To evaluate the activating capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI, and allocated to two groups: 56 oocytes were activated by incubation with ionomycin (ICSI-FDSa) and 52 were not activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14-16 h after ICSI in fixed presumptive zygotes. Only artificially activated oocytes developed into blastocysts after ICSI. In the present study, freeze-dried ram spermatozoa induced blastocyst development following ICSI at a relatively high proportion, providing evidence that sperm lyophilization is an alternative, low cost storage option for biodiversity preservation of domestic species.