Optimizing Rat In Vitro Fertilization for Rat Model Cryo-Resuscitation from Frozen–Thawed Sperm

TL;DR

This study improves rat in vitro fertilization (IVF) to replace the costly ICSI method for recovering rat models from frozen sperm, making the process more accessible and efficient.

Contribution

The study optimizes rat IVF protocols to enable reliable cryo-resuscitation of rat models using frozen sperm, reducing reliance on ICSI.

Findings

Modified IVF procedures can be completed within a 9-hour workday with acceptable embryo development rates.

Cleavage and blastocyst rates from frozen sperm IVF are sufficient for rat model cryo-resuscitation.

Strain-specific differences in oocyte maturation timing exist but adjusting protocols for LE rats did not improve outcomes.

Abstract

Rat is one of the most important models in biomedical research. Like mice, rat models are also maintained as frozen sperm, frozen embryos or both. However, the recovery of rat models from frozen sperm must rely on costly and difficult procedure-intracytoplasmic sperm injection (ICSI) in contrast to mouse model’s recovery through in vitro fertilization (IVF), which is relatively simple and cost-effective. Recent advances in rat IVF have made it possible in replacement of ICSI, though significant variation still exists among different strains/stocks. The present study aimed at optimizing the current IVF protocol so that it can be used as a reliable, cost-effective alternative to ICSI for rat model recovery. The results showed that satisfactory cleavage and blastocyst rates can be obtained with limited repetition of IVF. By adjusting the IVF procedures, IVF can be accomplished within a regular 9 h workday schedule. Modified superovulation protocol based on the timing of first polar body extrusion in LE strain did not result in significant improvement in IVF outcomes. Current results demonstrate that IVF can be routinely used for cryorecovery of rat lines with ICSI as a back-up procedure in situations where repeated IVF failures occur.

Optimizing cryo-resuscitation from frozen sperm would improve access to cryopreserved rat models. In this study, the possibility of replacing intracytoplasmic sperm injection (ICSI) with in vitro fertilization (IVF) for model cryo-resuscitation from frozen–thawed sperm was investigated. Rat IVF protocol was modified to allow the procedures to be performed during a 9 h workday. The possibility of genetic background-specific modification of the superovulation protocol for the improvement in IVF outcomes was explored. Wild-type and genetically modified Sprague Dawley (SD), Long Evans (LE) and Fischer 344 (F344) rats were used. Sperm freezing and IVF were conducted as previously described. Cleavage, blastocyst formation and hatching of the resulting embryos were used to assess their developmental potential in vitro. The results showed that, with limited repetitions, current sperm freezing and IVF protocols resulted in cleavage rates ranging from 58 ± 11% to 87 ± 7% and blastocyst rates ranging from 21 ± 25% to 54 ± 23%, which are acceptable for the cryo-resuscitation of rat models. With slight modifications, the procedure can be fit into a 9 h workday (SD: 48 ± 35%; F344: 36 ± 13%). Strain/stock-specific differences in oocyte maturation timing were observed: LE females had a two-hour delay compared to SD and F344 rats in response to the same superovulation protocol. However, modifying the protocol for LE rats did not significantly improve IVF outcomes (34 ± 6 vs. 32 ± 12%). Overall, while IVF with frozen–thawed sperm is a promising alternative to ICSI, significant variability remains across strains/stocks and protocols. Continued research is necessary to advance our understanding of factors affecting the efficiency and repeatability of rat sperm freezing and IVF.

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1. Introduction

The rat has been one of the major experimental model organisms in biomedical research for more than a century because of its genetic and physiological similarities to humans and has played an important role in understanding mechanisms of human diseases, being invaluable in developing new treatments and therapeutics [1]. The creation of rat models with targeted mutations has been greatly accelerated due to the development of embryonic stem cell lines and the ability to make genetic modifications to its genome using genome editing tools, such as Zinc-Finger Nucleases, Transcription Activator-Like Effector Nucleases, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) [2,3,4,5]. In particular, the simplicity and target-specificity of the CRISPR/Cas9 system have already enabled the generation of rat models at an even faster rate.

Animal models are usually cryo-archived as frozen germplasm (sperm, embryos or both). For example, models with single gene mutations on a commercially available strain/stock background are usually cryo-archived as frozen sperm as it is simple, cost-effective, and does not require many animals. Efficient and cost-effective methods to resuscitate animal models from frozen sperm are essential for the biomedical community to readily access animal models for which only cryo-archived sperm is available. Most mouse models cryo-archived in the form of frozen sperm can be efficiently resuscitated by in vitro fertilization (IVF). However, this is not true for rat models since rat IVF lacks consistency and repeatability. Intracytoplasmic sperm injection (ICSI) has been the only reliable method to resuscitate rat models from frozen sperm at the Rat Resource and Research Center (RRRC). The ICSI procedure requires expensive equipment and extensive technical training. Therefore, optimization of rat IVF and the ability to implement it as a reliable, cost-effective alternative to ICSI for cryo-resuscitation would be hugely beneficial to the biomedical community.

Improvements in both rat sperm freezing and IVF have been accomplished in recent years [6,7,8]. However, significant variation still exists among different strains/stocks, within a strain among different males and even with different straws of frozen sperm from the same male [7]. Moreover, the current published IVF protocol using frozen–thawed sperm must be conducted over an approximately 12 h period due to the time required to process frozen sperm and perform sperm pre-IVF incubation for capacitation and sperm–oocyte co-incubation [6,7,9]. In addition, it is well-documented that oocyte maturation is important for oocyte-controlled events needed for successful fertilization. The objective of the studies presented here was to optimize the current IVF protocol so that it can be used as a reliable, cost-effective alternative to ICSI for cryo-resuscitation of rat models from frozen sperm. The followings were examined: (1) the general efficiency of IVF using frozen sperm from different strains/stocks, (2) the possibility of modifying the IVF procedures with frozen–thawed sperm such that they can be accomplished in a 9 h period which is more compatible with a typical workday schedule, and (3) the strain/stock differences in the timing of oocyte maturation in vivo for potential optimization of strain/stock-specific superovulation to improve IVF outcomes.

2. Materials and Methods

2.1. Animals

Wild-type Sprague Dawley (SD), Fischer 344 (F344) and Long Evans (LE) rats were obtained from Charles River Laboratories (Wilmington, MA, USA). Male rats of the following strains/stocks, namely two SD-Tg(S334ter)3Lav (RRID:RRRC_00643) [10], one LE-Tg(Drd1a-iCre)3Ottc (RRID:RRRC_00767) [11], one LE-Drd2 ^em1Rrrc^(RRID:RRRC_00979) [12], and one LE-Fxn^em2^/FaraRrrc (RRID:RRRC_00961), were obtained from the RRRC (www.rrrc.us). All rats were housed in microisolator caging on ventilated racks in an environmentally controlled room with a temperature of 22 °C, 12 h light/12 h dark cycle (lights on at 6:30 am) with access to food (Purina 5008) and water ad libitum. This study was conducted in strict accordance with the recommendations in the Guide for Animal Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for animal care and surgical procedures was approved by the Animal Care and Use Committee of the University of Missouri.

2.2. Sperm Cryopreservation

Unless specifically stated, all chemicals were purchased from MilliporeSigma (St. Louis, MO, USA). Cauda epididymal sperm from mature (10-week to 10-month-old) male rats were cryopreserved using a protocol described previously with slight modifications [6,7]. Prior to animal euthanization, each 0.25 mL French straw (Minitube USA, Verona, WI, USA) was connected to a 1 mL plastic syringe. A total of 15 straws per male was prepared and then each straw was filled with ~30 µL modified human tubal fluid (mHTF) [13,14] followed by ~5 mm air. The mHTF used here contained 4 mg/mL bovine serum albumin (BSA) (mHTF-4 mg/mL BSA). Males were euthanized by first lightly anesthetizing by isoflurane inhalation and then performing cervical dislocation. For each male, two cauda epididymes were excised and then placed into 3 mL freezing solution (8% (w/v) lactose, 23% (v/v) chicken egg yolk, 0.7% Equex STM (Minitube USA) and 0.1% ATP (adenosine 5′-triphosphate disodium salt hydrate Grade II) with a pH of 7.4 adjusted with 10% tris aminomethane solution) in a 35 mm Petri dish at room temperature. Each cauda epididymis was cut ten to twelve times using sterile surgical scissors to let sperm swim out. To assess pre-freezing sperm motility and concentration, a 10 µL aliquot of the sperm solution that was taken 3–5 min into the swim out process was diluted into 990 µL pre-equilibrated mHTF-4 mg/mL BSA (100× dilution) in a 37 °C incubator with 5% CO_2_ and maximal humidity and assessed by the Hamilton Throne Computer-Assisted Sperm Analysis (CASA) and the Integrated Visual Optical System (IVOS) II (Hamilton Throne Inc., Beverly, MA, USA). Only sperm samples with total fresh sperm motility equal or greater than 60% and a concentration equal or greater than 15 × 10^6^ sperm/mL were selected for freezing.

After 10 min of sperm dispersion in the freezing solution, each straw was filled with ~150 µL sperm suspension. After all fifteen straws per male were filled, the straws were sealed by heating the other end using an Runruii impulse heat sealer (Runrui Ltd., Shaoxing, Zhejiang, China). Straws were then slowly cooled from 22 °C to 5 °C using a CRYSALYS cryocontroller PTC-9500 (Biogenics Inc., Harriman, TN, USA). Cooled straws were frozen using liquid nitrogen (LN_2_) vapor by placing straws on a steel rack at approximately 3 cm above LN_2_ inside a styrofoam box for 10 min. Straws were subsequently plunged into LN_2_ for longer term storage.

2.3. In Vitro Fertilization

IVF was conducted as described previously with slight modifications [6,7]. The medium used for IVF was mHTF supplemented with 40 mg/mL BSA (mHTF-40 mg/mL BSA) [7]. IVF dishes were made the day before or first thing in the morning prior to performing IVF. One IVF dish was prepared for every three donor females from which oocytes were collected. To make an IVF dish, one drop of 200 µL mHTF-40 mg/mL BSA was placed at the center of a 35 mm Petri dish. After the drops were overlayed with mineral oil, the IVF dishes were equilibrated at 37 °C with 5% CO_2_ in air with maximal humidity in an incubator. In the meantime, two 35 mL Petri dishes were filled with ~4 mL mineral oil and placed in an incubator for oviduct collection.

For each IVF dish, one straw of frozen sperm was used. Straws of frozen sperm were thawed at 37 °C in a water bath for 15 min. The sperm solution from each straw was released into 2 mL mHTF-4 mg/mL BSA in a 15 mL conical tube pre-warmed in a 37 °C incubator. Without disturbing, thawed sperm were allowed to swim up for 30 min by gently laying the 15 mL conical tubes on an incubator shelf at an approximately 30-degree angle. At the end of the swim up procedure, sperm were pelleted by centrifugation at 300× g for 2 min using a benchtop centrifuge (Thermo Scientific, Waltham, MA, USA). The sperm pellet was washed once with 2 mL mHTF-4 mg/mL BSA. After a second centrifugation, a 50 µL sediment from each 15 mL tube was taken using a pipette with a wide-mouth tip and gently laid at the center of one 200 µL mHTF-40 mg/mL BSA drop in a 35 mm Petri dish. After collections of 50 µL sediments from all thawed samples, the IVF dishes were returned to the incubator. After 30 min of incubation, clumps of dead sperm were removed by withdrawing 125 µL medium from the mHTF drop(s) under a Leica stereomicroscope (Leica Microsystems, Wetzlar, Germany). The IVF plates were then returned to the incubator to incubate for an additional 90 min to allow sperm capacitation. Motility of sperm was assessed by a CASA IVOS II (Hamilton Throne Inc.). Those IVF drop(s) having equal or greater than 10% progressive and 30% total motility were used for IVF.

Immature female rats (5 weeks of age) were superovulated by intraperitoneal administration of 300 IU/kg pregnant mare serum gonadotropin (PMSG) (ProSpec, Rehovot, Israel) around 10:00 am followed by intraperitoneal administration of 300 IU/kg human chronic gonadotropin (HCG) (Calbiochem, San Diego, CA, USA) approximately 54–55 h after administration of PMSG. At the end of the 90 min sperm capacitation process, donor females were euthanized by first lightly anesthetizing using isoflurane inhalation followed by cervical dislocation. It has been demonstrated that rat oviducts need to be dissected promptly after euthanization to ensure high and stable fertilization [6,7,15]. Therefore, the donor females were euthanized in groups of no more than two to ensure oviducts were excised into pre-warmed ~4 mL mineral oil in a 35 mL Petri dish within one minute. The Petri dish with excised oviducts was quickly brought to a Leica stereomicroscope. One oviduct was placed into the mineral oil section in a fertilization dish near the fertilization drop. The clutch of cumulus–oocyte complex (COC) was released from the oviduct by tearing the swollen ampulla using fine forceps and an insulin needle. The clutch of COC was directly introduced into the preincubated IVF drop from the mineral oil section without rinsing. This process was performed within three minutes [7,15]. The same process was repeated until the COCs were collected from all the donor females. Sperm and COCs were co-incubated in an incubator at 37 °C CO_2_ in air with maximal humidity for 8 h [16].

2.4. Embryo Culture and Development Assessment

At the end of sperm–COC co-culture, presumptive zygotes were washed three times with a potassium simplex-optimized medium for rat (KSOM-R) [17]. A maximum of 50 zygotes per 500 µL KOSM-R in NUNC a four-well culture plate under mineral oil were cultured in an incubator with 37 °C, 5% CO_2_ in air with maximal humidity. Embryonic development was assessed at the following time points: cleavage at 24 h, blastocysts at 120 h and hatching at 144 h by visualization under Leica M165C stereomicroscope (Leica Microsystems, Wetzlar, Germany).

2.5. General Efficiency of IVF Using Frozen Sperm from Different Strains/Stocks

To establish baseline data for performing IVF, frozen–thawed sperm from two transgenic lines (SD-Tg(S334ter)3Lav (two males) and LE-Tg(Drd1a-iCre)3Ottc) (one male) and two knock-in lines (LE-Drd2^em1Rrrc^(one male) and LE-Fxn^em2^/FaraRrrc) (one male) were used. These lines were chosen because SD and LE are two outbred genetic backgrounds that are commonly used in biomedical research. In addition, these lines were chosen because sperm from genetically modified stocks is commonly cryopreserved. Sperm freezing, donor superovulation, frozen–thawed sperm pre-treatment and IVF were conducted as described above. The developmental potential of the resulting fertilized oocytes was monitored by their ability to cleave, develop into blastocysts, and hatching in vitro.

2.6. IVF Within Conventional Workday Schedule

The standard rat IVF with frozen–thawed sperm takes up to a few hours beyond the conventional workday schedule [6,7,9]. We used wild-type SD and F344 rats to investigate the possibility to fit it into a conventional laboratory workday schedule (9 h). Donor superovulation, frozen–thawed sperm pre-treatment and IVF were conducted as described above but with the reduction in sperm pre-IVF incubation time from 90 min to 30 min after the removal of the dead sperm. There was also shortening of the sperm–COC co-culture time from 8 h to 6 h (SD) to prolong sperm–COC co-culture time up to 21 h (F344). The cleavage, blastocyst and hatching rates were assessed for their developmental potential in vitro.

2.7. Timing of Oocyte Maturation and IVF

The strain differences in timing of oocyte maturation in vivo using the same superovulation protocol in SD, LE and F344 rats were examined. Immature (5 weeks of age) female rats were superovulated as described above. Approximately 15 h after hCG administration, oocytes were collected between 8:00 and 9:00 am the next morning. After removal of cumulus cells by incubation in 1 mg/mL hyaluronidase in mFHM [18], oocytes were cultured in groups of 30 oocytes/50 µL KSOM-R drops. The oocytes were examined hourly, from 9:00 am to 2:00 pm, for the extrusion of the polar body. IVF was conducted as described previously for potential improvement in fertilization and embryo development based on the timing of oocyte maturation.

2.8. Statistical Analysis

Each experiment was repeated at least three times. The results are expressed as mean ± standard deviation. Data were analyzed using one-way ANOVA with rankings (Kruskal–Wallis test) or two-sample T-test after arcsine square root transformation. p < 0.05 was regarded as statistically significant.

3. Results

3.1. Rat IVF with Frozen–Thawed Sperm

The cleavage, blastocyst, and hatching rate results from IVF are shown in Table 1. There was a difference in cleavage rates between the SD and LE stocks (p < 0.05). However, there were no differences in blastocyst rates and hatched blastocyst rates between the two stocks (p > 0.05).

3.2. Procedure Modifications to Perform Rat IVF Within a Typical Workday Schedule

The current IVF protocol with frozen–thawed sperm spans approximately 12 h. One of our goals was to identify steps in the protocol that could potentially be shortened to make the performance of the IVF procedure more compatible with a typical workday schedule (~9 h). We investigated the impact on embryo survival and development when (1) sperm capacitation time was shortened from 90 min to 30 min after the removal of the dead sperm, and (2) sperm and COC co-culture time was shortened from 8 h to 6 h. This approach with shorter sperm capacitation (30 min) and sperm–oocyte co-culture time (~6 h) was not successful for inbred F344 males based on the lack of or low cleavage rates seen in oocytes following IVF. Therefore, we extended the sperm–oocyte co-culture time to 21 h (from 11 am to 8 am the next day). This provided an improvement in outcomes when using sperm from one of the four F344 males tested. The modifications to each step of the published IVF protocol and its corresponding time frame are summarized in Table 2. The cleavage, blastocyst and hatching rate results from IVF using frozen–thawed sperm from SD and F344 males are shown (Table 3). The modifications to SD and F344 IVF resulted in similar cleavage and blastocyst rates (p > 0.05).

3.3. Effect of Oocyte Maturation on IVF Success

We noticed that the blastocyst rate was lower in the LE lines than in the SD line (Table 1). It is known that oocyte maturation is important for oocyte-controlled events needed for successful fertilization. Therefore, the timing of oocyte maturation (extrusion of the first polar body) after superovulation was examined in wild-type SD, LE and F344 stocks/strains to investigate the stock/strain differences in oocyte maturation (Figure 1). Following the traditional superovulation protocol [6,7], oocytes were collected between 8 and 9 am. The timing of extrusion of the first polar body in SD and F344 was similar and peaked around 10 am. A total of 88% SD oocytes (136 out of 155) and 96% (179 out of 187) F344 oocytes displayed the first polar body (Figure 1a–c). However, the first polar body extrusion in LE oocytes was spread across a three-hour window and only 42% oocytes (73 out of 174 oocytes) displayed the first polar body. The peak for LE first polar body extrusion had a two-hour delay compared to what was seen in SD and F344 (Figure 2).

After moving the PMSG injection time two hours earlier (~8:00 am) in LE rats, 88% oocytes (138 out of 157 oocytes) displayed the first polar body over a two-hour window (Figure 1d). The extrusion of the first polar body in LE oocytes peaked one hour earlier (Figure 3) than the LE oocytes from females that received PMSG administration around 10 am as seen in Figure 2.

We then tested if altering the PMSG administration time in LE rats to account for the 2 h delay in first polar body extrusion improved IVF results. IVF was conducted with COCs from immature LE females using the standard and the new PMSG administration time, and the results are shown in Table 4. There was no significant improvement in cleavage, blastocyst and hatching rates between the two groups (p > 0.05).

4. Discussion

In the present study, the feasibility of using IVF for the cryo-resuscitation of rat models from frozen sperm was examined. The possibility of modifying the rat IVF with frozen–thawed sperm protocol [6,7,9] so it could be performed within a 9 h workday was also investigated. Lastly, we examined the extrusion of the first polar body from three strains/stocks in response to superovulation. The results demonstrated that there was significant variation in cleavage between SD and LE lines. However, there were no differences in blastocyst and hatched blastocyst rates. By reducing the pre-IVF sperm incubation time from 90 min to 30 min after dead sperm removal, and adjusting sperm and COC co-culture to 6 h (SD) or to 21 h (F344), we were able to customize the IVF process for different strains/stocks and allow the procedure to be accomplished within the confines of what is considered a regular workday schedule. We also found that the first polar body extrusions delayed by 2 h in LE compared to SD and F344 rats when the same superovulation protocol was used for LE rats. However, there was no significant improvement in IVF outcomes when the modified superovulation protocol that increased the time between PMSG and hCG administration by two hours was used in the LE rats.

The first successful rat IVF was published by Toyota and Chang [19]. However, rat IVF has remained difficult to perform, has low success rates and requires the procedure to be performed for long periods of time, often at unusual hours [8,20,21,22,23]. The efficiency and repeatability have been significantly improved in recent years through shortening sperm capacitation and oocyte collection time, modified sperm freezing protocols and altered capacitation conditions [6,7,9,15], which makes practical application a possibility. In this study, we aimed to optimize the current rat IVF protocol to replace ICSI for the rederivation of rat models from frozen sperm. Though variations exist among different genetically engineered rat lines and even different IVF attempts within the same rat line, in experiments where replicates were performed, we achieved blastocyst rates ranging from 46% to 71% in all four lines tested. Our results show that enough viable blastocysts can be produced to perform embryo transfers to recover live animals, but it may require multiple attempts. In the context of cryo-resuscitation to revive a strain/stock, only a few animals are needed as breeders so the relatively modest numbers recovered could be sufficient to reestablish a colony.

Another barrier to widespread use of current rat IVF protocols is the timing of steps that cannot be accomplished within a standard workday. We demonstrated that by reducing sperm capacitation time after dead sperm removal from 90 min to 30 min and limiting sperm–COC co-culture time to 6 h, we were able to achieve success in two of the four IVF attempts using frozen–thawed SD sperm (Table 2). IVF in inbred F344 was more challenging. Reducing sperm–COC co-culture time to 6 h resulted in little to no fertilization in the F344 strain. By extending the sperm–COC co-culture time from 6 h to ~21 h (overnight), IVF with sperm from one of the four F344 males resulted in satisfactory fertilization and blastocyst rates. However, few embryos were able to hatch in vitro. Continued optimization of F344 IVF with frozen–thawed sperm is needed.

Since the fertilization rates were significantly lower in LE rats than SD rats (see Table 1). We speculated that one factor might be the strain differences in oocyte maturation in response to superovulation. It is well-documented that oocyte maturation is important for oocyte-controlled events for successful fertilization [24,25]. Therefore, we examined the strain differences in timing of oocyte maturation in vivo using the same superovulation protocol in SD, LE and F344 rats. We showed that there are strain/stock differences in the peak of first polar body extrusion in LE compared to SD and F344 but adjusting for the 2 h delay seen in LE did not result in significant improvement in fertilization, cleavage and blastocyst formation. There are probably other biological factors affecting the IVF outcome in LE strain that if identified could further improve IVF success in this stock.

5. Conclusions

In conclusion, we have introduced some modifications to existing IVF protocols that represent incremental improvements in rat IVF. Given the level of variation inherent in current protocols, more optimization is required, and investigators must be cognizant of the fact that ultimately, development of individual-strain/stock-specific protocols may be necessary. However, we are optimistic that more routine implementation of the use of IVF for cryo-resuscitation will be possible with frozen–thawed rat sperm. Once this is achieved, the future goal would be to use rat IVF routinely for cryorecovery of cryopreserved rat lines and only rely on ICSI as a back-up procedure in situations where repeated IVF failures occur.