Long term maintenance of frozen mouse spermatozoa at −80 °C

Raspa, Marcello; Fray, Martin; Paoletti, Renata; Montoliu, Lluı́s; Giuliani, Alessandro; Emma,; Scavizzi, Ferdinando

doi:10.1016/j.theriogenology.2017.10.036

Cited by 11 publications

(8 citation statements)

References 36 publications

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“…This phase shift should probably proceed rapidly enough to avoid cryodamage to the spermatozoa 24 , and plastic straws might be better for this. Indeed, it was reported that mouse spermatozoa could be frozen and stored in plastic straws at −80 °C for 1 year, but this required preparation of a sperm suspension and a 3% skim milk/18% raffinose solution 25 . Alternatively, the efficiency of the EQ method can be improved if LN 2 is available such that the microtubes can be frozen by placing them up to 1 cm below the LN 2 surface, as we have shown previously 12 .…”

Section: Discussionmentioning

confidence: 99%

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains

Mochida

Hasegawa

Shikata

et al. 2021

Sci Rep

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Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a −80 °C freezer. The frozen–thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3 months (25%) with the C57BL/6 J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at −80 °C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.

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Section: Discussionmentioning

confidence: 99%

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains

Mochida

Hasegawa

Shikata

et al. 2021

Sci Rep

View full text Add to dashboard Cite

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“…As previously reported, 12 IVF was performed using the protocol described by Nakagata et al 13 and modified by Li et al 14 Before thawing, the straws stored in liquid nitrogen (control) or a −80°C freezer were transferred into a LN 2 Dewar flask for at least 10 min. That was performed to prove that spermatozoa can be transferred back to LN 2 for permanent storage.…”

Section: Methodsmentioning

confidence: 99%

The impact of five years storage/biobanking at −80°C on mouse spermatozoa fertility, physiology, and function

Raspa

Putti

Paoletti³

et al. 2021

Andrology

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Background We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a −80°C freezer, maintaining their ability to fertilize mouse eggs. Objectives The main objective here was to evaluate the effects of five years at −80°C compared to liquid nitrogen storage (LN2, control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. Materials and methods Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR‐14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3‐AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. Results Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at −80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2. Discussion Long‐term mouse sperm storage at −80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. Conclusion It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a −80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.

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“…Freezing mouse spermatozoa at ˗80 o C, without liquid nitrogen, and subsequent long-term exposure to ˗80 o C did not affect the potency of frozen spermatozoa, regardless of the cryoprotective agent used (Raspa et al, 2018). The kind and concentration of the cryoprotectant agents for germplasm cryostorage, have to be considered for all cryopreservation protocol.…”

Section: Introductionmentioning

confidence: 97%

“…There are real attempts to apply cryopreservation of gametes to as low as ˗80 o C, because storage at ˗80 o C has several advantages versus to LN2; dedicated space/room is not required, it is cheaper than a LN2 tank with constant refills and it just needs electrical support. Moreover, ˗80 o C freezer engaging area is easy to be provided in any laboratories (Raspa, et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

The Effect of Cryopreservation Temperature and Time on Viability and In Vitro Maturation of Sheep Oocytes

Abdel-Aal

2021

Journal of Animal and Poultry Production

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This study aim to compare the effects of freezing techniques on viability and in vitro maturation of sheep oocytes. This experiment was carried out in Animal Production Research Institute. Sheep ovaries were obtained from slaughterhouse. A total of 695 oocytes were used in this study, 114 oocytes as control and 581 were exposed to LN2 vapor then divided into 3 groups: G1(190); directly plunged into LN2, G2 (198): cryopreserved in ˗80 o C freezer and, G3 (193), plunged into LN2 for 10 min and then transferred to-80 o C freezer. Oocytes in each group were cryopreserved for (24h, and 7days). There were high significantly differences (P<0.01) between the percentage of damage for cryopreserved oocytes, where zona dissolution was the main damage for (G1and G3) oocytes. Whereas, zona rupture was the highest damage occurred in (G2) oocytes. Survival rate of G1 oocyte was higher significantly (P<0.05) than G3 and G2, the same results were obtained pattern, either after 24h or 7 days storage. Highly significant differences (P<0.01) between maturation rate achieved with the control oocytes (63%) compared with the other groups. After 24 h of storage, G1 recorded higher MII rate followed by G3 and G2 (47, 37 and 28%, respectively), MII % decreased at 7 days storage in G1, G3 and G2 (44%, 34% and 31%, respectively). In conclusion, Cryopreservation of sheep oocytes (G3) in-80 °C freezer achieved acceptable survival and in vitro maturation rates. The-80°C freezer provide a simple and cheap tool to maintain frozen sheep oocytes.

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Long term maintenance of frozen mouse spermatozoa at −80 °C

Cited by 11 publications

References 36 publications

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains

Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains

The impact of five years storage/biobanking at −80°C on mouse spermatozoa fertility, physiology, and function

The Effect of Cryopreservation Temperature and Time on Viability and In Vitro Maturation of Sheep Oocytes

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