Freeze-drying is a novel technique that permits the storage of semen at room temperature for long time periods, retaining their fertilizing capacity. The main objective of this work was to compare the fertilization ability of frozen-thawed (FT) and freeze-dried (FD) ram semen following intracytoplasmic sperm injection (ICSI) of invitro-matured (IVM) sheep oocytes. Oocytes were recovered by slicing the ovaries of slaughtered sheep. Selected cumulus-oocyte complexes (COCs) were IVM for 24h in tissue culture medium 199 (TCM-199) supplemented with 10% heat-treated oestrous sheep serum (ESS), 0.36mM pyruvate, FSH (1IUmL−1), and luteinising hormone (LH; 1IUmL−1) under mineral oil in a humidified atmosphere of 5% CO2, at 38.5°C. Semen was collected from fertile adult rams using an artificial vagina and processed for (1) freezing and thawing (Khalifa and Lymberopoulos, 2013 Cell Tissue Bank 14, 687-698; https://doi.org/10.1007/s10561-012-9357-6) or (2) freeze-drying and rehydration according to Arav et al. (2018 J. Assist. Reprod. Genet. 35, 1149-115; https://doi.org/10.1007/s10815-018-1145-1) protocols. For FD protocol, sperm samples were diluted in a sugar solution of trehalose and sorbitol (LyoB) and dehydrated for 24h. Later, the samples were rehydrated in a warming solution and diluted in TCM-199 before ICSI. After maturation, metaphase II (MII) oocytes with a polar body were injected with FT or FD sperm. Briefly, oocytes were transferred into groups of six in an ICSI dish containing 6-µL drops of holding medium (TCM-199 + 5% fetal bovine serum) and 3-µL drops of PVP for the sperm samples. Injection was carried out with an inverted microscope (Olympus IX73) connected to a micromanipulation system (Narishige) using ICSI pipettes with 7-µm internal diameter. Within 1h, ICSI oocytes were activated with 5 µM ionomycin for 4min and invitro cultured in modified synthetic oviductal fluid medium (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817; https://doi.org/10.1071/RD11023). After 17-21h, injected oocytes were fixed and stained in a solution of ethanol Hoechst 33342 and classified as FPN (one female pronucleus and one condensed sperm head), MPN (one male pronucleus and one MII), 2PN (two pronuclei, male and female), 3PN (three or more pronuclei), and NPN (no pronuclei). Data were analysed using analysis of variance (two-way ANOVA) followed by Tukey post hoc test with SAS software, version 9.4. The ICSI-FD group had a higher number of NPN and a lower number of 2PN than did the ICSI-FT group (P<0.05). We think that more technical advances in the FD process as well as the rehydration procedure are necessary to improve the application of FD ovine semen for invitro fertilization by ICSI in sheep, but in any case these results have showed that FD could be a useful tool for the future of invitro embryo production.
Table 1.Pronuclear formation at 17-21h post-injection1
Treatment
n
FPN
MPN
2PN
3PN
NPN
FT
71
9.66±4.12
4.26±1.48
48.13±2.79a
5.97±4.16
31.98±6.75a
FD
65
6.16±2.26
1.39±1.39
20.15±4.14b
10.57±6.59
61.73±6.89b
a,bValues in the same column with different superscript letters differ significantly (P<0.05).
1Data are presented as mean±s.e.m. FPN=female pronucleus, MPN=one male pronucleus and one metaphase II oocyte, 2PN=two pronuclei, male and female, 3PN=three or more pronuclei, NPN=no pronuclei.
Funding was provided by Spanish MINECO Grant AGL2017-85837-R, Spanish MECD pre-doctoral grant FPU15/00773, and Spanish MECD mobility grant EST18/00472 to Irene Menéndez Blanco.
Failure of pronuclear migration and repeated divisions of polar body nuclei associated with MTOC defects in polo eggs of Drosophila
