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. 2019 Apr 17;9(1):6219.
doi: 10.1038/s41598-019-42712-x.

Prevention of Hatching of Porcine Morulae and Blastocysts by Liquid Storage at 20 °C

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Free PMC article

Prevention of Hatching of Porcine Morulae and Blastocysts by Liquid Storage at 20 °C

Cristina A Martinez et al. Sci Rep. .
Free PMC article

Abstract

Vitrification is the ideal method for long-lasting storage of porcine embryos. However, both strict airline regulations for transport of liquid nitrogen dewars and the technical problems experienced when vitrified embryos are transferred using non-surgical procedures have led to the introduction of alternative storage methods, such as preserving embryos in liquid state. This study evaluated whether a pH-stable medium containing high concentrations of either foetal calf serum (FCS; 50%) or BSA (4%) combined with storage at temperatures of 17 °C or 20 °C maintained in vivo-derived morulae and blastocysts alive and unhatched (a sanitary requirement for embryo transportation) during 72 h of storage. Neither FCS nor BSA supplements were able to counteract the negative effect of low temperatures (17 °C) on embryonic survival after storage. At 20 °C, the protective effect of FCS or BSA depended on embryo stage. While FCS successfully arrested embryo development of only blastocysts, BSA arrested the development of both morulae and blastocysts. Over 80% of BSA arrested embryos restarted development by conventional culture and progressed to further embryonic stages, including hatching. In conclusion, porcine morulae and blastocysts can survive and remain unhatched during at least 72 h when stored at 20 °C in a BSA-containing medium.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Embryonic progression of in vivo-produced porcine morulae stored for 72 h at different temperatures and medium supplementations. (A) Bar graphs showing the proportion of embryos that reached each developmental stage per group after 72 h of storage at 17 °C and after 24 h and 48 h of conventional culture (NCSU-23 culture medium supplemented with 0.4% BSA and 10% FCS at 38.5 °C in humidified air with 5% CO2). (B) Bar graphs showing the proportion of embryos that reached each developmental stage per group after 72 h of storage at 20 °C and after 24 h and 48 h of conventional culture. BL: blastocyst.
Figure 2
Figure 2
Collapse and re-expansion of blastocysts. (A) The proportion of blastocysts that spontaneously collapsed after 72 h of storage at different temperatures and in different medium supplementations and the re-expansion ability of the collapsed blastocysts after 24 h of conventional culture (NCSU-23 culture medium supplemented with 0.4% BSA and 10% FCS at 38.5 °C in humidified air with 5% CO2). a,bDifferent letters shown per variable (collapsed or reformed) indicate significant differences (P < 0.05). BL: blastocyst. (B) Representative pictures of blastocyst collapse and re-expansion. (a) blastocysts just after collection; (b) selected blastocysts collapsed after 72 h of storage; (c) re-expansion of blastocysts after 24 upon conventional culture. Scale bar is 100 µm.
Figure 3
Figure 3
Progression stages of in vivo-produced porcine blastocysts stored for 72 h at different temperatures and in different medium supplementations. (A) Bar graphs showing the proportion of embryos that reached different developmental stages per group after 72 h of storage at 17 °C and after 24 h and 48 h of conventional culture (NCSU-23 culture medium supplemented with 0.4% BSA and 10% FCS at 38.5 °C in humidified air with 5% CO2). (B) Bar graphs showing the proportion of embryos that reached different developmental stages per group after 72 h of storage at 20 °C and after 24 h and 48 h of conventional culture. BL: blastocyst.
Figure 4
Figure 4
Preservation of porcine in vivo-derived morulae. (A) Representative images showing morulae at 0 h and 72 h of storage in medium containing 4% BSA at 20 °C or after 48 h upon conventional culture (NCSU-23 culture medium supplemented with 0.4% BSA and 10% FCS at 38.5 °C in humidified air with 5% CO2). (B) Representative images showing morulae at 0 h and 48 h of conventional culture (controls). Scale bar is 100 µm.

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References

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