Abstract
In the scientific interchange of genetically-modified mouse strains the transportation of refrigerated epididymis has several advantages over the transportation of live animals, especially with regard to the 3R (replacement, reduction and refinement) principles. The major limiting factor is the duration of the transportation. Previous reports have shown that sperm collected from transported epididymis maintained their fertility for around 72 h, but there are no published data with longer transportation times, and this window of time may be too short, especially for international shipments and where locations are not well connected. In this study live pups were born using frozen–thawed sperm after up to 192 h (8 days) of transportation, using a special in vitro fertilization design which resulted in a fertilization rate of 10.5%.
Keywords
In 1997 Jishage et al. reported that mouse epididymal sperm preserved in paraffin oil at 4℃ could maintain fertility after 24 h. 1 A new method was later published that demonstrated the feasibility of cold epididymis transportation within 48 h. 2 Recently a transport system for refrigerated mouse epididymal sperm was described. 3 In all these studies, most of the data published were obtained from results where the epididymis was stored in a refrigerator at 4℃,1–6 and only a few experiments were from results using actual transportation and for a maximum journey time of 72 h.3,5 This is a critical point for several reasons: maintaining cold temperatures during transportation can be complicated, 7 and in some circumstances, especially after 72 h, it may be necessary to cryopreserve the sperm after arrival, and previous studies have shown that the fertilization potential decreases significantly for frozen–thawed sperm after 48 h of cold storage. 5
For all these reasons, travel duration is the main limitation for shipping epididymides. With the aim of studying the feasibility of shipments exceeding 72 h, we imported the two knock-in strains from the University of Hong-Kong which is situated 10,423.8 km from our location, with an estimated delivery time of 144 h.
All procedures for animal experimentation were approved by the Spanish competent authority. The C57BL/6JRccHsd wild-type mice (Envigo, The Netherlands) used as oocyte donors in the experiments were 3–4 weeks old. The donor sperm came from males of the congenic strains with C57BL/6J backgrounds. Outbred ICR pseudopregnant females were used as embryo transfer recipients.
The first strain (congenic 1) was imported over the summer with hot weather conditions (23–34℃) and which needed two shipments, while the second strain (congenic 2) was imported as one shipment over the winter (2–24℃). The transportation was by commercial couriers using their economy service which did not provide extra cooling. Four epididymides (2 males) were packaged for each shipment following the protocol described by Takeo et al. 3 The second shipment was packaged using a NanoCool® device (NanoCool, Albuquerque, NM, USA) which evaporates water over time, maintaining a constant 2–8℃ environment inside the package for up to 96 h.
The epididymides were first evaluated for gross abnormalities under a stereomicroscope before use, and then washed twice with 2 mL of Research Vitro Fert (K-RVFE-50; Cook Medical, Bloomington, IN, USA) in 35 mm dishes. They were then dried on filter paper, and the sperm were collected and cryopreserved following the procedure by Takeo and Nakagata, 8 and eight straws were obtained per male. Sperm motility was assessed after thawing the test straw. A scale ranging from 0 (no motility) to 5 (very high motility) was used.
The first shipment of congenic 1 strain arrived on time after 144 h. There was no refrigerating provision inside the package (room temperature). The epididymides appeared to be slightly dark and matt, and their walls were not transparent.
The second shipment and the shipment of congenic 2 strain arrived late, after 168 and 192 h, respectively, but the samples remained cold. The epididymides appeared to be brighter, and the sperm could be seen through their walls. These samples obtained ‘1’ point in the motility assessment after thawing, suggesting that most parts of the sperm were non-motile, and it was difficult to observe any with a good forward movement.
To perform in vitro fertilization (IVF), females were superovulated with 5 IU of pregnant mares’ serum gonadotropin (PMSG) (Folligon; Intervet, Boxmeer, The Netherlands), followed by 5 IU of human chorionic gonadotropin (hCG) (Veterin Corion, Divasa, Barcelona, Spain) 48 h later.
The preincubation and fertilization dishes: one drop of preincubation medium (KYD-002-EX, Cosmo Bio, Tokyo, Japan) and one drop of CARD (KYD-003-EX; Cosmo Bio) with three drops of Vitro Fert, respectively covered with paraffin oil (NidOil; Nidacon, Mölndal, Sweden) were prepared the same day of the IVF and were incubated (37℃, 5% CO2) for at least 30 min before use.
We modified the IVF procedure described by Takeo and Nakagata, 9 with the rationale of avoiding problems due to the expected high number of damaged or dead sperm as a consequence of the long transportation time and injuries caused by the chilling. These cells released factors that inhibit IVF capacity. 10 Therefore, at 12 h post-hCG, seven sperm straws were thawed in order to have as much sperm as possible to enable a better selection. The sperm were preincubated in seven different drops for at least 30 min and then a careful selection of the motile sperm from the edge of each drop was performed, avoiding collecting non-motile/dead sperm. Two rounds of sperm selection were carried out, with 5 µL of sperm chosen from each preincubation drop per round. This way we collected motile sperm for up to four IVF drops.
Females were killed by cervical dislocation at approximately 13 h post-hCG. The cumulus–oocyte complexes (COCs) were removed and transferred into the CARD fertilization drop. Next we added the selected sperm suspension, completing the whole procedure within 13.5 h post-hCG. After 10 min, if the COCs were not well dissolved, more sperm were added to the fertilization drop by selecting, as many times as required, 5 µL of sperm with better sperm movement from the preincubation drops. In the end the final volume of the sperm suspension added to the IVF drop ranged from 20 to 30 µL. After 5 h of co-incubation of the gametes, the zygotes were washed into two drops of Vitro Fert and then placed in the last clean drop where they were left to incubate overnight.
Detailed results of the in vitro fertilization (IVF) experiments, and the embryo transfers.
Up to now, 192 h was considered to be the maximum time for recovering embryos (but not live pups) from epididymal sperm cold-stored in the refrigerator, and 72 h for obtaining live pups from frozen–thawed sperm after cold epididymis transportation.3,6 A previous report 7 also suggested that refrigerated epididymis transportation was only useful for transportation times of up to three days.
In our study, we demonstrate that by including sperm selection in IVF treatment it is possible for live pups to be produced using frozen–thawed sperm after 192 h of cold epididymis transportation. Also using epididymis transported for 168 h and later frozen and thawed we obtained a better fertilization rate than that reported 3 for epididymis stored in a refrigerator for the same time and used fresh for IVF (15.6% vs 5.8%).
In conclusion, enhanced transport conditions and careful sperm selection can substantially increase the time over which epididymis remains a suitable alternative to the transport of live animals or the use of dry liquid nitrogen tanks.
Footnotes
Acknowledgements
The authors would like to thank Dr Jorge Sztein and Dr Fernando Benavides for their editing and comments on the original manuscript.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.