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

Abstract

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.

DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development

“…Ejaculated spermatozoa: EJ-sperm was collected as described previously [ 15 ]. Briefly, semen was collected from three adult fertile rams using an Artificial Vagina (AV) filled with warm water (40–44°C) and connected to a 15-ml tube.…”

“…Epididymal spermatozoa: Samples in 15-ml Falcon tubes were cooled to 4–5ºC at ~0.3ºC min –1 by submerging the tubes in an isothermal water bath and then placing them in a refrigerator (4ºC). Once chilled, each sample was packaged into four pre-chilled 8-ml polypropylene tubes, with 2 ml in each, and frozen in a directional freezing machine (MTG-550, IMT, Ness Ziona, Israel) as previously described [ 15 , 16 ]. All frozen samples were stored under liquid nitrogen pending lyophilization and evaluation.…”

“…Ejaculated spermatozoa: EJ-sperm was lyophilized as described previously [ 15 ]. Briefly, Spermatozoa were lyophilized, using a freeze-dryer (SP Scientific-VirTis, 2.0 BenchTop, Warminster, PA, USA) following the protocol used by Wakayama and Yanagimachi [ 1 ].…”

Exploring dry storage as an alternative biobanking strategy inspired by Nature

“…Combining the available knowledge from these diverse fields might help reach the moment of Eureka we are all waiting for. [197] Sheep Freeze-drying MPN formation [198] Sheep Partial freezedrying Sperm motility [199] Sheep Freeze-drying Blastocyst formation [90,200] Weasel Freeze-drying MPN development in mouse oocyte [150] MPN: male pro-nucleus…”

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze‐drying rabbit (Oryctolagus cuniculus) spermatozoa

“…In recent years, the development of an alternative method to sperm cryopreservation, such as freeze‐drying (FD) or lyophilization, has facilitated the storage and maintenance of genetic merit of several species, including rabbits (Keskintepe & Eroglu, ; Liu et al, ), and bio‐banking of endangered species (Anzalone at al., ). FD is a method in which frozen material is dried by ice sublimation, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase (Keskintepe & Eroglu, ).…”