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Defined oocyte collection time is critical for reproducible in vitro fertilization in rats of different strains
Theriogenology 144 (2020) 146e151
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Defined oocyte collection time is critical for reproducible in vitro fertilization in rats of different strains Chihiro Hino**, Jun Ueda*, Hiroshi Funakoshi 1, Seiji Matsumoto Center for Advanced Research and Education, Asahikawa Medical University, Asahikawa, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2019 Received in revised form 1 December 2019 Accepted 5 January 2020 Available online 7 January 2020
In vitro fertilization (IVF) is an established technology that is widely used in reproductive engineering. However, in rats, successful application of IVF is difficult to achieve, and it has had poor reproducibility. In a previous study on the critical issues associated with successful IVF in Wistar rats, we investigated the influence of oocyte collection duration on fertilization rates by dividing the procedure into three steps (oviduct extraction from euthanized animals, oocyte collection from the ampullae of oviducts, and oocyte preincubation until insemination), and identified the appropriate times for each. Here we show that use of the same defined duration for oviduct extraction from superovulated Wistar rats and for oocyte collection from the oviducts also produced highly reproducible fertilization rates of more than 90% in other rat strains. Furthermore, the versatility of these criteria was demonstrated using another IVF protocol. Thus, this simple procedure has enabled the standardization of IVF in rats and will enhance further experimental studies. © 2020 Elsevier Inc. All rights reserved.
Keywords: In vitro fertilization Rat Cryopreservation Embryo transfer
1. Introduction In vitro fertilization (IVF) is a well-established technique in reproductive biology that enables the efficient production of fertilized embryos. Since its establishment [1], IVF has been applied to many mammalian species and has revolutionized experimental studies in animals and the treatment of infertile couples [2e4]. In mice, in particular, IVF has been put to practical use in many experimental and breeding facilities worldwide. For example, it has contributed greatly to the preservation of numerous unique strains and genetic mutations and has also been of value for establishing specific-pathogen-free mice. Additionally, the use of IVF makes it easier and less costly to transport embryos of desired mouse strains (including those with genetic modifications) among different facilities. By contrast, IVF is still not widely used for rats, and many facilities use natural mating to obtain embryos. However, the need for robust IVF protocols is increasing, as genetically modified rats are now being produced actively by genome editing technologies
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C. Hino), [email protected] (J. Ueda). 1 Current address: Department of Advanced Medical Science, Asahikawa Medical University, Asahikawa, Japan. https://doi.org/10.1016/j.theriogenology.2020.01.006 0093-691X/© 2020 Elsevier Inc. All rights reserved.
[5,6]. In the rat, IVF was first reported by Miyamoto et al., in 1973 using sperm collected from the uterus of mated females [7]. Subsequently, in 1974, Toyoda et al. succeeded in performing IVF using sperm collected from the cauda epididymidis [8]. Following these initial reports, a number of studies have attempted to optimize IVF in rats. Factors such as the droplet volume for insemination [8], composition of the medium [9,10], sperm preincubation time and concentration [11,12], and mechanisms of sperm capacitation [13,14] have been modified to identify optimal conditions for rat IVF. However, obtaining high reproducibility of IVF in rats has been found to be more difficult than in other species, and it was reported that factors ‘most probably related to technical improvement’ were associate with the reproducibility of this technology [2,15]. Thus far, the critical procedures that affect fertilization rates in rat IVF are still unknown. To date, most studies on IVF in rats have focused on methods for optimizing the fertilization capacity of the sperm suspensions, and few studies have examined the influence of oocyte factors on IVF success. Studies in mice have shown that the interval between euthanasia of female donors and oocyte collection affects the survival rate of oocytes [16], and in some mouse strains, longer intervals reduce the rate of fertilization because of hardening of the zona pellucida [17]. The Center for Animal Resources and Development in Kumamoto University, which conducts mouse
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reproductive technology training worldwide, emphasizes the importance of the time spent in collecting oocytes from the oviduct and recommends 30 s or less for this process (http://card.medic. kumamoto-u.ac.jp/card/english/sigen/manual/mouseivf.html). On the basis of this information from mouse models, we postulated that the duration for oocyte collection from the oviduct might have a critical influence on the success rates of IVF in rats. We, therefore, investigated the influence of this factor on IVF success rate by examining the influence of the duration of each step from euthanasia to oocyte collection. Previously, we showed that highly reproducible and successful IVF in Wistar rats can be achieved by defining the duration for oocyte collection [18]. Here, we tested whether this factor might also be important in other rat strains and in when using another IVF protocol. 2. Materials and methods
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2.4. Collection of oocytes and insemination Immature female rats (4e7 weeks of age) [8,20,21] were induced to superovulate using an intraperitoneal injection of 150e300 IU/kg pregnant mare serum gonadotropin (PMSG; Serotropin, ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) followed approximately 48 h later by 150e300 IU/kg human chorionic gonadotropin (hCG; Gonatropin, ASKA Pharmaceutical Co., Ltd.). At 17e19 h after hCG administration, the superovulated rats were lightly anesthetized using isoflurane inhalation and euthanized by cervical dislocation, except as otherwise indicated. The oviducts were dissected from each female and placed into oocyte collection droplets covered with sterile liquid paraffin oil. The ampulla of each oviduct was disrupted, and the oocyte mass was transferred to HTF droplets and kept at 37 C under 5% CO2 in humidified air. Then, sperm suspensions that had been preincubated for 15e60 min were added to the fertilization droplet at a final concentration of 4.5e5.0 105 sperm/mL for insemination.
2.1. Animals
2.5. Duration of oocyte collections
Wistar (Slc:Wistar) and SD-Tg CAG-EGFP [19] rats were purchased from Japan SLC Inc. (Hamamatsu, Japan), and Jcl:SD and F344/Jcl rats were purchased from CLEA Japan, Inc. (Tokyo, Japan). The breeding stocks were established at the Animal Laboratory for Medical Research, Asahikawa Medical University. For SD-Tg CAGEGFP rats, transgene heterozygote animals were used in this study and genotypes were verified by enhanced green fluorescence protein (EGFP) signals detected by light-emitting diode (LED) emissions using the appropriate filter (Cat. No. LED470-3WOF, OptoCode Co., Ltd., Tokyo, Japan). Rats were maintained in an environment of 22 ± 1 C and 20e80% humidity with 12/12 h light/dark switching at 07:00 and 19:00. All experiments were carried out according to the Guidelines of Animal Experiments of Asahikawa Medical University and all efforts were made to minimize suffering. The animal experiment protocols were approved by the Institutional Animal Care and Use Committee at Asahikawa Medical University (No. 17155, 17164, 17165, 18141).
The oocyte collection process was divided into three time steps: Step 1, from euthanasia (cervical dislocation) to extraction of the oviduct; Step 2, oocyte collection from the oviduct; and Step 3, incubation of oocytes until insemination (Fig. 1A). The times taken for each step were measured.
2.2. Culture medium HTF (Human tubal fluid) medium (ARK Resource Co., Ltd., Kumamoto, Japan) was used for sperm preincubation, fertilization, and embryo transfer. For sperm preincubation, a 200 mL droplet was used. For oocyte collection and IVF, a 100 mL volume droplet was used. Embryos were washed by passing through four such droplets. Each droplet was placed on a 35 mm culture dish (Corning® Cat. No. 430588, Thermo Fisher Scientific Inc., Waltham, MA, USA), covered with liquid paraffin oil (Nacalai Tesque, Inc., Kyoto, Japan), and kept at 37 C under 5% CO2 in humidified air overnight. 2.3. Sperm preincubation Mature male rats (outbred rats aged 10 weeks to 10 months, and inbred rats aged 14 weeks to 10 months) were anesthetized by inhalation of isoflurane (Forane Inhalant Liquid AbbVie Inc., North Chicago, IL, USA) from an anesthesia bottle and euthanized by cervical dislocation. After laparotomy, the epididymides were extracted, and fat and blood were removed by thorough washing. Each cauda epididymidis was covered with liquid paraffin oil for sperm preincubation, cut open with micro-spring scissors, and the sperm were collected and transferred to a droplet of HTF medium and incubated at 37 C under 5% CO2 in humidified air for preincubation.
2.6. Culture and selection of 2-cell stage embryos Approximately 6.5 h after insemination, the oocytes were washed three times with HTF medium and cultured at as above. At 7e8 h after insemination, the oocytes were checked for sperm penetration or pronuclear formation under an inverted microscope to identify any polyspermic fertilization or parthenogenetic embryos (about 6.5% of the total). After culturing for a further 20 h, the numbers of 2-cell stage embryos were counted; these were defined as fertilized embryos. Two-cell stage embryos were used for cryopreservation and later for transfer into the oviducts of pseudopregnant recipient female rats (see below). 2.7. Cryopreservation and thawing of embryos Cryopreservation and thawing of 2-cell stage embryos were performed using established procedures with some modifications [22]. Briefly, embryos were treated with 1.0 M dimethyl sulfoxide (DMSO) in PB1 medium (ARK Resource Co., Ltd., Kumamoto, Japan) [23]; they were first placed in an 80 mL droplet for 2e3 min at room temperature, and then transferred to a second 80 mL droplet for 3e5 min. The embryos, in 5 mL 1.0 M DMSO/PB1, were then transferred to a 1.2 mL cryogenic vial (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and kept at 0 C for 5 min. Ninety-five microliters of DAP213 (2 M DMSO, 1 M acetamide, 3 M propylene glycol) (ARK Resource Co., Ltd., Kumamoto, Japan) were added at 0 C to each vial, and after 5 min at 0 C, the vials were placed into liquid nitrogen at 196 C. Embryos were stored frozen for 1e3 weeks. To thaw the embryos, the vials were removed from the liquid nitrogen and allowed to thaw at room temperature for 60 s. Then, 0.9 mL 0.25 M sucrose in PB1 (0.25 M sucrose, ARK Resource) (preheated to 37 C) were gently added to the cryogenic vial to warm the embryos. The embryo suspension was transferred to a 35 mm culture dish; the embryos were recovered and then washed three times in 100 mL droplets of HTF covered with liquid paraffin oil at 37 C under 5% CO2 as above. The embryos were kept in the first droplet for 5 min, the second droplet for 3 min, and the third droplet very briefly. Thawed embryos were transferred into the oviducts of
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Fig. 1. Effect of the duration of oocyte collection from the oviduct on fertilization rates of Wistar rats. (A) Schematic diagram of the oocyte collection process from superovulated female rats. This process was divided into three time steps: oviduct extraction from euthanized rats (Step 1); oocyte collection from the oviduct ampulla (Step 2); and oocyte incubation until insemination (Step 3). (B) Fertilization rates under different timings of Step 1. (C) Fertilization rates under different timings of Step 2. (D) Fertilization rates under different timings of Step 3. Data are shown in box-and-whisker plots and are modified from previous results [18]. *P < 0.05; N.S., not significant; n indicates the number of female rats used in each experiment; symbols indicate outlier data points. The numbers within the brackets denote the following: total number of 2-cell stage embryos, total number of unfertilized eggs, and total number of fragmented embryos, respectively. Fragmented embryos were not included in calculating fertilization rates. Twenty male rats were used in these experiments.
pseudopregnant recipient female rats (see below). 2.8. Transfer of embryos into oviducts Embryo transfer was performed using previously described procedures with some modifications [24]. Briefly, fresh or frozenethawed embryos produced by IVF were placed in HTF
droplets (without liquid paraffin oil covering) and kept in a 37 C and 5% CO2 incubator with humidified air until use. Pseudopregnant female rats that had been mated with vasoligated males were anesthetized with isoflurane using an inhalation anesthesia machine. Then, the ovaries and oviducts were exposed and fat tissue near the ovaries was clamped for fixation. Next, the embryos (sandwiched by air bubbles) were drawn into a capillary. The
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oviduct wall near the ovarian follicle was cut using micro-spring scissors, the capillary was inserted, and the embryos were transferred into the ampulla. The clamps were removed, and the ovaries and oviducts were returned to their original positions. The rats gave birth 20e21 days after surgery, and the numbers of pups were counted. 2.9. Statistical analysis Statistical significance was calculated with the Steel test and the data item located at the left end in each graph shown in the figures was used for comparison. P < 0.05 was considered statistically significant. In the figures and legends, statistical values are indicated as follows: *P < 0.05; and N.S., not significant. 3. Results 3.1. Effect of the duration of oocyte collection from the oviduct on fertilization rates Our first experiment largely repeated that reported previously using Wistar rats [18], but included an extra time point to obtain more conclusive results. The oocyte collection process for IVF is shown in Fig. 1A. This process was divided into three time steps: from euthanasia (cervical dislocation) to extraction of the oviduct (Step 1); during oocyte collection from the oviduct (Step 2); and during incubation of oocytes until insemination (Step 3). The effects of varying the length of time in each step on IVF success rates are shown in Fig. 1BeD. When Step 1 was performed within 1 min, fertilization rates of 93.8e100% were obtained; the rate of fertilization varied widely from 2.0% to 100% if Step 1 was extended to 1e2 min, from 0% to 90.2% for 2e3 min, and 0%e46.2% for 10 min (Fig. 1B). Next, the influence of Step 2 was examined when Step 1 was fixed at <1 min, as this duration resulted in lower variation in fertilization rates than with longer intervals. Fertilization rates were highest (93.1e100%) when Step 2 was performed within 3 min but when the time was increased to 3e10 min, the fertilization rates declined (65.5e98.6%; Fig. 1C). The influence of Step 3 was examined when Step 1 was within 1 min and Step 2 was carried out within 3 min. Fertilization rates of 91.7e100% were obtained, indicating that Step 3 had no obvious effect on fertilization rates. 3.2. Effect of different methods of euthanasia of female rats on fertilization rates We next investigated how the method of euthanasia of superovulated female rats affected IVF rates. The euthanasia methods examined were consistent with the “AVMA Guidelines for the Euthanasia of Animals (2013 edition)” recommended by the Japanese Association of Laboratory Animal Facilities of National University Corporation [25]. It takes about 20 s to lightly anesthetize by isoflurane, and 20e30 s by carbon dioxide, to render the animal unconscious for cervical dislocation. On the other hand, it takes about 3e4 min to reach respiratory arrest by isoflurane overdose, and 4e5 min by 100% carbon dioxide method. When euthanization was performed by cervical dislocation under light anesthesia, fertilization rates of 87.2e95.7% were obtained (Fig. 2). However, cervical dislocation of rats rendered unconscious by carbon dioxide gave fertilization rates of 71.1e90.6%; euthanasia by isoflurane overdose gave fertilization rates of 8.3e92.1%; and euthanasia using the 100% carbon dioxide method [26] with a flow rate of 20% of the chamber volume per min resulted in fertilization rates of 0e21.6% (Fig. 2).
Fig. 2. Effect of different methods of euthanasia on fertilization rates of Wistar rats. Fertilization rates were compared among four euthanasia methods in female rats. Data are shown in box-and-whisker plots. *P < 0.05; N.S., not significant; n indicates the number of female rats used in each experiment. The numbers within the brackets denote the following: total number of 2-cell stage embryos, total number of unfertilized eggs, and total number of fragmented embryos, respectively. Fragmented embryos were not included in calculating fertilization rates. Five male rats were used in these experiments.
3.3. Effect of time to sperm collection after euthanasia on fertilization rates The influence of the duration for collecting epididymal sperm suspensions on IVF rates was investigated. When the cauda epididymidis was excised immediately after euthanasia and IVF was performed, a fertilization rate of 95.8% was obtained. When the cauda epididymidis was excised 10 min after euthanasia, the fertilization rate was 96.5%, indicating that the duration to collect epididymal sperm suspensions did not have a significant effect on IVF rates (Table 1). 3.4. Cryopreservation and transfer of embryos produced by IVF To confirm that the embryos produced by IVF were capable of generating live pups, 2-cell stage embryos were first cryopreserved and then transferred after thawing into the oviducts of pseudopregnant recipient female rats. A survival rate of 96.0% was found for cryopreserved embryos after thawing. When fresh and frozenethawed embryos were transferred into pseudopregnant recipient rats, 63.3% and 25.0% developed to healthy pups, respectively (Table 2). 3.5. Reproducibility of IVF using another protocol and various rat strains Finally, to test whether the factors identified above would also be relevant to other IVF protocols and rat strains, we performed IVF using the protocol described by Toyoda et al. [8] and the rat strains Jcl:SD, F344/Jcl, and SD-Tg CAG-EGFP. Toyoda et al. used mKRB medium instead of HTF, and different droplet sizes for sperm preincubation and insemination. The IVF conditions that produced reproducible high fertilization rates in Wistar rats (the duration for oocyte collection in IVF; within 1 min for euthanasia to oviduct extraction and within 3 min for oocyte collection from oviducts) were tested. Applying the criteria determined in the Wistar rat experiments above resulted in similar or better fertilization rates using the other protocol and rat strains. Thus, IVF with the protocol of Toyoda et al. [8] resulted in fertilization rates of 87.8e100%. IVF
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Table 1 Effect of time from euthanasia of Wistar male rats to dissection of the cauda epididymidis on success of IVF. One epididymis was excised within 1 min of euthanasia and that on the other side was excised after 10 min. Time
No. of rats
No. of 2-cell embryos/Total no. of embryos
Fertilization rate (%)
1 min 10 min
3 3
136/142 109/113
95.8 96.5
Table 2 Survival and birth rates with/without cryopreservation and transfer of IVF embryos into the oviducts of recipient Wistar rats. Cryopreserved embryos
Fresh embryos
No. of surviving embryos/Total no. of Survival rate No. of embryos (%) recipients
No. of offspring/Total no. of embryos
Birth rate (%)
No. of recipients
No. of offspring/Total no. of embryos
Birth rate (%)
191/199
31/121
25.0
2
19/30
63.3
96.0
6
Fig. 3. Fertilization rates in three other rat strains are comparable to those of Wistar rats. Data are shown in box-and-whisker plots. N.S., not significant; n indicates the number of female rats used in each experiment; symbols indicate outlier data points. The numbers within the brackets denote the following: total number of 2-cell stage embryos, total number of unfertilized eggs, and total number of fragmented embryos, respectively. Fragmented embryos were not included in calculating fertilization rates. Twelve male rats of different strains were used: five Wistar; two SD; three SD-Tg; and two F344/N.
using the other rat strains also resulted in high fertilization rates: Jcl:SD, 96.8%; F344/Jcl, 90.3%; and SD-Tg CAG-EGFP, 96.0% (Fig. 3). Embryos were also tested for cryopreservation and transfer into the oviducts of pseudopregnant recipient female rats for the production of pups. The survival rates of embryos after thawing and birth rates were similar to those found in Wistar rats survival rates Jcl:SD, 93.6%; F344/Jcl, 98.3%; and SD-Tg CAG-EGFP 95.0%; and birth rates: Jcl:SD, 21.0%; F344/Jcl, 14.9%; and SD-Tg CAG-EGFP, 23.4% (Table 3). 4. Discussion We found that the duration of oocyte collection is a key for achieving reproducibly high success rates in IVF in rats. Thus,
oviduct extraction needs to be conducted within 1 min of euthanasia by cervical dislocation (Fig. 1B), and oocyte collection from the ampulla needs to be performed within 3 min (Fig. 1C). If these criteria are followed, reproducible fertilization rates of more than 90% could be achieved. These criteria were also found to be applicable in another rat IVF protocol and to other strains. Various factors that might affect the fertilizing ability of sperm have been examined extensively in the past; however, less attention has been paid to factors that affect oocytes, except for the maturation stage of the oocytes and the appropriate number of oocytes for IVF [15]. To our knowledge, no previous reports have described criteria that critically affect fertilization rates in rat IVF. The oocyte collection time after euthanasia had a critical influence on oocyte survival and fertilization rates in mice [16,27]. Here, the factor with the largest influence on fertilization rate was the duration for dissecting out the oviducts after euthanizing the animal; this could easily take more than 1 min if a time restraint is not imposed. By contrast, in male rats, even when the cauda epididymidis was removed up to 10 min after euthanasia, there was no decrease in fertilization rates (Table 1). From these results, we conclude that oocytes are more sensitive to time after euthanasia than are epididymal sperm suspensions. As the duration after euthanasia of female rats affected fertilization rates, we also investigated whether the method of euthanasia itself might influence IVF success. In the mouse, euthanasia prior to IVF is usually performed by cervical dislocation [28,29]. However, as rats are much larger animals, euthanasia by cervical dislocation alone is technically difficult and is not recommended in some countries [25]. We examined fertilization rates after different euthanization procedures. In these rats, cervical dislocation under light anesthesia by isoflurane gave the highest fertilization rates (Fig. 2). In consistent with our results, there is a report that premature cortical granule exocytosis and F-actin distribution occurred when euthanasia was performed by carbon dioxide, consequently leading to a decreased IVF rate in mice [30]. On the other hand, there is also a report that euthanasia by isoflurane overdose induced the formation of morphologically abnormal oocytes in mice (such as increased number of fragmented, cleaved, and atretic oocytes) [31]. Thus, it might also be possible that
Table 3 Survival and birth rates in three rat strains after cryopreservation and transfer of IVF embryos into oviducts of recipient rats. Strain
No. of surviving embryos/Total no. of embryos
Survival rate (%)
No. of recipients
No. of offspring/Total no. of embryos
Production rate (%)
Jcl:SD F344/Jcl SD-Tg CAG-EGFP
176/188 117/119 114/120
93.6% 98.3% 95.0%
4 4 5
13/62 11/74 22/94
21.0% 14.9% 23.4%
C. Hino et al. / Theriogenology 144 (2020) 146e151
euthanasia by carbon dioxide or isoflurane overdose in rats might have impaired oocyte qualities. The results obtained in this study have confirmed that the duration from euthanasia to oocyte collection critically affects fertilization rates in rat IVF; moreover, this was true for Wistar rats and the other rat strains tested. Our analysis also confirmed the most appropriate method of euthanasia for rat IVF. Our experiments have provided information on factors affecting rat oocytes during IVF, for which limited information was available previously; we have also provided one objective index for ‘technical improvement’, namely limiting the duration for oocyte collection, which was not defined precisely in the past. However, it is worth noting that even if the time limits suggested by our experimental analyses are exceeded, fertilization rates do not decrease necessarily. We speculate that fertilization rates might also be affected by changes inside the body after death, which could have an impact on oocyte quality. Our analyses have clarified the conditions required for reproducibly high fertilization rates; further investigations will be needed to clarify other factors that affect fertilization rates during rat IVF. In recent years, advances in genome editing technology have made it possible to produce genetically modified rat strains with greater ease [5,6,32]. Therefore, it is very likely that the demands for rat IVF will increase in the future. We believe that the simple criteria identified here will benefit standardization of rat IVF protocols and contribute to the reproducibility of this technology. Author contributions section Chihiro Hino: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Roles/Writing - original draft, Writing - review & editing. Jun Ueda: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Roles/Writing original draft, Writing - review & editing. Hiroshi Funakoshi: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing - review & editing. Seiji Matsumoto: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing. Acknowledgements We express our greatest gratitude to Dr Naomi Nakagata for his encouragement, guidance, and support throughout this work. We would also like to thank Dr Tomoji Mashimo for providing mKRB medium, and Drs Fumiaki Itoi and Michihiro Hashimoto for critically reading the manuscript. This work was supported by the Japan Society for the Promotion of Science KAKENHI grants [JP16H01319, JP16K07099, and JP19K06452 to J.U.]; the Takeda Science Foundation (J.U.); Kato Memorial Bioscience Foundation (J.U.); Akiyama Life Science Foundation (J.U.); Grant for Joint Research Program of the Institute for Genetic Medicine, Hokkaido University (J.U., S.M.); Asahikawa Medical University Fund (C.H.) and Grant for Innovative Research in Life Science, Asahikawa Medical University (H.F.). References [1] Yanagimachi R, Chang MC. Fertilization of hamster eggs in vitro. Nature 1963;200:281e2.
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Defined oocyte collection time is critical for reproducible in vitro fertilization in rats of different strains Chihiro Hino**, Jun Ueda*, Hiroshi Funakoshi 1, Seiji Matsumoto Center for Advanced Research and Education, Asahikawa Medical University, Asahikawa, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2019 Received in revised form 1 December 2019 Accepted 5 January 2020 Available online 7 January 2020
In vitro fertilization (IVF) is an established technology that is widely used in reproductive engineering. However, in rats, successful application of IVF is difficult to achieve, and it has had poor reproducibility. In a previous study on the critical issues associated with successful IVF in Wistar rats, we investigated the influence of oocyte collection duration on fertilization rates by dividing the procedure into three steps (oviduct extraction from euthanized animals, oocyte collection from the ampullae of oviducts, and oocyte preincubation until insemination), and identified the appropriate times for each. Here we show that use of the same defined duration for oviduct extraction from superovulated Wistar rats and for oocyte collection from the oviducts also produced highly reproducible fertilization rates of more than 90% in other rat strains. Furthermore, the versatility of these criteria was demonstrated using another IVF protocol. Thus, this simple procedure has enabled the standardization of IVF in rats and will enhance further experimental studies. © 2020 Elsevier Inc. All rights reserved.
Keywords: In vitro fertilization Rat Cryopreservation Embryo transfer
1. Introduction In vitro fertilization (IVF) is a well-established technique in reproductive biology that enables the efficient production of fertilized embryos. Since its establishment [1], IVF has been applied to many mammalian species and has revolutionized experimental studies in animals and the treatment of infertile couples [2e4]. In mice, in particular, IVF has been put to practical use in many experimental and breeding facilities worldwide. For example, it has contributed greatly to the preservation of numerous unique strains and genetic mutations and has also been of value for establishing specific-pathogen-free mice. Additionally, the use of IVF makes it easier and less costly to transport embryos of desired mouse strains (including those with genetic modifications) among different facilities. By contrast, IVF is still not widely used for rats, and many facilities use natural mating to obtain embryos. However, the need for robust IVF protocols is increasing, as genetically modified rats are now being produced actively by genome editing technologies
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C. Hino), [email protected] (J. Ueda). 1 Current address: Department of Advanced Medical Science, Asahikawa Medical University, Asahikawa, Japan. https://doi.org/10.1016/j.theriogenology.2020.01.006 0093-691X/© 2020 Elsevier Inc. All rights reserved.
[5,6]. In the rat, IVF was first reported by Miyamoto et al., in 1973 using sperm collected from the uterus of mated females [7]. Subsequently, in 1974, Toyoda et al. succeeded in performing IVF using sperm collected from the cauda epididymidis [8]. Following these initial reports, a number of studies have attempted to optimize IVF in rats. Factors such as the droplet volume for insemination [8], composition of the medium [9,10], sperm preincubation time and concentration [11,12], and mechanisms of sperm capacitation [13,14] have been modified to identify optimal conditions for rat IVF. However, obtaining high reproducibility of IVF in rats has been found to be more difficult than in other species, and it was reported that factors ‘most probably related to technical improvement’ were associate with the reproducibility of this technology [2,15]. Thus far, the critical procedures that affect fertilization rates in rat IVF are still unknown. To date, most studies on IVF in rats have focused on methods for optimizing the fertilization capacity of the sperm suspensions, and few studies have examined the influence of oocyte factors on IVF success. Studies in mice have shown that the interval between euthanasia of female donors and oocyte collection affects the survival rate of oocytes [16], and in some mouse strains, longer intervals reduce the rate of fertilization because of hardening of the zona pellucida [17]. The Center for Animal Resources and Development in Kumamoto University, which conducts mouse
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reproductive technology training worldwide, emphasizes the importance of the time spent in collecting oocytes from the oviduct and recommends 30 s or less for this process (http://card.medic. kumamoto-u.ac.jp/card/english/sigen/manual/mouseivf.html). On the basis of this information from mouse models, we postulated that the duration for oocyte collection from the oviduct might have a critical influence on the success rates of IVF in rats. We, therefore, investigated the influence of this factor on IVF success rate by examining the influence of the duration of each step from euthanasia to oocyte collection. Previously, we showed that highly reproducible and successful IVF in Wistar rats can be achieved by defining the duration for oocyte collection [18]. Here, we tested whether this factor might also be important in other rat strains and in when using another IVF protocol. 2. Materials and methods
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2.4. Collection of oocytes and insemination Immature female rats (4e7 weeks of age) [8,20,21] were induced to superovulate using an intraperitoneal injection of 150e300 IU/kg pregnant mare serum gonadotropin (PMSG; Serotropin, ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) followed approximately 48 h later by 150e300 IU/kg human chorionic gonadotropin (hCG; Gonatropin, ASKA Pharmaceutical Co., Ltd.). At 17e19 h after hCG administration, the superovulated rats were lightly anesthetized using isoflurane inhalation and euthanized by cervical dislocation, except as otherwise indicated. The oviducts were dissected from each female and placed into oocyte collection droplets covered with sterile liquid paraffin oil. The ampulla of each oviduct was disrupted, and the oocyte mass was transferred to HTF droplets and kept at 37 C under 5% CO2 in humidified air. Then, sperm suspensions that had been preincubated for 15e60 min were added to the fertilization droplet at a final concentration of 4.5e5.0 105 sperm/mL for insemination.
2.1. Animals
2.5. Duration of oocyte collections
Wistar (Slc:Wistar) and SD-Tg CAG-EGFP [19] rats were purchased from Japan SLC Inc. (Hamamatsu, Japan), and Jcl:SD and F344/Jcl rats were purchased from CLEA Japan, Inc. (Tokyo, Japan). The breeding stocks were established at the Animal Laboratory for Medical Research, Asahikawa Medical University. For SD-Tg CAGEGFP rats, transgene heterozygote animals were used in this study and genotypes were verified by enhanced green fluorescence protein (EGFP) signals detected by light-emitting diode (LED) emissions using the appropriate filter (Cat. No. LED470-3WOF, OptoCode Co., Ltd., Tokyo, Japan). Rats were maintained in an environment of 22 ± 1 C and 20e80% humidity with 12/12 h light/dark switching at 07:00 and 19:00. All experiments were carried out according to the Guidelines of Animal Experiments of Asahikawa Medical University and all efforts were made to minimize suffering. The animal experiment protocols were approved by the Institutional Animal Care and Use Committee at Asahikawa Medical University (No. 17155, 17164, 17165, 18141).
The oocyte collection process was divided into three time steps: Step 1, from euthanasia (cervical dislocation) to extraction of the oviduct; Step 2, oocyte collection from the oviduct; and Step 3, incubation of oocytes until insemination (Fig. 1A). The times taken for each step were measured.
2.2. Culture medium HTF (Human tubal fluid) medium (ARK Resource Co., Ltd., Kumamoto, Japan) was used for sperm preincubation, fertilization, and embryo transfer. For sperm preincubation, a 200 mL droplet was used. For oocyte collection and IVF, a 100 mL volume droplet was used. Embryos were washed by passing through four such droplets. Each droplet was placed on a 35 mm culture dish (Corning® Cat. No. 430588, Thermo Fisher Scientific Inc., Waltham, MA, USA), covered with liquid paraffin oil (Nacalai Tesque, Inc., Kyoto, Japan), and kept at 37 C under 5% CO2 in humidified air overnight. 2.3. Sperm preincubation Mature male rats (outbred rats aged 10 weeks to 10 months, and inbred rats aged 14 weeks to 10 months) were anesthetized by inhalation of isoflurane (Forane Inhalant Liquid AbbVie Inc., North Chicago, IL, USA) from an anesthesia bottle and euthanized by cervical dislocation. After laparotomy, the epididymides were extracted, and fat and blood were removed by thorough washing. Each cauda epididymidis was covered with liquid paraffin oil for sperm preincubation, cut open with micro-spring scissors, and the sperm were collected and transferred to a droplet of HTF medium and incubated at 37 C under 5% CO2 in humidified air for preincubation.
2.6. Culture and selection of 2-cell stage embryos Approximately 6.5 h after insemination, the oocytes were washed three times with HTF medium and cultured at as above. At 7e8 h after insemination, the oocytes were checked for sperm penetration or pronuclear formation under an inverted microscope to identify any polyspermic fertilization or parthenogenetic embryos (about 6.5% of the total). After culturing for a further 20 h, the numbers of 2-cell stage embryos were counted; these were defined as fertilized embryos. Two-cell stage embryos were used for cryopreservation and later for transfer into the oviducts of pseudopregnant recipient female rats (see below). 2.7. Cryopreservation and thawing of embryos Cryopreservation and thawing of 2-cell stage embryos were performed using established procedures with some modifications [22]. Briefly, embryos were treated with 1.0 M dimethyl sulfoxide (DMSO) in PB1 medium (ARK Resource Co., Ltd., Kumamoto, Japan) [23]; they were first placed in an 80 mL droplet for 2e3 min at room temperature, and then transferred to a second 80 mL droplet for 3e5 min. The embryos, in 5 mL 1.0 M DMSO/PB1, were then transferred to a 1.2 mL cryogenic vial (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and kept at 0 C for 5 min. Ninety-five microliters of DAP213 (2 M DMSO, 1 M acetamide, 3 M propylene glycol) (ARK Resource Co., Ltd., Kumamoto, Japan) were added at 0 C to each vial, and after 5 min at 0 C, the vials were placed into liquid nitrogen at 196 C. Embryos were stored frozen for 1e3 weeks. To thaw the embryos, the vials were removed from the liquid nitrogen and allowed to thaw at room temperature for 60 s. Then, 0.9 mL 0.25 M sucrose in PB1 (0.25 M sucrose, ARK Resource) (preheated to 37 C) were gently added to the cryogenic vial to warm the embryos. The embryo suspension was transferred to a 35 mm culture dish; the embryos were recovered and then washed three times in 100 mL droplets of HTF covered with liquid paraffin oil at 37 C under 5% CO2 as above. The embryos were kept in the first droplet for 5 min, the second droplet for 3 min, and the third droplet very briefly. Thawed embryos were transferred into the oviducts of
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Fig. 1. Effect of the duration of oocyte collection from the oviduct on fertilization rates of Wistar rats. (A) Schematic diagram of the oocyte collection process from superovulated female rats. This process was divided into three time steps: oviduct extraction from euthanized rats (Step 1); oocyte collection from the oviduct ampulla (Step 2); and oocyte incubation until insemination (Step 3). (B) Fertilization rates under different timings of Step 1. (C) Fertilization rates under different timings of Step 2. (D) Fertilization rates under different timings of Step 3. Data are shown in box-and-whisker plots and are modified from previous results [18]. *P < 0.05; N.S., not significant; n indicates the number of female rats used in each experiment; symbols indicate outlier data points. The numbers within the brackets denote the following: total number of 2-cell stage embryos, total number of unfertilized eggs, and total number of fragmented embryos, respectively. Fragmented embryos were not included in calculating fertilization rates. Twenty male rats were used in these experiments.
pseudopregnant recipient female rats (see below). 2.8. Transfer of embryos into oviducts Embryo transfer was performed using previously described procedures with some modifications [24]. Briefly, fresh or frozenethawed embryos produced by IVF were placed in HTF
droplets (without liquid paraffin oil covering) and kept in a 37 C and 5% CO2 incubator with humidified air until use. Pseudopregnant female rats that had been mated with vasoligated males were anesthetized with isoflurane using an inhalation anesthesia machine. Then, the ovaries and oviducts were exposed and fat tissue near the ovaries was clamped for fixation. Next, the embryos (sandwiched by air bubbles) were drawn into a capillary. The
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oviduct wall near the ovarian follicle was cut using micro-spring scissors, the capillary was inserted, and the embryos were transferred into the ampulla. The clamps were removed, and the ovaries and oviducts were returned to their original positions. The rats gave birth 20e21 days after surgery, and the numbers of pups were counted. 2.9. Statistical analysis Statistical significance was calculated with the Steel test and the data item located at the left end in each graph shown in the figures was used for comparison. P < 0.05 was considered statistically significant. In the figures and legends, statistical values are indicated as follows: *P < 0.05; and N.S., not significant. 3. Results 3.1. Effect of the duration of oocyte collection from the oviduct on fertilization rates Our first experiment largely repeated that reported previously using Wistar rats [18], but included an extra time point to obtain more conclusive results. The oocyte collection process for IVF is shown in Fig. 1A. This process was divided into three time steps: from euthanasia (cervical dislocation) to extraction of the oviduct (Step 1); during oocyte collection from the oviduct (Step 2); and during incubation of oocytes until insemination (Step 3). The effects of varying the length of time in each step on IVF success rates are shown in Fig. 1BeD. When Step 1 was performed within 1 min, fertilization rates of 93.8e100% were obtained; the rate of fertilization varied widely from 2.0% to 100% if Step 1 was extended to 1e2 min, from 0% to 90.2% for 2e3 min, and 0%e46.2% for 10 min (Fig. 1B). Next, the influence of Step 2 was examined when Step 1 was fixed at <1 min, as this duration resulted in lower variation in fertilization rates than with longer intervals. Fertilization rates were highest (93.1e100%) when Step 2 was performed within 3 min but when the time was increased to 3e10 min, the fertilization rates declined (65.5e98.6%; Fig. 1C). The influence of Step 3 was examined when Step 1 was within 1 min and Step 2 was carried out within 3 min. Fertilization rates of 91.7e100% were obtained, indicating that Step 3 had no obvious effect on fertilization rates. 3.2. Effect of different methods of euthanasia of female rats on fertilization rates We next investigated how the method of euthanasia of superovulated female rats affected IVF rates. The euthanasia methods examined were consistent with the “AVMA Guidelines for the Euthanasia of Animals (2013 edition)” recommended by the Japanese Association of Laboratory Animal Facilities of National University Corporation [25]. It takes about 20 s to lightly anesthetize by isoflurane, and 20e30 s by carbon dioxide, to render the animal unconscious for cervical dislocation. On the other hand, it takes about 3e4 min to reach respiratory arrest by isoflurane overdose, and 4e5 min by 100% carbon dioxide method. When euthanization was performed by cervical dislocation under light anesthesia, fertilization rates of 87.2e95.7% were obtained (Fig. 2). However, cervical dislocation of rats rendered unconscious by carbon dioxide gave fertilization rates of 71.1e90.6%; euthanasia by isoflurane overdose gave fertilization rates of 8.3e92.1%; and euthanasia using the 100% carbon dioxide method [26] with a flow rate of 20% of the chamber volume per min resulted in fertilization rates of 0e21.6% (Fig. 2).
Fig. 2. Effect of different methods of euthanasia on fertilization rates of Wistar rats. Fertilization rates were compared among four euthanasia methods in female rats. Data are shown in box-and-whisker plots. *P < 0.05; N.S., not significant; n indicates the number of female rats used in each experiment. The numbers within the brackets denote the following: total number of 2-cell stage embryos, total number of unfertilized eggs, and total number of fragmented embryos, respectively. Fragmented embryos were not included in calculating fertilization rates. Five male rats were used in these experiments.
3.3. Effect of time to sperm collection after euthanasia on fertilization rates The influence of the duration for collecting epididymal sperm suspensions on IVF rates was investigated. When the cauda epididymidis was excised immediately after euthanasia and IVF was performed, a fertilization rate of 95.8% was obtained. When the cauda epididymidis was excised 10 min after euthanasia, the fertilization rate was 96.5%, indicating that the duration to collect epididymal sperm suspensions did not have a significant effect on IVF rates (Table 1). 3.4. Cryopreservation and transfer of embryos produced by IVF To confirm that the embryos produced by IVF were capable of generating live pups, 2-cell stage embryos were first cryopreserved and then transferred after thawing into the oviducts of pseudopregnant recipient female rats. A survival rate of 96.0% was found for cryopreserved embryos after thawing. When fresh and frozenethawed embryos were transferred into pseudopregnant recipient rats, 63.3% and 25.0% developed to healthy pups, respectively (Table 2). 3.5. Reproducibility of IVF using another protocol and various rat strains Finally, to test whether the factors identified above would also be relevant to other IVF protocols and rat strains, we performed IVF using the protocol described by Toyoda et al. [8] and the rat strains Jcl:SD, F344/Jcl, and SD-Tg CAG-EGFP. Toyoda et al. used mKRB medium instead of HTF, and different droplet sizes for sperm preincubation and insemination. The IVF conditions that produced reproducible high fertilization rates in Wistar rats (the duration for oocyte collection in IVF; within 1 min for euthanasia to oviduct extraction and within 3 min for oocyte collection from oviducts) were tested. Applying the criteria determined in the Wistar rat experiments above resulted in similar or better fertilization rates using the other protocol and rat strains. Thus, IVF with the protocol of Toyoda et al. [8] resulted in fertilization rates of 87.8e100%. IVF
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Table 1 Effect of time from euthanasia of Wistar male rats to dissection of the cauda epididymidis on success of IVF. One epididymis was excised within 1 min of euthanasia and that on the other side was excised after 10 min. Time
No. of rats
No. of 2-cell embryos/Total no. of embryos
Fertilization rate (%)
1 min 10 min
3 3
136/142 109/113
95.8 96.5
Table 2 Survival and birth rates with/without cryopreservation and transfer of IVF embryos into the oviducts of recipient Wistar rats. Cryopreserved embryos
Fresh embryos
No. of surviving embryos/Total no. of Survival rate No. of embryos (%) recipients
No. of offspring/Total no. of embryos
Birth rate (%)
No. of recipients
No. of offspring/Total no. of embryos
Birth rate (%)
191/199
31/121
25.0
2
19/30
63.3
96.0
6
Fig. 3. Fertilization rates in three other rat strains are comparable to those of Wistar rats. Data are shown in box-and-whisker plots. N.S., not significant; n indicates the number of female rats used in each experiment; symbols indicate outlier data points. The numbers within the brackets denote the following: total number of 2-cell stage embryos, total number of unfertilized eggs, and total number of fragmented embryos, respectively. Fragmented embryos were not included in calculating fertilization rates. Twelve male rats of different strains were used: five Wistar; two SD; three SD-Tg; and two F344/N.
using the other rat strains also resulted in high fertilization rates: Jcl:SD, 96.8%; F344/Jcl, 90.3%; and SD-Tg CAG-EGFP, 96.0% (Fig. 3). Embryos were also tested for cryopreservation and transfer into the oviducts of pseudopregnant recipient female rats for the production of pups. The survival rates of embryos after thawing and birth rates were similar to those found in Wistar rats survival rates Jcl:SD, 93.6%; F344/Jcl, 98.3%; and SD-Tg CAG-EGFP 95.0%; and birth rates: Jcl:SD, 21.0%; F344/Jcl, 14.9%; and SD-Tg CAG-EGFP, 23.4% (Table 3). 4. Discussion We found that the duration of oocyte collection is a key for achieving reproducibly high success rates in IVF in rats. Thus,
oviduct extraction needs to be conducted within 1 min of euthanasia by cervical dislocation (Fig. 1B), and oocyte collection from the ampulla needs to be performed within 3 min (Fig. 1C). If these criteria are followed, reproducible fertilization rates of more than 90% could be achieved. These criteria were also found to be applicable in another rat IVF protocol and to other strains. Various factors that might affect the fertilizing ability of sperm have been examined extensively in the past; however, less attention has been paid to factors that affect oocytes, except for the maturation stage of the oocytes and the appropriate number of oocytes for IVF [15]. To our knowledge, no previous reports have described criteria that critically affect fertilization rates in rat IVF. The oocyte collection time after euthanasia had a critical influence on oocyte survival and fertilization rates in mice [16,27]. Here, the factor with the largest influence on fertilization rate was the duration for dissecting out the oviducts after euthanizing the animal; this could easily take more than 1 min if a time restraint is not imposed. By contrast, in male rats, even when the cauda epididymidis was removed up to 10 min after euthanasia, there was no decrease in fertilization rates (Table 1). From these results, we conclude that oocytes are more sensitive to time after euthanasia than are epididymal sperm suspensions. As the duration after euthanasia of female rats affected fertilization rates, we also investigated whether the method of euthanasia itself might influence IVF success. In the mouse, euthanasia prior to IVF is usually performed by cervical dislocation [28,29]. However, as rats are much larger animals, euthanasia by cervical dislocation alone is technically difficult and is not recommended in some countries [25]. We examined fertilization rates after different euthanization procedures. In these rats, cervical dislocation under light anesthesia by isoflurane gave the highest fertilization rates (Fig. 2). In consistent with our results, there is a report that premature cortical granule exocytosis and F-actin distribution occurred when euthanasia was performed by carbon dioxide, consequently leading to a decreased IVF rate in mice [30]. On the other hand, there is also a report that euthanasia by isoflurane overdose induced the formation of morphologically abnormal oocytes in mice (such as increased number of fragmented, cleaved, and atretic oocytes) [31]. Thus, it might also be possible that
Table 3 Survival and birth rates in three rat strains after cryopreservation and transfer of IVF embryos into oviducts of recipient rats. Strain
No. of surviving embryos/Total no. of embryos
Survival rate (%)
No. of recipients
No. of offspring/Total no. of embryos
Production rate (%)
Jcl:SD F344/Jcl SD-Tg CAG-EGFP
176/188 117/119 114/120
93.6% 98.3% 95.0%
4 4 5
13/62 11/74 22/94
21.0% 14.9% 23.4%
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euthanasia by carbon dioxide or isoflurane overdose in rats might have impaired oocyte qualities. The results obtained in this study have confirmed that the duration from euthanasia to oocyte collection critically affects fertilization rates in rat IVF; moreover, this was true for Wistar rats and the other rat strains tested. Our analysis also confirmed the most appropriate method of euthanasia for rat IVF. Our experiments have provided information on factors affecting rat oocytes during IVF, for which limited information was available previously; we have also provided one objective index for ‘technical improvement’, namely limiting the duration for oocyte collection, which was not defined precisely in the past. However, it is worth noting that even if the time limits suggested by our experimental analyses are exceeded, fertilization rates do not decrease necessarily. We speculate that fertilization rates might also be affected by changes inside the body after death, which could have an impact on oocyte quality. Our analyses have clarified the conditions required for reproducibly high fertilization rates; further investigations will be needed to clarify other factors that affect fertilization rates during rat IVF. In recent years, advances in genome editing technology have made it possible to produce genetically modified rat strains with greater ease [5,6,32]. Therefore, it is very likely that the demands for rat IVF will increase in the future. We believe that the simple criteria identified here will benefit standardization of rat IVF protocols and contribute to the reproducibility of this technology. Author contributions section Chihiro Hino: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Roles/Writing - original draft, Writing - review & editing. Jun Ueda: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Roles/Writing original draft, Writing - review & editing. Hiroshi Funakoshi: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing - review & editing. Seiji Matsumoto: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing. Acknowledgements We express our greatest gratitude to Dr Naomi Nakagata for his encouragement, guidance, and support throughout this work. We would also like to thank Dr Tomoji Mashimo for providing mKRB medium, and Drs Fumiaki Itoi and Michihiro Hashimoto for critically reading the manuscript. This work was supported by the Japan Society for the Promotion of Science KAKENHI grants [JP16H01319, JP16K07099, and JP19K06452 to J.U.]; the Takeda Science Foundation (J.U.); Kato Memorial Bioscience Foundation (J.U.); Akiyama Life Science Foundation (J.U.); Grant for Joint Research Program of the Institute for Genetic Medicine, Hokkaido University (J.U., S.M.); Asahikawa Medical University Fund (C.H.) and Grant for Innovative Research in Life Science, Asahikawa Medical University (H.F.). References [1] Yanagimachi R, Chang MC. Fertilization of hamster eggs in vitro. Nature 1963;200:281e2.
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