Journal of Embryo Transfer 2016; 31(4): 367-374
Published online December 29, 2016
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Dan-Bi Lee1,#, Kee Sang Park2, and Byoung Boo Seo1,3,†
*This research was supported by the Daegu University Research Grant, 2014.
Correspondence to: Correspondence: Byoung Boo Seo,
This study was conducted to evaluate the effect of transfer temperature of epididymis on survival rate of semen and development ability of B6D2F1 mice embryos. No significant differences were noted in the survival rate of semen (59.0% ± 0.1 vs. 47.6% ± 0.1), in vitro fertilization rate (90.7% ± 0.1 vs. 90.7% ± 0.1), developmental rate (90.0% ± 0.1 vs. 90.0% ± 0.1), and blastocysts formation rate (53.1% ± 0.2 vs. 52.3% ± 0.2) between groups. (
Taken altogether, it is expected to achieve the best developmental ability of B6D2F1 mice embryos in the transfer temperature of epididymis. Also these results can provide fundamental data to maximize culture condition for
Keywords: B6D2F1 mice, epididymis,
Since it was first introduced in canine by Spallanzani in 1780, many
In the current study, hybrid B6D2F1 mice (maternal line: C57BL/6NCrIBR; paternal line: DBA/2CrIBR) were used. Female mice were 5~8 weeks old whereas male was 9~13 weeks old (OrientBio Co., City, South Korea). Upon arrival, experimental animals were acclimated to breeding conditions over a week to minimize stress in advance to implement experiments. For animals, lights were maintained on a 12:12 h light/dark cycle (Light: 7:00~19:00, Dark: 19:00~7:00). Temperature and humidity were maintained at 22~24°C and 40~50%, respectively. Mice were fed ad libitum and animal handling and experiments followed the guideline of Daegu University Animal Experiment Ethics Committee (DUIACC-2014 -02-0509-101).B6D2F1 mice culture media and oil
The G1 plus culture media (Vitrolife, Sweden) and the Cleavage medium (CM) (COOk Medical, Austraila) were used up to 2PN~8 cell stages. After, the G2 plus culture media (Vitrolife, Sweden) and the Blastocyst medium (BM) (COOk Medical, Australia) were utilized from 8 cell stage through blastula stage. Suspension culture media for sperms was the GIVF (Vitrolife, Sweden), while capacitation sperm and in vitro fertilization culture media was the fertilization medium (FM, COOk Medical, Australia). Last, the OVOIL (Vitrolife, 10029, Sweden) and the OIL (Sage, 4008-5P, USA) were used to coat culture media.Preparation of mice oocytes
To achieve more oocytes, animals were subjected to superovulation in which female B6D2F1 mice (5~8 weeks old) were injected with 7.5 IU of pregnant mare's serum gonadotropin (PMSG, SIGMA, USA) 48 hr prior to oocyte maturation experiments. In order to improve experimental accuracy and minimize variations, the hormone was injected at 4:00 in the afternoon. After 48 hr of hormone injection, animals were killed through dislocation of the cervical vertebrae to collect ovaries. Upon collection, excessive blood and adipose tissues of collected ovaries (or epididymis) were removed using sterilized gauze and then transferred to the laboratory. Experiments were performed in the Infertility Clinic of Obstetrics and Genecology, Kyungpook National University Hospital. Ovaries were placed in Eppendorf test tubes containing DPBS (Dulbecco's Phosphate-Buffered Saline, Gibco, USA) + 10% FBS to be transferred. The test tubes were covered with aluminum foil to block light and then further covered with sterilized gauze; to maintain the ovaries at specific temperature (i.e., 37°C), the test tubes were directly attached to body while transporting. On the other hand, epididymis samples were stored in Eppendorf test tubes containing mineral oil (Sigma, USA) and transferred at either at 4 °C or 37°C. To keep the temperature at 4°C, test tubes were stored in a Styrofoam box containing ice packs (Fig. 1) whereas the other case, 37°C, was similar to that of ovary transport. Transferred ovaries were moved to culture dishes (Falcon, USA) containing 2 ml of PBS + 10% FSB and folliculus were ruptures using a syringe needle under a dissecting microscope to collect immature oocytes. Likewise, release of sperm pellet was induced from epididymis in GIVF culture media.
Prepare of organ transport.
A: Preparing for the transport eppendorf tube.
B: 4°C epididymis transportation methods by using styrofoam box.
Collected B6D2F1 mice oocytes were washed once more using PBS buffer supplemented with 20% FBS and then subjected to maturation culture. As depicted in the Figure 1, oocytes were classified into 1) Cumulus-oocyte complexes (COC), 2) Cumulus-oocyte partial contact (CO), or 3) Denuded oocyte (O). The COC was defined if oocytes were densely and uniformly covered by cumulus cells. The CO and O were defined in which oocytes were either partially covered or not covered by the cumulus cells, respectively. In order to compare effects of types of oils, the OVOIL or OIL was applied to 50 uL of droplet to perform in vitro maturation of COC. Approximately after 19 hours of in vitro maturation, dilated cumulus cells around oocytes were removed and the maturation degree was determined as follow: 1) metaphase II (MII) where the first polar body was released, 2) anaphase I (AI) where the first polar body has not been released, or 3) metaphase I (MI) where the first polar body has not been formed. Selected COC were subjected to further in vitro fertilization experiments.
As shown in the Figure 1, remaining mineral oil on transferred epididymis was removed using sterilized gauze. In order to induce suspension, sperms were transferred in 100 uL of sperm suspension culture media (GIVF), spread with the OVOIL; and, under a dissecting microscope, epididymis was ruptured using a dissecting forceps and syringe needle to release sperm pellets. After, sperm suspension was induced by placing samples in an incubator maintaining 37°C and 6% CO2 (Heraeus BB6620, Germany) for 15 minutes. Suspension solution (100 uL) was then transferred to the capacitation culture media (FM) and incubated over 1.5 hours to perform sperm capacitation. While capacitating, sperm concentrations and their activity were monitored using a sperm counter (Makler counting chamber, Sefi Medical Instruments, Israel) and inverted microscope (Olympus, BX-50, Japan), respectively. To investigate effects of transfer temperature of epididymis, sperms acquired from different temperature conditions (i.e., 4°C vs 37°C) were compared. The sperm was applied into fertilization culture media at a concentration of 2 × 106/mL and the fertilization experiment was carried out over 4 ~ 6 hours.
Differential fluorescence staining of B6D2F1 mouse blastocyst (x200).
A: blastocyst from B6D2F1 mouse; B: stained blastocyst by propidium iodide and bisbenzimide, TE cells stained by propidium iodide (PI, Red), ICM cells stained by bisbenzimide (Blue).
In order to remove any potential bias, experiment batches and observations were planned in a completely randomized design. Experimental results were expressed as percentage and standard deviation of discontinuous variables and statistical significance were tested using the SAS program (Statistics Analytical System, version 9.4, USA). Statistical significance between groups was examined using the LSD and Duncan’s multiple range tests and p-value less than 0.05 was considered as statistically significant.
This experiment was done to confirm the survival rate of sperm in response to different transfer temperature of epididymis (4°C vs. 37°C). The survival rates of B6D2F1 mouse sperms are summarized in the Table 1. It was 59.0% ± 0.1 and 47.6% ± 0.1 in the 4°C and 37°C group, respectively. Although the survival rate was slightly higher in the 4°C group yet no statistical significance was noted between groups.
In order to find if transfer temperature of B6D2F1 mouse epididymis influence on fertilization rate, cleavage rate, and blastocyst rate, immature oocytes were matured in vitro and, after 24 hr of fertilization, completely cleaved 2-cell stage embryos were used for the experiment. In each group, a total of 100 embryos were included and respective effects on fertilization rate, cleavage rate, and blastocyst rate are shown in the Table 2. As shown, depending on the transfer temperature (4°C vs. 37°C), the fertilization rate and cleavage rate were 90.7% ± 0.1 vs. 90.7% ± 0.1 and 90.0% ± 0.1 vs. 90.0% ± 0.1, respectively. Further the blastocyst rate was 53.1% ± 0.2 and 52.3% ± 0.2 in the 4°C and 37°C group respectively. There was no statistical significance noted between groups. Even though it was not statistically significant, the blastocyst rate in the 4°C group tends to be slightly higher compared to that of 37°C group.
On the 5th day of in vitro fertilization, blastocysts were subjected to differential fluorescence staining and then cell numbers were compared between groups (i.e., 4°C vs. 37°C; Table 3). Regardless of the transfer temperature, there was no difference in ICM of blastocysts, developed from fertilized B6D2F1 mouse oocytes (17.0 ± 7.8 vs. 14.6 ± 8.6), TE cells (55.8 ± 29.8 vs. 64.0 ±24.4), total cell numbers (72.7 ± 31.6 vs. 62.0 ± 36.6) and ICM:TE ratio (1:4.2 ± 4.1 vs. 1:6.4 ± 7.2). Although ICM of blastocysts were higher in the 4°C group TE cell numbers were higher in the other group. On the other hand, % ICM was significantly higher in the 4°C group (27.0% ± 0.1) compared to the 37°C group (18.3% ± 0.1;
To find effects of different transfer temperature of B6D2F1 mouse epididymis on zona hatched rate, B6D2F1 mouse embryos were fertilized and then cultured for 7 days. Results are summarized in the Figure 3. As shown, the hatched rate of B6D2F1 mouse blastocysts was higher in the 4°C group compared to the 37°C group (47.8% ± 0.1 vs. 25.6% ± 0.1;
Effect of epididymis transport temperatures on zona hatched rate of B6D2F1 mice blastocysts.
Each value is mean±standard deviation.
a,b: Means with different superscript within a row were significantly
Over last several decades, human in vitro fertilization and embryos culture techniques have been rapidly improved including its related culture techniques, culture apparatus as well as culture materials. However, origins of raw materials and their quality, used for human in vitro fertilization and embryo culture can slightly vary depending on manufacturers. And, oocytes and embryos are continuously exposed to coated oil and plastic dishes. For instance, it was addressed in multiple previous reports that negative effects might be shown by toxic substances from culture media; further secreted oil from cumulus cells may influence on absorptivity as well (
In the present study, the authors examined effects of transfer temperature (4°C vs. 37°C) of B6D2F1 mouse epididymis on sperm survival rate, and embryonic development after in vitro fertilization. As aforementioned, even though the survival rate of sperms was slightly higher in the 4°C group compared to the 37°C group yet no statistical significance was shown. In contrast, the blastulation rate was shown to be slightly higher in the 37°C group than the 4°C group.
No differences in ICM cell numbers of blastocysts, TE cells, total cell counts, and ICM:TE ratio were demonstrated between difference temperature, tested in the study. Of note, although ICM cell numbers of blastocysts were slightly higher in the 4°C group yet TE cells were higher in the other group. Lastly, %ICM and zona hatched rate of blastocysts were higher in the 4°C group (
Taken altogether, transportation of epididymis at 4°C might be better in later development of mouse blastocysts as opposed to 37°C. Given our results herein, it might be also applicable for long-distance transportation of epididymis spermatozoa as well. Further, our results may provide important preliminary data for not limited to optimizing B6D2F1 mouse in vitro fertilization y