Published: July 7th, 2023
The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.
For successfully maintaining pregnancy with embryo transfer or artificial insemination, female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.
Assisted reproduction technologies are used for the production of genetically modified mouse models, as well as the recovery of strains from cryopreservation, the rederivation of strains from a compromised health status, and strategic vivarium management, including the production of age-matched cohorts. All assisted reproduction techniques in mice require the use of pseudopregnant female recipients for embryo development. Historically, pseudopregnant recipients have been generated by mating with sterile males, which are either surgically vasectomized or genetically infertile, and the presence of a copulation plug is assessed the following morning1. Recently, a protocol for sonic stimulation in mice has been developed for the surgical transfer of pronuclear or two-cell mouse embryos2. We have also developed a cervical manipulation (CM) protocol for use with artificial insemination and the non-surgical embryo transfer of blastocysts. The rationale for the use of the procedure is to provide a 3Rs reduction in the number of animals required (no longer requiring male mice) and a refinement of the techniques used (no longer necessitating the surgical vasectomy procedure for male mice). The description of this protocol includes the associated assisted reproduction technique to aid in the integration of CM into normal workflows. The overall goal of the CM method is to replace the use of male mice in the generation of pseudopregnant females for assisted reproduction techniques, including artificial insemination and embryo transfer.
The CM protocol described here was first developed to assist with artificial insemination in mice. The artificial insemination protocol, as originally described, achieved a pregnancy rate of 50%, with an average litter size of 7 pups3. CD1 recipient mice were estrous synchronized with a low dose of hormones, including pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), at a 47 h interval prior to insemination. An advantage of estrous synchronization was that it permitted the use of the protocol during normal working hours. Females were paired with vasectomized males immediately after artificial insemination, and mating was confirmed by the presence of a copulation plug. Inconsistency in the mating rate with recipients was reported as a difficulty in the procedure. Therefore, alternatives to mating were sought for the induction of pseudopregnancy.
The present study presents a standardized CM technique to increase the efficiency of producing pseudopregnant females. For females in estrus or proestrus, the blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is performed on a wire-topped cage. No anesthesia or analgesia is required. The CM technique is convenient for producing pseudopregnant females, which can produce litters after non-surgical artificial insemination without the need for mating with vasectomized males. CM can also be used for the production of pseudopregnant females as recipients of embryo transfer. Specifically, the CM technique can be paired with non-surgical embryo transfer, as described here. Non-surgical methods have been shown to be effective for the embryo transfer of blastocyst stage embryos in mice4,5and rats6,7. As this non-surgical method is an effective alternative to surgical methods, it is considered a 3Rs refinement of the technique. Based on previous research, fecal corticosterone levels, as a measure of stress, indicate that the non-surgical nature of the procedure does not increase stress levels in rodents7,8. The procedures are less technically challenging than surgical embryo transfer and are much faster to perform. As the embryos are transferred to the uterus, embryos of the correct stage for uterine development must be transferred. For mice, blastocysts are transferred to 2.5 days post coitum (dpc) pseudopregnant recipients.
For the two non-surgical techniques described here, timing of the hormone administration and the CM technique differs. The timing of the CM procedure relative to estrus is important for success, as it replaces natural mating for the production of pseudopregnant recipients. By eliminating the need for vasectomized males to induce pseudopregnancy, this procedure provides 3Rs benefits by both reducing the number of animals required and eliminating the necessity for surgically altered males. The procedure itself is quick (30 s) and does not require anesthesia or analgesia. The technique greatly increases the reliability and predictability of producing pseudopregnant females for assisted reproduction.
All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the ParaTechs Corporation Institutional Animal Care and Use Committee and conducted under the standards dictated by the Office of Laboratory Animal Welfare, National Institutes of Health, Public Health Service, United States Department of Health and Human Services. Male and female CD1(ICR) and female C57Bl/6J mice aged >8 weeks old were used for the present study. The animals were obtained from commercial sources (see Table of Materials).
1. Cervical manipulation procedure for use with artificial insemination in CD1 mice
Figure 1: The mouse-holding technique for cervical manipulation. The mouse rests on a wire cage top and is stabilized at the tail and on both sides at the front of the hind legs. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated by contact with a trimmer. Please click here to view a larger version of this figure.
2. Non-surgical embryo transfer procedure for use with cervical manipulation in CD1 mice
As electrical9 and sonic10 cervical stimulation have been used to induce pseudopregnancy in rats, this work presents a standardized mechanical procedure that can be used in mice. Vaginal cytology can aid in the identification of females in various stages of estrous. To confirm pseudopregnancy in females, this same method was employed. First, cytology profiles for females were compared for CD1 and C57Bl/6 mice throughout estrous cycles, during pregnancy, and during pseudopregnancy induced by mating or CM. Vaginal swabs were taken from the females and stained using a Papanicolaou staining system (see Table of Materials). The cells were observed under 100x magnification with bright-field illumination. The cells observed included leukocytes, nucleated epithelial cells, and anucleate cornified epithelial cells. The determination of the estrous cycle stage was based on the relative percentage of each cell type11,12. Estrus is characterized by the predominance of cornified epithelial cells. As estrus ends, metestrus begins, and leukocytes begin to appear, while cornified epithelial cells become less evident. Diestrus has moderate to low cellularity, with leukocytes predominating and nucleated epithelial cells beginning to appear. Proestrus is distinguished by the loss of leukocytes, an increase in nucleated epithelial cells, and the appearance of cornified epithelial cells. After proestrus, estrus begins, and the cycle continues.
To develop a baseline profile, vaginal cytology was recorded for at least two full estrous cycles for each female (N = 20 for CD1, N = 20 for C57Bl/6) prior to mating. The cycle lengths and individual profiles varied among the mice; however, the expected general trends were observed. The average length of an estrous cycle for the CD1 and C57Bl/6 mice was 3.8 days, with a range of 3-5 days. The day before natural mating occurred, all the female mice were in proestrus. After mating, cytology was performed again 1.5 days post coitum (dpc) until estrous cycling resumed. Figure 2 shows the cytological profile for pseudopregnant CD1 and C57Bl/6 females mated with vasectomized males.
Figure 2: Cytological profile for pseudopregnant female mice. The average percentages of each cell type for leukocytes, nucleated epithelial cells, and cornified epithelial cells are shown as a function of days post coitum (DPC) for (A) CD1 (N = 20) and (B) C57Bl/6 (N = 20) mice. Please click here to view a larger version of this figure.
In this work, a CM technique has been standardized to increase the efficiency of producing pseudopregnant females. For females in estrus or proestrus, the blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is performed on a wire-topped cage. No anesthesia or analgesia is required. To determine the effectiveness of the CM procedure, vaginal cytology was compared for female CD1 (N = 20) and C57Bl/6 (N = 20) mice after mating with vasectomized males and after CM (total N = 40). The cytological profile of pseudopregnancy induced by CM was similar to the profile of pseudopregnancy induced by mating, as shown in Figure 3.
Figure 3: Cytological profile for pseudopregnant female mice after cervical manipulation (CM). The percentage of each cell type for leukocytes, nucleated epithelial cells, and cornified epithelial cells are shown as a function of days post coitum (DPC) or days post cervical manipulation (DPCM). The average cell type percentages are shown for the CD1 and C57Bl/6 mice (N = 40) (A) mated with vasectomized males or (B) after CM. Please click here to view a larger version of this figure.
To determine if the CM technique is sufficient to establish pregnancy for assisted reproduction, CM was performed as part of a non-surgical artificial insemination (NSAI) protocol in CD1 females. The artificial insemination protocol included estrus synchronization of the females with a low dose of the hormones PMSG and hCG prior to sperm transfer. CM was performed just prior to the sperm transfer. For CD1 mice, the efficiency of pseudopregnancy induction using CM for females in estrus (N = 76) with this technique was 83%. In a control experiment, only 38% of females in estrus were plugged by vasectomized males (N = 24). The artificial insemination recipients receiving cervical manipulation (N = 76) had a 72% pregnancy rate and an average litter size of 8.3 pups. Thus, induction of pseudopregnancy by CM is a convenient and efficient replacement to mating with a vasectomized male when performing NSAI. Using CM to prepare CD1 recipients (N = 4) in estrus for the non-surgical embryo transfer of fresh CD1 blastocysts resulted in a 100% pregnancy rate. The transfer of 9-15 embryos yielded three healthy litters with a 45% birth rate and an average litter size of 6 pups. In comparison, the CD1 recipients (N = 20) that were mated with vasectomized males to induce pseudopregnancy had an 80% pregnancy rate and a 46% birth rate after the transfer of 20 fresh B6C3F2 blastocysts.
Results for assisted reproduction techniques will likely be strain-specific, as variables such as the dose and timing of hormone administration for superovulation have been found to be strain-dependent13. In addition, factors such as recipient age and weight can affect reaction to hormones14. In this work, when performing the CM procedure for artificial insemination, only females in late proestrus or estrus were found to be responsive. In general, to increase the number of available recipients in a population, the females are first estrus-synchronized with a low dose of hormones3. Estrus synchronization with 2.5 IU of PMSG and hCG was 78% effective for 11-14 week old CD1 females (N = 27) and 60% effective for 18-32 week old C57Bl/6 females (N = 22) in this work. The efficiency of pseudopregnancy induction using cervical manipulation on CD1 females was 83% for females in estrus (N = 76) and 82% (N = 100) for C57Bl/6 females in estrus with this technique.
The 3Rs is an ethical framework for animal use in research, as described in 1959 by Russel and Burch in "The Principles of Humane Experimental Technique"15. The 3Rs represent replacement, reduction, and refinement in animal use. The protocols highlighted here are in alignment with the 3Rs. The cervical manipulation technique reduces the number of animals needed by no longer requiring the use of males to produce pseudopregnant females. The technique also eliminates the need to perform vasectomies on the males, thus providing refinement by reducing pain and distress. The assisted reproduction techniques described here (artificial insemination and embryo transfer) are non-surgical, and thus, both provide a 3Rs refinement by reducing the pain and distress8caused by their surgical alternatives.
The use of pseudopregnant females is necessary for the recovery of pups when performing assisted reproduction in mice1. The CM procedure is an effective method for producing pseudopregnant females, but the synchronization of the phase of the estrous cycle of the recipient females is a critical first step in the process. Estrous synchronization can drastically reduce the number of females needed in the colony to prepare potential recipients and aids in producing timed pseudopregnant females on demand. Using a low dose of hormones does not seem to cause any deleterious effects on the recovery of live litters in CD1 mice. Care must be taken with other strains to find the hormone and concentration combination that produces the best-quality recipient females for the embryos or sperm transferred. Synchronization can be achieved using PMSG and hCG16, but doses that produce superovulated females may not be appropriate for a sustained pregnancy17.
To determine if a female is in estrus, a cytological evaluation was performed in this work. The estrous phase can also be evaluated by the observation of the vaginal opening11,18. While this method is extremely helpful and can be used by itself or as confirmation, it is more subjective than the use of cytology. Vaginal cytology without staining is both rapid and effective for choosing females in estrus because cornified epithelial cells can be easily identified. In this protocol, the cytological evaluation is performed prior to CM to determine potential recipients. It is important to perform cytology prior to CM, as the procedure tends to fragment the cells sloughed from the vaginal area, thus making identification difficult. Cytological evaluation for pseudopregnancy or pregnancy can be performed at 3.5-11.5 days post CM (dpcm) for 3 consecutive days. The profile of an estrous cycling female should have at least 1 day with considerable infiltration of cornified epithelial cells. Pseudopregnant/pregnant females should display a diestrus profile (mostly leukocytes with potentially low cell numbers) for 3 consecutive days.
Through the development of the CM technique, some mice were found to be more receptive to the procedure than others. CD1 female mice are excellent candidates because of their calm nature and excellent nurturing instincts. This strain is easy to handle and performs well during the CM and non-surgical assisted reproduction techniques. C57Bl/6 mice tend to be more aggressive and less nurturing. While this protocol effectively produced pseudopregnant C57Bl/6 females using CM, they were less likely to be consistently permissive of the procedure. This seemed to correlate somewhat with the estrous phase during CM. Females in estrus or proestrus were more receptive. The use of an enrichment tube for the animal to enter allowed access to the vagina for the procedure and helped calm the female. The procedure itself does not fully restrain the female, so the animal can pull away at any time. If this occurs, the animal can be repositioned, and the procedure can then be continued. The timing of the procedure stops if the female walks away and resumes when the procedure is resumed. Critical to the success of the procedure are the phase of the estrous cycle (late proestrus and estrus) and the contact of the rod with the cervix. The vibration of the trimmer provides standardized CM. To ensure contact with the cervix, gentle pressure is applied to the rod, and the positioning of the rod against the cervix is assured with small back-and-forth movements of the rod.
The use of CM has improved the NSAI protocol, as females in the correct phase of the estrous cycle can be chosen prior to sperm transfer, and the protocol is no longer contingent upon mating with vasectomized males. The artificial insemination estrous cycle synchronization is timed such that oocyte maturation corresponds to sperm transfer on the morning of day 4. Critical to the success of the protocol is the adaptation of the timing of ovulation such that fertilization can occur. Care must be taken to administer hCG 15-17 h before expected sperm transfer, as is suggested for the timings used for in vitro fertilization1. The quality of the sperm sample will directly affect the outcome of artificial insemination. Fresh sperm that have been capacitated will perform best. Cryopreserved sperm of good quality can produce fertilized embryos in vivo. However, care should be taken with the direct transfer of thawed sperm, as residual cryoprotectants transferred to the uterine horn may inhibit implantation (unpublished observations).
The use of CM in conjunction with embryo transfer is conceptually an easy adaptation. Estrous cycle synchronization reduces the number of females necessary for producing the recipient pool. Determining the estrous stage prior to CM increases the likelihood of obtaining pseudopregnant recipients. One drawback of the method is that the cytology of the recipients at the time of embryo transfer is in a stage of flux. All cell types are present if the female is transitioning from estrus to the pseudopregnancy profile, and pseudopregnancy becomes obvious only if the cytology is tracked for several days. Based on the success (>80%) of the transition from estrus to pseudopregnancy for CD1 and C57Bl/6 mice, this method is expected to be suitable for embryo transfer recipients. The preliminary results show good success with limited non-surgical embryo transfer. In general, the efficiency of non-surgical embryo transfer is comparable to that of the surgical technique4,5, and non-surgical transfer can replace surgical embryo transfers at the blastocyst stage. For earlier-stage embryos, embryo culture to the blastocyst stage is required. However, if a surgical transfer is preferred, it is possible to adapt the CM technique to the correct timing needed for appropriate pseudopregnant recipients2. In general, the embryo recipients are 1 day less advanced than the embryo. For example, blastocysts are harvested at 3.5 dpc from donors and transferred to 2.5 dpc recipients. Therefore, CM will need to be performed such that the recipient is in a less developed pseudopregnant state than the embryos.
In conclusion, the CM technique outlined here shows excellent promise for integration with other assisted reproduction techniques for mice. We have provided successful protocols for artificial insemination and embryo transfer using non-surgical techniques. In combination, the CM technique provides 3Rs advantages, including (1) a reduction in the number of animals by eliminating the need for vasectomized males and (2) a refinement of techniques by replacing surgical techniques with non-surgical alternatives.
Barbara Stone and Sarah Srodulski are both employed at ParaTechs Corporation, Lexington, KY, USA. ParaTechs holds exclusive licensing rights to manufacture the mNSET device for mice. ParaTechs Corp has developed and sells the mNSET and mC&I devices, which can be used for non-surgical embryo transfer and non-surgical artificial insemination, respectively.
The research reported in this publication was supported by the Office of the Director, Office of Research Infrastructure Programs, of the National Institutes of Health under Award Number R43OD020304 and the National Institute of Mental Health of the National Institutes of Health under Award Number R44MH122117. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
|Blastocyst stage embryos
|CARD Fertiup Preincubation Medium (PM)
|For sperm capacitation
|Embryo handling pipette
|Flexipet is available in various diameters
|Embryo handling pipette assembly
|Female mice, Crl:CD1(ICR)
|Charles River Laboratories
|>8 weeks old
|Fine Science Tools
|Toothed, for dissection
|Fine Science Tools
|Curved, for dissection
|Fine Science Tools
|Fine, for dissection
|human Chorionic Gonadotropin (hCG)
|For estrus synchronization
|Incubator, 37 °C 5% CO2
|Incubator, 37 °C, benchtop
|Embryo handling medium
|Male mice, Crl:CD1(ICR)
|Charles River Laboratories
|>8 weeks old
|For sperm transfer, specula included
|Smooth, blunt, with a diameter @3 mm
|20x and 40x magnification with transmitted and reflected illumination source for embryo work and dissections
|100x magnification with bright field illumination
|For embryo transfer, specula included
|Needles, 26 G
|Papanicolaou Staining System
|Fits the C&I device for sperm transfer
|Fits the NSET device for embryo transfer
|Pregnant Mare Serum Gonadotropin (PMSG)
|For estrus synchronization
|American Weigh Scales
|Fine Science Tools
|Fine Science Tools
|For vaginal cytology
|Syringes, 1 cc
|Becton Dickenson and Company
|Tissue culture dishes, 35 mm
|Tissue culture dishes, 60 mm
|Wire bar topped mouse cage
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