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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Lab Anim. 2015 Nov 12;50(5):335–343. doi: 10.1177/0023677215616530

Refinements for embryo implantation surgery in the mouse: Comparison of injectable and inhalant anaesthesias---tri-bromoethanol, ketamine and isoflurane--on pregnancy and pup survival

William B Norton 2, Ferdinando Scavizzi 4, Chad N Smith 1, Wenli Dong 3, Marcello Raspa 4, Jan V Parker-Thornburg 1,*
PMCID: PMC4865456  NIHMSID: NIHMS731774  PMID: 26566637

Abstract

An essential aspect of Genetically Engineered Mutant (GEM) mice is the ability to produce live animals after the appropriate injection procedure. Animals are produced by implantation of manipulated embryos into pseudo-pregnant females for gestation, parturition, and growth to weaning stage. We wished to test whether the anesthesia used during surgery could affect the number of pups produced. Anesthetics commonly used for implant surgery include tri-bromoethanol (Avertin) delivered by intraperitoneal (IP) injection, IP-injected ketamine:xylazine or medetomidine mix, and inhaled isoflurane. To determine if the anesthesia used would affect the number of animals produced, we tested each in implant surgeries and assessed the numbers of pups produced using both wild-type and GEM embryos. Parallel studies were conducted in institutions in the EU and in the US. Based on a direct comparison of pregnancy status, number of pups born and number of pups weaned for each agent, we found no statistical differences among the three anesthetics. We conclude that all three anesthetic agents tested are equally useful for implantation surgery.

Introduction

The surgical implantation of mouse embryos into recipient dams is an essential procedure for any genetically engineered mouse (GEM) program. The embryo transfer procedure is used to generate animals containing modified genes (1), expand colonies by implantation of embryos fertilized in vitro (2), and eliminate endemic murine pathogens by rederivation of contaminated mouse colonies (3, 4). While institutional facilities have generated many GEM, it is the advent of large, world-wide initiatives such as the IKMC/IMPC (www.mousephenotype.org/) and Infrafrontier-EMMA (www.infrafrontier.eu) that has exponentially increased the demand for GEM models. Additionally, the recent development of the CRISPR-Cas9 endonuclease approach in animal transgenesis is defining a new era for biology (5-7). Dedicated GEM programs must optimize time and resources to meet increased demand. Procedures must be done efficiently and in a manner that reduces the risk of complication to either the recipient dam or the embryos. Metrics such as number and quality of sperm and oocytes produced, in vivo and in vitro fertilisation rates, embryo yield, and pup viability are essential to determine areas where refinement may enhance production.

Manipulators disagree on the effect that anesthetic agents may have on recipient dams or embryo viability. Historically, embryo transfer techniques often used 2,2,2-tribromoethanol (TBE) [previously known as Avertin] to anesthetize dams for implantation (8-11). TBE rapidly induces a depth of anesthesia for durations suitable to complete the procedure, approximately 15-20 minutes at 250 mg/kg (12, 13). Some have reported success administering a second dose if necessary (14, 15), while others have reported an increased incidence of mortality (16).

The length of time to recover ambulation after anesthesia with TBE is prolonged (30-90 minutes). Also, degradation can occur in the presence of heat or light, producing toxic by-products that can cause peritoneal adhesions (17), peritoneal and visceral necrosis near the site of injection, and increased risk of mortality (18). Currently, use of TBE has become problematic, as the Eighth edition of The Guide for the Care and Use of Laboratory Animals (37) discourages the use of non-pharmaceutical grade compounds in laboratory species. Despite this, many transgenic facilities in the United States have successfully lobbied their local animal care and use committees to approve use of TBE, as they fear alternative anesthetics will affect the numbers of pups produced. As well, similar restrictions for TBE use have been established by EU country legislatures.

One popular alternative is the combination of ketamine with xylazine (K/X), or with medetomidine (K/M), components that are available in pharmaceutical grade. At a dose of 120:16 mg/kg (K/X) or 75:1 mg/kg (K/M) respectively, intraperitoneal injections of the ketamine:xylazine/medetomidine mix will induce anesthesia for approximately 20-30 minutes, with time to recover ambulation requiring an additional half-hour (17). Unlike TBE, ketamine is a controlled substance, requiring a DEA license and drug log for use in the USA. In Italy, ketamine is classified as a psychotropic drug; as such, the user must maintain authorized acquisition and use records, and must store the agent in a locked cabinet. There is evidence to suggest that use of K/X or K/M allows for comparable embryo yields as does TBE without inducing the same adverse effects (19). However, we have observed that the margin of safety appears to be narrower than for TBE. The use of medetomidine allows for partial reversal of anesthesia by administration of atipamezole (1 mg/kg).

A third alternative involves the use of inhalant isoflurane. Unlike injectable forms of anesthesia, the depth of anesthesia is easily adjusted. Induction is rapid, and suitable anesthetic depth is easy to achieve and maintain (20). The time to recover ambulation after cessation of anesthesia is much shorter, approximately 3-5 minutes. A precision vaporizer is recommended to obtain a calibrated dose of anesthetic. Oxygen tanks, hosing, vaporizers, and scavenger systems are all recommended to facilitate administration. Regular maintenance is necessary to ensure consistent dosing and to reduce personnel exposure. Thus, inhalant anesthetic delivery systems can require substantially more setup and maintenance than injectable anesthetics. Positioning of the patient can also be problematic, as the manipulator must ensure that the dam’s nose is adequately exposed to the source of the gas at all times; alternatively, endotracheal intubation may help to maintain proper flow rate.

Specific to embryo transfer and recovery of pups, relatively little in vivo research on the effects of different anesthetics has been performed. Multiple studies have documented the detrimental effects of isoflurane on two-stage embryos in vitro (21-23) and another study examined the effects on pups conceived by dams administered different anesthetics during gestation (24) .

Ultimately, the goal of embryo implantation is to obtain live offspring. This study examines these common anesthetic regimens for their effects on pregnancy rate and pup yield. Direct comparison of these methods revealed no statistical differences among the three anesthetic agents in their effects on pregnancy rate or pup production.

Materials and Methods

Animals

M.D. Anderson Cancer Center (MDA)

Female (36 – 45 g, 2 – 5 months of age) and male (2 – 6 months of age) CD-1@ mice were obtained from Charles River Laboratories (Wilmington MA, USA). Males were housed singly and females housed in groups of 5 in ventilated microisolator cages (Techniplast USA, Exton, PA, USA) at a temperature of 20 +/− 2 °C and a humidity of 55 +/− 15% with 12-15 air exchanges per hour, on corncob bedding (Bed O’Cob, Andersons Lab Bedding, Maumee, OH). All mice were provided filtered and chlorinated water ad libitum. Males and unmanipulated females were given irradiated Purina PicoLab Rodent Diet #5053 (Purina LabDiet, St. Louis, MO, USA). Surgically treated females were singly housed and given irradiated Harlan-Teklad Breeder Diet #7926 (Harlan Laboratories, Inc, Madison, WI, USA). Animals were maintained on a 12:12 light/dark cycle. Based on sentinel health surveillance, the housing area is free of Mouse parvovirus, Minute virus of mice, Mouse hepatitis virus, Mouse norovirus, GDVII, Enzootic diarrhea of infant mice, Sendai virus, Pneumonia virus of mice, Reo virus, Mycoplasma pulmonis, Lymphocytic Choriomeningitis virus, Mouse adenovirus, Ectromelia virus, K virus, Polyoma virus and Helicobacter spp. Sentinels are screened annually by bacterial culture or PCR for Bordetella bronchiseptica, Corynebacterium kutscheri, Salmonella sp., Pseudomonas sp., and Citrobacter rodentium. Noncontact sentinels are screened quarterly for Syphacia obvelata, Aspiculuris tetraptera, Myocoptes musculinus, Myobia musculi, and Radfordia affini. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Texas M. D. Anderson Cancer Center (an AAALAC accredited institution).

CNR- Institute of Cell Biology and Neurobiology (CNR)

For recipients, female (30 – 35 g, 2 – 4 months of age) CD-1 mice were supplied by the CNR-EMMA-Infrafrontier unit (Monterotondo Scalo, Rome, Italy). As embryo donors, GEM of four different backgrounds (C57BL6/NCnrm, C57BL6/JCnrm, BALB/cByJCnrm, FVB/NJCnrm) were used by the EMMA/Infrafrontier Network. Mice were kept in IVC racks and containment HEPA biosafety cabinets (Tecniplast, Gazzada, Italy), at a temperature of 20 +/− 2 °C and a humidity of 55 +/− 15% with 12-15 air exchanges per hour using a 12:12 light/dark cycle. Certified wooden dust-free spruce particles (scobis one, Mucedola, Settimo Milanese, Milano, Italy) were provided as bedding. Mice were fed a standardised diet, or a diet enriched with fat and protein (4RFN and Emma 23, Mucedola, Settimo Milanese, Italy) depending on strain. All mice were provided filtered and chlorinated water ad libitum. Experimental and sentinel mice were maintained free of excluded pathogens according to FELASA recommendations (25).

Superovulation, Collection of germplasm, In vitro fertilisation (CNR only) And Cryopreservation

MDA

Superovulation and cryopreservation

CD-1@ females (>35 g, 3-6 months of age) that had been scheduled for removal from the colony were used as embryo donors. Females were super-ovulated with 5 IU of Pregnant Mare’s Serum Gonadotropin (PMSG) (Harbor-UCLA Medical Center, Torrance CA, USA) followed 47-49 hours later by 5 IU of Human Chorionic Gonadotropin (HCG) (Sigma-Aldrich, St. Louis, MO, USA) (26). Females were mated to proven stud males immediately following HCG injection. Females were humanely euthanised by cervical dislocation the following morning for embryo isolation. Personnel have ≥ 25 years of experience performing cervical dislocation, and have demonstrated their proficiency to an institutional veterinarian. Embryos were isolated and cultured for 24 hours in KSOM embryo culture media (Millipore, Billerica MA, USA) under embryo-tested mineral oil (Sigma-Aldrich, St. Louis MO, USA) at 37°C in 5% CO2 . Upon reaching the 2-cell stage, embryos were cryopreserved in propylene glycol. Cryopreserved embryos were thawed the morning of surgery and cultured in KSOM as noted above.

CNR

Sperm isolation

Males were humanely euthanised by cervical dislocation. CNR personnel have ≥ fifteen years of experience performing cervical dislocation, and have demonstrated their proficiency as per requirements from the Animal Welfare & Ethical Review Body. Each cauda epididymis and vas deferens was placed in the sperm collection dish containing 500 μl of Mouse Vitro Fert medium (MVF; Cook Medical; Brisbane, Australia) covered with oil. Sperm were allowed to disperse from the tissue for 10 min and subsequently frozen using cryoprotective medium (CPM) of 18% raffinose (w/v), 3% skim milk (w/v) and 477 μM monothioglycerol (MTG) (27). Four aliquots of 12 μl of sperm+CPM were loaded into 0.25 ml French straws (IMV Technologies, France) and the straws were loaded into cassettes. Cassettes were placed onto a polystyrene raft LN2 vapors for 10 min before being plunged in LN2.

Oocyte isolation and in vitro fertilisation

In vitro fertilisation was performed using the protocol described by Ostermeier et al. (27). Sperm samples were thawed in a 37°C water bath for ~30 sec. Approximately 12 μl of the CPM+sperm was added to 500 μl of equilibrated MVF medium (Cook Medical; Brisbane, Australia) and incubated for ≥ 40 min under 5% CO2. Three-four week old female mice Hsd: athymic nude-nu (nu/nu) and C57BL/6N were used as oocyte donors. Donors were superovulated by IP injections of 5 IU PMSG (Intervet, Milan, Italy), followed 48 hours later by 5 IU HCG (Intervet). At 12-14 hours post-HCG, females were humanely euthanized by cervical dislocation. Oocytes were isolated and added to the IVF drop of 500 μl of MVF medium + sperm (Cook Medical; Brisbane, Australia). After 4 hrs of co-incubation, the presumptive zygotes were washed through drops of 150 μl of MVF medium. Those appearing normal were cultured overnight in 150 μl MVF. Approximately 18 hrs later, two-cell embryos were cryopreserved in 1.5 M propylene glycol (28). Cryopreserved embryos were thawed when needed the morning of surgery and cultured in KSOM.

Embryo Implant Surgery

CD-1@ females (26 – 33 g, < 5 months of age) [CRL (MDA); EMMA/Infrafrontier, Italy (CNR)] for implant surgery were identified by the presence of a copulation plug after mating to vasectomized CD-1@ males [CRL (MDA); EMMA/Infrafrontier, Italy (CNR)]. At the MDA site, implants took place over six separate days; at the CNR site, implants took place on four separate days. Females were anesthetized for surgery by either 2.5% Isothesia@ [isoflurane gas] to effect (after induction at 4-5% isoflurane) [Butler Schein Animal Health, Dublin OH, USA (MDA); IsoFlo Abbott Laboratories LTD, UK (CNR)], ketamine:xylazine/medetomidine [Fort Dodge Animal Health, Fort Dodge IA, USA; Lloyd Laboratories, Shenandoah IA, USA (MDA); Imalgene1000 Merial Italia and Domitor, Orion Pharma, Finland (CNR)] 100:10 mg/kg (K/X) and 75:1 mg/kg (K/M), or a tribromoethanol:tert-amyl alcohol mix [obtained from M. D. Anderson Pharmacy Research Laboratory (MDA); Merck Italia (CNR)] at 250 mg/kg tribromoethanol (29). A 2% working solution of tribromoethanol was prepared by adding sterile water to a 50x stock solution at 37°C and then mixed thoroughly. 250 mg/kg of the working solution was administered and the remainder immediately discarded. The stock solution was stored at 4°C in the dark and was used within six months after receipt. No discoloration was observed during the experimental period.

After anesthetic induction, the surgical site was prepared by shaving. Females were moved to a 37°C warming plate during surgery and the site prepared with chlorhexadine:alcohol scrubs. A 1 cm skin incision was made over the spine and the surgical window was moved laterally to visualize the ovary. A 3-5 mm incision was made into the peritoneum and the ovary/oviduct complex was extracted. The fascia covering the oviduct was torn and the infundibulum located. Fifteen (CNR) or thirty (MDA) embryos in a minimal amount of KSOM (Millipore, Billerica MA, USA) were implanted unilaterally (CNR) or bilaterally (MDA) into the ampulla of the oviduct. [Numbers of embryos implanted varied 2-fold between CNR and MDA. The embryo numbers implanted were based on results observed over the past five years at each facility.] The oviduct was returned to the peritoneum and the central wound closed using suture or surgical staples. (2, 29). Surgical duration was less than ten minutes per animal, after which the animal was moved to a warming cage (29, 30). After surgery, the animal was given Buprenex@ /Temgesic ®(31)[Reckitt Benckiser Pharmaceuticals Inc., Richmond VA, USA (MDA); Intervet, Italy (CNR)] at 0.1 mg/kg, placed on a 37°C warming plate, and monitored until ambulatory. At CNR, atipamezole (1mg/kg) (Elanco Animal Health Italy) was administered to reverse the clinical effects of the medetomidine. Females were singly housed for gestation either immediately post-surgery (MDA) or after becoming visibly pregnant (CNR). [Single housing to assess pup numbers for each implanted female was required for statistical purposes in this study.] At MDA, two bacon-flavored meloxicam tablets (0.0125 mg/tablet)(Bio-Serv, Flemington NJ, USA) were placed in the housing cage for ongoing analgesia. Animals were monitored daily for any complications. Housing cages were given identification numbers that did not indicate the anesthetic used, and the key was maintained in confidence by the surgeon.

Data Collection

Data collection was performed by participants blinded to the anesthetic modality received by the implanted females. Pregnancy status was assessed by trained animal technicians at 4-5 days prior to the expected delivery date, and positive status was denoted by appearance. Births were noted, when possible without disturbing the cage, on the expected delivery date. Pup numbers were obtained at 5 days after birth and again at weaning.

Statistical Methods

Group size of recipients was determined by statistical analysis of 5-year historical birth numbers. Based on one-way ANOVA method, with a sample size of 18 mice per group, the experiment has 80% power to detect an effect size of 0.2 for the number of pups among the anesthetic groups, assuming a type one error rate of 0.05 (nQuery Advisor 7.0). The effect size is the variance of the means divided by the square of the common standard deviation.

Mean and standard error (SE) were provided for continuous variables (eg: number of pups at 5 days and number of pups at weaning). The one-way ANOVA procedure (F test) was used to examine overall differences among the means of the multiple groups. Before performing ANOVA analysis, we checked data distributions and the assumptions of the ANOVA analysis. The data of pup numbers at five days and pup numbers at 21 days were normally distributed (p values = 0.10 and 0.13 based on normality test). The residuals from the ANOVA models were also normally distributed (p values > 0.10 based on normality test). For pair-wise comparisons, the differences between means were provided. To control the overall type I error rate, Tukey’s honestly significant difference (HSD) test was used for pair-wise comparisons to determine differences among groups. Frequencies and percentages were reported for categorical variables (eg: treatment group and pregnancy status). Fisher’s exact test was used to evaluate the association between treatment and pregnancy status. All tests were two-sided. P values less than 0.05 were considered statistically significant. All analyses were conducted using SAS (version 9.3, Cary, NC). Due to the different types of embryos used for implantation at MDA and CNR, statistical analyses were conducted separately for each institution.

Results

Each implant day, pseudopregnant females were collected in multiples of three, ranging from a group of three to a group of 15 [Table 1]. Females were distributed into equal groups to receive one of three anesthetic agents during implant surgery. A total of 18-20 females were used for each agent to achieve statistical significance.

Table 1. Implant results for each anesthetic.

Eighteen (MDA) or 20 (CNR) females per anesthetic agent were implanted with embryos over four (CNR) or six (MDA) different days. 15 embryos (CNR) or 30 embryos (MDA) were implanted per female. Equal numbers of females for all three anesthetics were implanted on the same day (N). Pregnancy status was evaluated 14-16 days post implant. The number of pups born was determined on the fifth day after the estimated date of delivery. Pup number was re-evaluated at weaning.

Anesthetic Day Number Implanted Number Pregnant
MDA CNR MDA CNR
TBE 1 4 5 4 4
2 3 5 3 3
3 2 5 2 4
4 3 5 3 3
5 5 5
6 1 1
Totals 18 20 18 14
Isoflurane 1 4 5 4 4
2 3 5 3 4
3 2 5 2 4
4 3 5 3 4
5 5 5
6 1 1
Totals 18 20 18 16
Ketamine a 1 4 5 3 4
2 3 5 2 3
3 2 5 2 3
4 3 5 3 2
5 5 5
6 1 0
Totals 18 20 15 12
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a

MDA: Ketamine:Xylazine (100:10 mg/kg); CNR: Ketamine:Medetomidine (75:1 mg/kg).

All surgeries were performed using the same protocol by surgeons experienced with all 3 anesthetic regimens. Implants were of either freshly harvested embryos (CNR only) or cryopreserved embryos (CNR, MDA), thawed just prior to implantation. Two weeks after implantation, the pregnancy status of the mice was recorded. At the MDA site, for both the TBE and isoflurane groups, 100% (18/18) of the mice were visibly pregnant, whereas at the CNR site, 14 of 20 implanted females in the TBE group, and 16 of 20 implanted females in the isoflurane group were visibly pregnant. In the K/X group (MDA), 87.3% (15/18) of mice were visibly pregnant and in the K/M group (CNR), 60% (12/20) of females were visibly pregnant. We found no statistically significant differences in pregnancy status among the three anesthetic groups [Table 2].

Table 2. Association between pregnancy status and anesthesia treatment.

Pregnancy status was compared for females treated with each different anesthesia. Treatments were evaluated for significant effect upon pregnancy using Fisher’s Exact Test. (Significance = p < 0.05).

STATUS TBE Isoflurane Ketaminea Fisher's Exact
Test P-value
(2-tail)
MDA (N = 18)
Pregnant 18 (100.0%) 18 (100.0%) 15 (83.3%) 0.099
Non-Pregnant 0 0 3 (16.8%)
CNR (N = 20)
Pregnant 14 (70.0%) 16 (80.0%) 12 (60.0%) 0.442
Non-Pregnant 6 (30.0%) 4 (20.0%) 8 (40.0%)
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a

MDA: Ketamine:Xylazine (100:10 mg/kg); CNR: Ketamine:Medetomidine (75:1 mg/kg). Statistical analyses were performed separately for each study site.

Three days prior to the estimated date of parturition, dams were placed in clean cages, and left undisturbed until five days after the estimated date of delivery [to reduce possible cannibalization]. On the fifth day after the predicted delivery date, pup number was determined. At 5 days, females in the TBE group had an average of 3.7 (CNR) and 5.2 (MDA) pups per dam, females in the isoflurane group averaged 5 (CNR) and 6.3 (MDA) pups per dam, and the K/M;K/X groups averaged 3.05 (CNR) to 6 (MDA) pups per dam [Table 3A]. A head-to-head comparison among the anesthetic groups indicated that differences in the distribution of the number of pups at day 5 were not statistically significant (p values > 0.05) [Table 3B].

Table 3. Comparison of pup numbers at five days.

A. The average number of pups was evaluated at 5 days after the expected date of birth. B. Pairwise comparisons were made between the mean number of pups for each anesthetic group to compare relative effects. (Significance = p < 0.05).

A. Average number of pups at five days.
Anesthetic MDA Pups at 5 days (SE) CNR Pups at 5 days (SE)
TBE 5.22 (0.74) 3.70 (0.80)
Isoflurane 6.33 (1.06) 5.00 (0.74)
Ketaminea 5.06 (0.77) 3.05 (0.70)
B. Pairwise comparison of number of pups at 5 days.
Treatment Comparison Difference between means Adjusted p-value
MDA CNR MDA CNR
TBE : Isoflurane −1.111 −1.300 0.641 0.442
TBE : Ketamine 0.167 0.650 0.990 0.813
Isoflurane: Ketamine 1.278 1.950 0.557 0.165
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a

MDA: Ketamine:Xylazine (100:10 mg/kg); CNR: Ketamine:Medetomidine (75:1 mg/kg). Statistical analyses were performed separately for each study site.

Pups were re-counted at weaning. At the CNR site, all pups survived to weaning. At the MDA site, all pups in one TBE cage had been cannibalized due to a defective water valve. As it was unlikely that the pup mortality in this cage was to the anesthetic agent used, it was not included for statistical comparisons. In another TBE cage (MDA), only eight of nine pups survived to weaning. This data was included in subsequent analysis. All pups from the remaining TBE cages at MDA survived from day 5 to day 21. All pups from isoflurane cages that were observed on day 5 survived to day 21. Similarly, all pups from ketamine:xylazine cages survived from day 5 to day 21 [Table 4A]. For both sites, the number of pups at weaning was not significantly different among the three anesthesia groups (p values > 0.05) [Table 4B].

Table 4. Comparison of pup numbers at weaning.

A. The average number of pups at weaning was evaluated. B. The effects of the anesthetic agents upon mean number of pups at weaning were compared one to one to evaluate their relative effects. (Significance = p < 0.05).

A. Average number of pups at 21 days (weaning).
Anesthetic MDA Pups at 5 days (SE) CNR Pups at 5 days (SE)
TBE 5.06 (0.76) 3.70 (0.80)
Isoflurane 6.33 (1.06) 5.00 (0.74)
Ketaminea 5.06 (0.77) 3.05 (0.70)
B. Pairwise comparison of number of pups at 21 days (weaning).
Treatment Comparison Difference between means Adjusted p-value
MDA CNR MDA CNR
TBE : Isoflurane −1.274 −1.300 0.569 0.442
TBE : Ketamine 0.003 0.650 1.000 0.813
Isoflurane: Ketamine 1.278 1.950 0.559 0.165
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a

MDA: Ketamine:Xylazine (100:10 mg/kg); CNR: Ketamine:Medetomidine (75:1 mg/kg). Statistical analyses were performed separately for each study site.

Discussion

Highly productive mouse transgenic facilities have detailed anesthetic protocols that reliably produce good pup numbers after surgery. However, new regulations and novel agents must be considered as they arise. Successful procreation is an important criterion for animal well-being; if pup numbers are already high, animal surgeons are often reluctant to change. Additionally, studies to identify and modify mouse genes have exponentially increased the international exchange of mouse mutant resources. Surgical refinements are crucial to saving animal lives; surgeons should consider flexible anesthetic approaches to achieve optimal results.

Our findings on the effects of tribromoethanol, ketamine, and isoflurane on embryo transfer suggest that comparable pregnancy rates and pup yields can be obtained regardless of anesthetic used. This is consistent with previous research in which recipient dams anesthetized with ketamine:xylazine yielded comparable numbers of pups as those anesthetized with tribromoethanol (19). Compared to this study, we observed a higher percentage of dams in the tribromoethanol group with surviving offspring at one site, but not the other, although this difference was not significant when compared to the other anesthetic cohorts. Additionally, the number of embryos implanted varied between sites in this study. While the numbers of pups obtained at the MDA site were generally 2-fold higher than at CNR, this was anticipated due to the 2X increase in number of embryos implanted. The non-significant differences observed among all of the studies, including this one, are likely due to housing, differences in surgical techniques, and improved chemical isolation techniques developed since 1998.

Notably, the ketamine cohort showed reduced pregnancy rates both at MDA and at CNR despite different housing conditions and different stock/strains of embryos (C57BL6/NCnrm, C57BL6/JCnrm, BALB/cByJCnrm, and FVB/NJCnrm embryos at CNR vs. CD-1 embryos at MDA). The regimen at CNR also used medetomidine that was reversed with atipamezole. As both institutions noted similar trends, these differences are unlikely to be the cause. However, while we noted an overall trend for ketamine use, the effect was not statistically significant. It would be worthwhile to examine this further, as ketamine is widely used for implant surgery.

We sought to determine whether isoflurane administration would result in the same number of pups as other, more widely used anesthetics. Isoflurane, combined with morphine, was shown to be effective for production of transgenic rats (32). Although previous research on murine fetal development demonstrated comparable rates of pregnancy using isoflurane versus TBE or K/X (24), our study marks the first time that the impact of isoflurane on live mouse pup yield has been compared head-to-head to either of these more common anesthetics. Notably, previous studies have identified adverse impacts of isoflurane on two-cell embryos in vitro (22, 23). In those studies, the gas was applied directly to the embryos, likely changing the pH of the solution and hampering embryo development. In this study, isoflurane was administered through a well-fitted nose-piece with a controlled rate vaporizer and utilizing the required scavenging. Anesthetic durations were kept consistent with what would be expected for each anesthetic (e.g., approximately 10 minutes for isoflurane and 30-90 minutes for K/X;K/M and TBE) rather than expose the dams to isoflurane for the extended periods of unconsciousness induced by TBE and K/X or K/M. Our results demonstrate that, when used in vivo and scavenged properly, isoflurane does not adversely affect the viability of embryos or the resulting pups.

The analgesics given at both institutions were uniform with the exception of meloxicam tablets given post-operatively at MDA. This difference should not influence pup yield at birth or weaning, as previous research has indicated that multimodal opioid-NSAID administration has no effect on these values relative to the administration of opioid alone(33). And, although meloxicam has been shown to affect fetal growth when administered to the dam at fertilization, it does not affect litter size (34).

Whereas previous studies have reported pup numbers at a single time point, we recorded numbers at five days post-delivery and at weaning. We had previously noted that disturbed new dams have a greater propensity to cannibalize offspring. Thus, we chose the 5-day post birth assessment as one that could be safely used to determine pup survival post-partum. Even so, we noted instances of positive pregnancy status followed by lack of pups at day five. Interestingly, three of five instances (MDA) were noted for dams treated with isoflurane, with the other two regimens having one instance each. We chose not to assess pup numbers by delivering via c-section, as we wished our study to be applicable to transgenic facilities, and this is not standard procedure for such facilities. Future studies could utilize c-section delivery or detection tools to record the number of live births, as well as determine the specific cause of death of deceased or cannibalized pups.

Previous research has identified stock/strain variations in response to different anesthetics (35, 36). For this reason, at MDA and CNR, we compared the impact of the anesthetic agents using just one recipient stock. CD-1@ is an outbred stock commonly used for embryo transfer in mouse transgenic facilities. At MDA, frozen/thawed CD-1@ embryos were implanted, thus, restricting the strain variation for both embryo and recipient. At CNR the embryos came from multiple strains (C57BL6/NCnrm, C57BL6/JCnrm, BALB/cByJCnrm, and FVB/NJCnrm) obtained from fresh and frozen batches, allowing comparison of different strains of embryos despite using only one stock of recipients. The statistical variation among anesthetic groups, examined separately for each institution, showed no differences, thus confirming that the anesthetic used had no significant effect on implantation success whether the embryos were matched to the recipient or came from unmatched strains/stock. Additional studies could incorporate additional recipient strains commonly used for implant surgery (e.g. B6D2F1, CBAD2F1, etc.)

Our results indicate that comparable numbers of pregnancies and pups should result regardless of whether tribromoethanol, ketamine, or isoflurane is chosen as the anesthetic for implant surgery. With this information, transgenic facilities can make more informed decisions regarding their choice of anesthetic for embryo transfer. For labs struggling to acquire or accommodate certain agents, alternatives can be sought, knowing that the impact to their production program will be minimal to nonexistent.

Acknowledgements

At MDA, we thank Earnessa Edison for animal care and data collection, Dr. Suzanne Craig for assistance with study design, Drs. Katherine Naff and Mary Robinson for edits and Dr. Peggy Tinkey for support for animal purchase and care. At CNR, we thank Dr. Renata Paoletti, the Cryo-Lab staff and Fabrizio Bonaventura who did most of the experiments at EMMA-Infrafrontier, Italy.

Funding

This work was supported in part by a Comprehensive Cancer Grant to MD Anderson Cancer Center, NCI #CA016672 (GEMF) and the INFRAFRONTIER-I3 project under the EU contract Grant Agreement Number 312325 of the EC FP7 Capacities Specific Programme at CNR Institute of Cell Biology and Neurobiology.

Abbreviations

IP

intraperitoneal

TBE

tri-bromoethanol

K/X

Ketamine:xylazine

K/M

Ketamine:medetomidine

US-DEA

United States Drug Enforcement Agency

MDA

M.D. Anderson Cancer Center

CNR

CNR Institute of Cell Biology and Neurobiology

Footnotes

Conflict of Interest

The Authors declare that there is no conflict of interest.

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