non-surgical embryo transfer procedure for use with cervical manipulation in cd1 mice

מניפולציה בצוואר הרחם גרמה לפסאודו-הריון בעכברים לצורך רבייה בסיוע

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,&#160;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.

מחבר זרקור: גרימת פסאודו-הריון בעכברות נקבות ללא צורך בזכרים שעברו כריתת כלי דם לפני החזרת עוברים לא ניתוחיים או הזרעה מלאכותית  

גרימת פסאודו-הריון בעכברות נקבות ללא צורך בזכרים שעברו כריתת כלי דם לפני החזרת עוברים לא ניתוחיים או הזרעה מלאכותית

Our primary research focus is the development of assisted reproduction techniques that provide refinements and reductions in keeping with the 3Rs of animal experimentation. We have developed non-surgical embryo transfer and non-surgical artificial insemination procedures for mice and a non-surgical embryo transfer procedure for rats. With the addition of the cervical manipulation procedure to provide pseudo pregnant females, we create 3Rs improvements and assisted reproduction in rodents.The protocols described here provide streamlined methods for recovering pups using non-surgical embryo transfer methods and artificial insemination in mice. The cervical manipulation protocol provides a refinement of procedures producing pseudo pregnant female recipients for assisted reproduction techniques. It reduces the number of females needed as pseudo pregnant recipients and it eliminates the need for vasectomized males.The protocols outlined here can be used for strategic vivarium management. The protocols can be used with cryo preservation, in vitro fertilization, rederivation, the production of age match cohorts and the production of genetically modified animals.

Watch this Scientific Journal Video about Non-Surgical Embryo Transfer Procedure for Use with Cervical Manipulation in CD1 Mice at JoVE.com

Non-Surgical Embryo Transfer Procedure for Use with Cervical Manipulation in CD1 Mice  

הליך החזרת עוברים לא ניתוחי לשימוש עם מניפולציה צווארית בעכברי CD1

התחל החזרת עוברים לא כירורגיים בעכבר CD1 ביום 6 על ידי הנחת טיפה של 20 מיקרוליטר של מדיום M2 על מכסה צלחת תרבית רקמה. השתמש בתאורה רפלקטיבית תחת מיקרוסקופ והעמיס 10 עד 20 עזרי פיצוץ לתוך הטיפה הבינונית M2 עם פיפטה סטנדרטית לטיפול בעוברים. יש לאבטח את המכשיר להחזרת עוברים ללא ניתוח על פיפטה P2 המוגדרת ל-1.8 מיקרוליטר ולהסיר את מכסה הצנתר המגן.לחץ על הבוכנה של פיפטה לתחנה הראשונה והורד את קצה הצנתר לתוך המדיום לפני שתמשוך באיטיות את העוברים לתוך קצה הצנתר של המכשיר. לאחר מכן הסר את הצנתר מהמדיום. לאחר מכן, הגדר את נפח פיפטה ל 2.0 מיקרוליטר כדי ליצור בועת אוויר קטנה בקצה הצנתר.הניחו בעדינות את פיפטה עמוסה בעוברים ליד כלוב העכבר. הניחו את הנקבה הנמענת על ראש כלוב מתלה התיל והחזיקו את העכבר במקומו על ידי אחיזה קרוב לבסיס הזנב באמצעות האצבע והאגודל, ואז סובבו את הזנב כלפי מעלה תוך ייצוב החיה. הכניסו ספקולום קטן לנרתיק והכניסו את צנתר מכשיר ההעברה לתוך הספקולום דרך צוואר הרחם ולתוך הרחם.ברגע שרכזת המכשיר יוצרת קשר עם הספקולום, שחררו את העוברים על ידי לחיצה על בוכנה פיפטה לתחנה הראשונה. הימנע מהעברת אוויר נוסף לקרן הרחם. הסר את המכשיר ואת ספקולום.

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JoVE Journal

Biology

Non-Surgical Embryo Transfer Procedure for Use with Cervical Manipulation in CD1 Mice

1.3K Views.  ParaTechs Corporation. התחל החזרת עוברים לא כירורגיים בעכבר CD1 ביום 6 על ידי הנחת טיפה של 20 מיקרוליטר של מדיום M2 על מכסה צלחת תרבית רקמה. השתמש בתאורה רפלקטיבית תחת מיקרוסקופ והעמיס 10 עד 20 עזרי פיצוץ לתוך הטיפה הבינונית M2 עם פיפטה סטנדרטית לטיפול בעוברים. יש לאבטח את המכשיר להחזרת עוברים ללא ניתוח ?...

Video: Non-Surgical Embryo Transfer Procedure for Use with Cervical Manipulation in CD1 Mice  

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;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.

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,&#160;female recipient mice must be induced into a pseudopregnant ...

Cervical Manipulation Procedure for Use with Artificial Insemination in CD1 Mice

To begin cytological evaluation for confirming estra cycle synchronization, gently collect the vaginal cells from the CD1 mouse by rolling the swab against the vaginal wall using a small pre moisten swab smear the collected vaginal cells into a 20 microliter drop of sterile water on a microscope slide and air dry. Under a microscope, use 100X magnification with bright field illumination to evaluate for the presence of cornified epithelial cells. For cervical manipulation, place a recipient female on the top of a cage with a wire rack approximately 0.5 hours before insemination allowing the mouse to grab the cage bar surface grasp near the base of the mouse's tail using the thumb and forefinger and angle the tail upward while stabilizing the animal.Insert the blunt end of a small plastic rod vaginally to contact the cervix and vibrate for 30 seconds by contact with the trimmer. To perform a non-surgical sperm transfer for artificial insemination, place the insemination device on a P200 pipette set to 40 microliters and remove the protective cover. Remove an aliquot of the capacitated sperm sample from the dish placed at 37 degrees Celsius and 5%carbon dioxide and transfer it to a 35 millimeter tissue culture dish without oil at 37 degrees Celsius, press the plunger of the pipette to the first stop.Lower the tip of the catheter into the sperm sample and slowly load 40 microliters of the sample into the transfer device. Remove residual oil from the exterior of the insemination device using an absorbent tissue before setting the pipette aside. Next place the recipient female on the wire rack cage top.Hold the female mouse in position by grasping near the base of the tail, using the thumb and forefinger and angle the tail upward while stabilizing the animal. Place the small speculum into the vagina and insert the insemination device catheter into the speculum through the cervix and into the uterus. Once the device hub contacts the speculum dispense the sperm by pressing the pipette plunger to the first stop.Once done, remove the device and speculum.

הליך מניפולציה צוואר הרחם לשימוש עם הזרעה מלאכותית בעכברי CD1

Begin non-surgical embryo transfer in the CD1 mouse on day 6 by placing a 20 microliter drop of M2 medium onto the lid of a tissue culture dish. Use reflective lighting under a microscope and load 10 to 20 blast assists into the M2 medium drop with a standard embryo handling pipette. Secure the non-surgical embryo transfer device onto a P2 pipette set to 1.8 microliters and remove the protective catheter cover.Press the plunger of the pipette to the first stop and lower the tip of the catheter into the medium before slowly pulling the embryos into the catheter tip of the device. Then remove the catheter from the medium. Next, set the pipette volume to 2.0 microliters to generate a small air bubble at the tip of the catheter.Gently lay the pipette loaded with the embryos near the mouse cage. Place the recipient female on the wire rack cage top and hold the mouse in position by grasping near the base of the tail using the forefinger and the thumb then angle the tail upward while stabilizing the animal. Place a small speculum into the vagina and insert the transfer device catheter into the speculum through the cervix and into the uterus.Once the device hub contacts the speculum dispense the embryos by pressing the pipette plunger to the first stop. Avoid the transfer of extra air into the uterine horn. Remove the device and speculum.