Innovative technologies for easier and more cost-effective research.
IMPORTANT! Before beginning the mNSET 60010 device technique, please read carefully the: mNSET 60010 Technical Support Letter, mNSET 60010 Instructions, mNSET 60010 Helpful Hints, and mNSET 60010 FAQs.
• mNSET 60010 Technical Support Letter [PDF only]
• mNSET 60010 Instructions [PDF]
• mNSET 60010 Helpful Hints [PDF]
• mNSET 60010 Frequently Asked Questions [PDF]
• mNSET 60010 Publications [PDF]
• mNSET 60010 Presentations
• mNSET 60010 Videos [Complete Demonstration] & [Quick Procedure Only]
• mNSET 60010 Webinar
The mNSET™ Device 60010 is manufactured in the USA, solely for ParaTechs Corporation, by an FDA Registered Medical Device Manufacturer and ISO 13485:2003 registered company and is EtO (Ethylene Oxide) sterilization processed. Patent Information: Non-Surgical Embryo Transfer Method and Apparatus, United States Patent 9,615,903. [PDF]
International shipments are subject to customs duties and taxes. The Recipient (Importer) of such goods is responsible for paying all related charges. Please read our Goods Customs Duties and Taxes Statement. [PDF]
Catalogue # 60010
EtO (Ethylene Oxide) Sterilization Processed
10 Devices per Box (Devices are individually packaged with 1 small and 1 large speculum in each sealed pouch.)
Patent Information: Non-Surgical Embryo Transfer Method and Apparatus, United States Patent 9,615,903. [PDF]
This device is used for ethical transcervical transfer of mouse embryos into recipient female mice. For research purposes only. Not intended for human or animal diagnostic or therapeutic uses.
OTHER USES: The mNSET 60010 device can also be used for artificial insemination (AI) or other material/pathogen transfer into recipient female mice. For larger volume transfer, please consider the mC&I device (60020) which has 20-40µl capacity.
Devices are single use only. Discard after use.
Prior to mNSET Embryo Transfer
For the production of mouse embryos for transfer and pseudopregnant females to serve as recipients, standard transgenic methodologies are used.¹ Matings are set up with male and female mice as in standard transgenic procedures. Female donors can be superovulated if desired. Embryos are incubated in EmbryoMax® KSOM media (Millipore#MR-106-D) or desired culture media. Embryos should be 3.5 days post-coitum (3.5 dpc) on the day of mNSET transfer; recipient female mice should be 2.5 dpc on the day of mNSET transfer.
The mNSET Demonstration Videos
The quick procedure and full procedure demonstration videos can be viewed and downloaded here. The quick procedure video demonstrates how easily mouse uterine embryo transfer can be accomplished using the mNSET™ (Non-Surgical Embryo and Sperm Transfer) Device for Mice 60010. The full procedure video details the entire setup and preparation, through the end of the actual procedure.
Embryo Transfer Procedure
1. Place a 15μl drop of culture medium onto the lid of a 60 mm tissue culture dish (Falcon 353002, or similar).
2. Load 12–20 blastocysts into the medium using a standard embryo handling pipette. (Note: optimal number of embryos to transfer will vary depending upon mouse strain and manipulations embryos have received.)
3. Place the mNSET device onto a P-2 Pipetman that has been set to 1.8μl. (Recommended pipettes are the Pipette Rainin Classic PR2, 0.1-2μl or Gilson Pipetman P2, 0.2-2μl.)
4. Press pipette plunger to first stop, lower tip of the mNSET device into medium and slowly pull embryos into the tip. Remove mNSET device tip from the medium.
5. Carefully set pipette to 2.0μl to create a small air bubble at mNSET tip to help ensure embryos stay inside device tip during insertion into the mouse. Gently lay pipette with loaded tip aside (near cage) for use in step #9. Avoid jostling the mNSET tip.
6. Place the unanesthetized recipient female on top of a cage with a wire rack, allowing the mouse to “grab” the cage bar surface. Grasp the base of the tail using thumb and forefinger and angle the tail upward while lightly pressing the base of the tail with the opposite edge of the hand. (See image below.)
7. Gently place small speculum into mouse’s vagina.
8. Optional: Remove small speculum and replace with the large speculum. If desired, use an adequate light source and visualize the cervix.
9. While holding the female mouse with one hand as described in step #6, carefully pick up the pipette and gently insert the mNSET device tip into the speculum and through the cervix. Once mNSET device hub contacts speculum, expel embryos by pressing plunger to the first stop.
10. Gently remove mNSET device without releasing pipette plunger and remove speculum. Return mouse to cage. No post-procedure monitoring is required.
¹Behringer R, Gertsenstein M, Nagy KV, Nagy A. 2014. Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press.
This product is intended for research purposes only.
CAUTION: Not intended for human or animal diagnostic or therapeutic uses.
Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product.
PARATECHS CORPORATION LIMITED WARRANTY
ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product. This warranty limits ParaTechs’ liability to replacement of the Product. PARATECHS MAKES NO OTHER WARRANTIES, EXPRESSED OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.
The following are hints and suggestions from our scientists, technicians and customers which have proved helpful.
1. Read all “Helpful Hints” and the mNSET for Mice 60010 instruction insert carefully before beginning your mNSET trials. The instruction insert can be found in each box of NSET.
2. Prior to actual experiments, practice the mNSET technique on 2.5 dpc pseudopregnant mice without embryos.
3. mNSET is designed to fit snugly on a Rainin Classic PR2, 0.1-2µl or Gilson Pipetman P2, 0.2-2µl pipette for loading embryos and precise measurement of media into the tip of the device.
4. We suggest using conscious, calm, and unagitated mice. This makes it easier to get the mouse in a natural position to find and enter the cervix. The female mouse in the video on our website is not sedated.
5. We use CD1 mice and highly recommend using this strain for your pseudopregnant recipients. We suggest using mice that weigh ≥26g and are at least 60 days old.
6. Embryos should be incubated to blastocyst stage (e3.5) since the device transfers them to one of the uterine horns and not the oviduct. Some end users have been successful using morula embryos.
7. Select a media you have used which gives you the best success in incubating your embryos. For example, use M2 or KSOM medium and transfer 12 to 20 embryos.
8. The mNSET device is only able to pass the cervix during certain phases of estrus and at 2.5 dpc in pseudopregnant mice. Therefore, we strongly recommend the use of 2.5 dpc pseudopregnant mice (after the plug has fallen out) for training purposes and embryo transfers using the mNSET device.
9. We find it relatively easy to keep the female still and reduce squirming by placing the mouse on top of the cage with a wire rack so she can grab the cage bar surface. Use the holding technique as described in #6 of the mNSET instructions and also demonstrated in the mNSET videos found on our webpage: https://paratechs.com/collections/art-devices/products/nset-device-formice#mnset-60010-videos.
10. We do not recommend the use of lubricants. You may use sterile water or culture media to moisten the specula then shake off excess before insertion into the vagina.
11. Gently insert small speculum into the vagina. The mouse will innately push the speculum out a little. Gently press it back in place so the mNSET device can pass through the cervix and into one of the uterine horns.
12. Optional: Remove small speculum and replace with large speculum. If desired, use an adequate light source such as a gooseneck light and visualize the cervix.
13. Be patient and do not apply too much pressure when finding and penetrating the cervix with themNSET tip. This could cause tissue damage and will likely bend the mNSET device tip making it nearly impossible to use. If the first attempt to insert the mNSET is not in the correct location, gently reposition the device and repeat.
14. Embryo loss may occur if tip gets bent due to too much pressure applied while finding the cervical opening. Again, gentle repeated attempts are pertinent to mNSET success.
15. You will know the device is properly inserted through the cervix into the uterus when the hub of the mNSET device touches the end of the speculum.
16. To expel your embryos, press the pipette plunger to the first stop. Count to 3. Do not release plunger.
17. Slowly remove mNSET without releasing pipette plunger. If plunger is released prior to removal, some embryos could be pulled back into the tip.
18. Inspection of the mNSET tip under a microscope after use is good practice. The clear tip mNSETallows visualization inside the tip.
The device is designed for a one-time use only. Repeated use will clog the mNSET tip with cervical tissue. Reuse may render the catheter pliable and no longer rigid enough to pass the cervix. Thus, potentially depositing embryos in the vagina and not the uterine horn as intended.
Contact us with any questions by phone or email firstname.lastname@example.org. We are always happy to help.
1. Can I reuse the mNSET device to transfer embryos into multiple mice?
ParaTechs does not recommend using the mNSET device for more than one transfer. Tissue from the mouse reproductive tract tends to clog the catheter. Reuse also renders the catheter pliable and no longer rigid enough to pass the cervix, thus depositing embryos in the vagina. When the device is used multiple times there may be a noticeable drop in the success rate of embryo transfer.
2. What mouse strain should I use as my embryo recipients?
ParaTechs recommends CD1 or ICR mice. For best results, the recipients should weigh ≥ 26g and be over 60 days old.
3. What day post coitum (dpc) should the recipient mice be?
We strongly recommend the use of 2.5 dpc pseudopregnant mice for both training purposes and embryo transfers using the mNSET device. It is possible for the mNSET device to pass through the cervix of a pseudopregnant mouse 1.5 dpc. However, the success rate of embryo transfer at that time has shown to be lower than using the recommended 2.5 dpc pseudopregnant female.
4. What developmental stage should my embryos be for transfer?
Embryos should be in a later developmental stage than the reproductive tract of the pseudopregnant female. For instance, blastocysts (e3.5 days after fertilization) should be transferred into a 2.5 dpc pseudopregnant recipient.
5. Is anesthesia required to perform the mNSET procedure?
No. ParaTechs does not recommend the use of anesthesia. A calm conscious animal can be positioned so that the mNSET device catheter can easily pass the cervix. The mouse in the demonstration video on our website is not anesthetized: https://paratechs.com/collections/art-devices/products/nset-device-for-mice#mnset-60010-videos. Using an unanesthetized mouse also makes the procedure faster and easier while eliminating the risks and stress of anesthesia.¹ Anesthesia may be helpful for training purposes but need not be used under ordinary conditions.
6. Should I use a lubricant during the mNSET procedure?
No. Lubricants can clog the mNSET catheter and prevent the correct placement of embryos in the uterine horn. The specula maybe moistened with sterile water or culture media prior to insertion, but even this is unnecessary. If moistening the specula, be sure to shake off any excess moisture before inserting the devices into the vagina.
7. I’m having trouble locating and passing through the cervix. What should I do?
Be sure that your recipient female is 2.5 dpc pseudopregnant. Use gooseneck lighting to inspect and locate the cervical opening before inserting the mNSET catheter, as this will help you position the device correctly. It may be helpful to practice passing through the cervix of 2.5 dpc pseudopregnant females before attempting embryo transfer.
8. How many embryos should I transfer into each recipient mouse?
For most transfers, ParaTechs recommends transferring 12-20 embryos to each recipient mouse. (Note: optimal number of embryos to transfer will vary depending upon mouse strain and manipulations embryos have received.)
9. I performed a non-surgical embryo transfer, but when I removed the mNSET device, the catheter was bent. What happened?
A bent catheter likely means that the catheter did not enter the cervix or that you applied too much pressure while trying to locate the cervical opening. If the catheter is bent, it is unlikely that the embryos were deposited into the uterine horn of the mouse. It is important to use gentle pressure when locating the cervical opening.
10. Can I use mice multiple times as embryo recipients?
Studies by ParaTechs have shown that it is possible to perform multiple mNSET procedures on a female recipient and obtain up to three litters. However, there was a decrease in pregnancy rate and embryo transfer efficiency after the first litter.
11. Can the mNSET device be used for Artificial Insemination (AI)?
Yes. The mNSET device can also be used to deliver sperm to a recipient female mouse to facilitate AI.² Please see the article below byStone et al. (2015). Please email us (email@example.com) if you would like to receive the protocol.
12. Does the mNSET device have other applications?
Yes. The mNSET device can also be used as a novel method for effective transfer of substances for studies of uterine physiology and bacterial infection.³ Please email us (firstname.lastname@example.org) if you would like to receive more information.
¹Steele KH, Hester JM, Stone BJ, Carrico KM, Spear BT, Fath-Goodin A. (2013). Non-surgical embryo transfer device (NSET) is less stressful than surgery for embryo transfer in mice. JAALAS. Jan; 52(1): 17-21. https://aalas.publisher.ingentaconnect.com/content/aalas/jaalas/2013/00000052/00000001/art00004.
²Stone BJ, Steele KH, Fath-Goodin A. (2015). A rapid and effective nonsurgical artificial insemination protocol using the NSETᵀᴹ device for sperm transfer in mice without anesthesia. Transgenic Research Associated with the International Society for Transgenic Technologies (ISTT) 2015 :9887DOI: 10.1007/s11248-015-9887-3. https://link.springer.com/article/10.1007/s11248-015-9887-3.
³GondekDC, Olive AJ, Stary G, Starnback MN. (2012). CD4+ T cells are necessary and sufficient to confer protection against Chlamydiatrachomatis infection in the murine upper genital tract. J Immunol. Sep; 189(5): 2441-9. Epub 2012 Aug 1. https://www.jimmunol.org/content/189/5/2441.
³Barrette VF, Adams MA, Croy BA. (2012). Endometrial decidualization does not trigger the blood pressure decline of normal early pregnancy in mice. Biol Reprod. Mar 8; 86(3):66. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380067/.
Albers RE, Kaufman MR, Natale BV, Keoni C, Kulkarni-Datar K, Min S, Williams CR, Natale DRC, Brown TL (2019) Trophoblast-Specific Expression of Hif-1α Results in Preeclampsia-Like Symptoms and Fetal Growth Restriction. Sci Rep. 9(1):2742.
doi: 10.1038/s41598-019-39426-5. PMID: 30808910; PMCID: PMC6391498. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391498/
Bin Ali R, van der Ahé F, Braumuller TM, Pritchard C, Krimpenfort P, Berns A, Huijbers IJ (2014) Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer. Transgenic Res 23(4):691-5. PMID: 24798251; PMCID: PMC4053600. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053600/
Bulut-Karslioglu A, Biechele S, Jin H, Macrae TA, Hejna M, Gertsenstein M, Song JS, Ramalho-Santos M (2016) Inhibition of mTOR induces a paused pluripotent state. Nature 540(7631):119-123. doi: 10.1038/nature20578. PMID: 27880763; PMCID: PMC5143278. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5143278/
Choi ES, Kawano K, Hiraya M, Matsukawa E, Yamada M (2019) E of pyruvate and dimethyl-α-ketoglutarate, either alone or in combination, on pre- and post-implantation development of mouse zygotes cultured in vitro. Reprod Med Biol 18(4):405-410. doi: 10.1002/rmb2.12288. PMID: 31607802; PMCID: PMC6780036. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780036/
Costa-Borges N, Spath K, Miguel-Escalada I, Mestres E, Balmaseda R, Serafín A, Garcia-Jiménez M, Vanrell I, González J, Rink K, Wells D, Calderón G. (2020) Maternal spindle transfer overcomes embryo developmental arrest caused by ooplasmic defects in mice.
Elife. 9:e48591. doi: 10.7554/eLife.48591. PMID: 32347793; PMCID: PMC7259950. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259950/
de Waal E, Mak W, Calhoun S, Stein P, Ord T, Krapp C, Coutifaris C, Schultz RM, Bartolomei MS (2014) In vitro culture increases the frequency of stochastic epigenetic errors at imprinted genes in placental tissues from mouse concepti produced through assisted reproductive
technologies. Biol Reprod 90(2):22. doi: 10.1095/biolreprod.113.114785. PMID: 4337315; PMCID: PMC4076403.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076403/
de Waal E, Vrooman LA, Fischer E, Ord T, Mainigi MA, Coutifaris C, Schultz RM, Bartolomei MS (2015) The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model. Hum Mol Genet 24(24):6975-85. doi: 0.1093/hmg/ddv400. PMID: 26401051; PMCID: PMC4654053. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654053/
Green MA, Bass S, Spear BT (2009) A device for the simple and rapid transcervical transfer of mouse embryos eliminates the need for surgery and potential post-operative complications. Biotechniques. 47:919-924. doi: 0.2144/000113257. PMID:20041845; PMCID: PMC4506771. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506771/
Herrick JR, Greene-Ermisch AF, Schoolcraft WB, Krisher RL. (2017) Exogenous growth factors do not affect the development of individually cultured murine embryos. J Assist Reprod Genet. 35(3):523-531. doi: 10.1007/s10815-017-1103-3. PMID: 29270871; PMCID: PMC5904071. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904071/
Huang CH, Chan WH (2017) Rhein induces oxidative stress and apoptosis in mouse blastocysts and has immunotoxic effects during embryonic development. Int J Mol Sci 18(9):2018. doi: 10.3390/ijms18092018. PMID: 28930172; PMCID: PMC5618666. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618666/
Huang CH, Chan WH (2017) Protective effects of liquiritigenin against citrinin-triggered, oxidative-stress-mediated apoptosis and disruption of embryonic development in mouse blastocysts. Int J Mol Sci 18(12):2538. doi: 10.3390/ijms18122538. PMID: 29186930; PMCID: PMC5751141. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751141/
Jimenez R, Melo EO, Davydenko O, Ma J, Mainigi M, Franke V, Schultz RM (2015) Maternal SIN3A regulates reprogramming of gene expression during mouse preimplantation development. Biol Reprod 93(4):89. doi: 0.1095/biolreprod.115.133504. PMID: 26353893; PMCID: PMC4711907. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711907/
Karimi H, Mahdavi P, Fakhari S, Faryabi MR, Esmaeili P, Banafshi O, Mohammadi E, Fathi F, Mokarizadeh A (2017) Altered helper T cell-mediated immune responses in male mice conceived through in vitro fertilization. Reprod Toxicol 69:196-203. doi: 10.1016/j.reprotox.2017.03.005. PMID: 28284725. https://europepmc.org/abstract/med/28284725/
Kaufman MR, Albers RE, Keoni C, Kulkarni-Datar K, Natale DR, Brown TL (2014) Important aspects of placental-specific gene transfer. Theriogenology 82(7):1043-8. doi:
10.1016/j.theriogenology.2014.07.010. PMID: 25110063; PMCID: PMC4167466.
Liu SJ, Sun JB, Hao X, Han Z, Wen X, Wang XY, Zhou CJ, Liang CG (2020) Blastocyst hatching site is regularly distributed and does not influence foetal development in mice. Scientific reports, 10(1):2475. doi: 10.1038/s41598-020-59424-2. PMID: 32051520; PMCID: PMC7015891. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015891/
Mainigi MA, Olalere D, Burd I, Sapienza C, Bartolomei M, Coutifaris C (2014) Peri-implantation hormonal milieu: elucidating mechanisms of abnormal placentation and fetal growth. Biol Reprod 90(2):26. doi: 10.1095/biolreprod.113.110411. PMID: 24352558; PMCID: PMC4076405. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076405/
Mainigi M, Rosenzweig JM, Lei J, Mensah V, Thomaier L, Talbot CC Jr, Olalere D, Ord T, Rozzah R, Johnston MV, Burd I. (2016) Peri-Implantation Hormonal Milieu: Elucidating Mechanisms of Adverse Neurodevelopmental Outcomes. Reprod Sci. 3(6):785-94. doi: 10.1177/1933719115618280. PMID: 26614264; PMCID: PMC5933150.
Martin NP, Myers P, Goulding E, Chen SH, Walker M, Porter TM, Van Gorder L, Mathew A, Gruzdev A, Romeo C (2018) En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida. Transgenic Res 27(1):39-49. doi: 10.1007/s11248-017-0056-8. PMID: 29442214; PMCID: PMC5990369. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990369/
Navarrete FA, Alvau A, Lee HC, Levin LR, Buck J, Leon PM, Santi CM, Krapf D, Mager J, Fissore RA, Salicioni AM, Darszon A, Visconti PE (2016) Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models. Sci Rep 6:33589. doi: 0.1038/srep33589. PMID: 27627854; PMCID: PMC5024339. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024339/
Navarrete FA, Aguila L, Martin-Hidalgo D, Tourzani DA, Luque GM, Ardestani G, Garcia-Vazquez FA, Levin LR, Buck J, Darszon A, Buffone MG, Mager J, Fissore RA, Salicioni AM, Gervasi MG, Visconti PE. (2019) Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies. Front Cell Dev Biol. 7:262. doi: 10.3389/fcell.2019.00262. PMID: 31750304; PMCID: PMC6848031. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848031/
Prasad P, Molla MR, Cui W, Canakci M, Osborne B, Mager J, Thayumanavan S (2015) Polyamide nanogels from generally recognized as safe components and their toxicity in mouse preimplantation embryos. Biomacromolecules 16(11):3491-8. doi: 10.1021/acs.biomac.5b00900. PMID: 26367020; PMCID: PMC4970214. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970214/
Racicot K, Aldo P, El-Guindy A, Kwon JY, Romero R, Mor G. (2017) Cutting Edge: Fetal/Placental Type I IFN Can Affect Maternal Survival and Fetal Viral Load during Viral Infection. J Immunol. 198(8):3029-3032. doi: 10.4049/jimmunol.1601824. PMID:
28264970; PMCID: PMC5633930. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633930/
Rahimi S, Martel J, Karahan G, Angle C, Behan NA, Chan D, MacFarlane AJ, Trasler JM. (2019) Moderate maternal folic acid supplementation ameliorates adverse embryonic and
epigenetic outcomes associated with assisted reproduction in a mouse model. Hum Reprod. 34(5):851-862. doi: 10.1093/humrep/dez036. PMID: 30989206; PMCID: PMC6505447. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505447/
Saben JL, Asghar Z, Rhee JS, Drury A, Scheaffer S, Moley KH (2016) excess maternal fructose consumption increases fetal loss and impairs endometrial decidualization in mice. Endocrinology 157(2):956-68. doi: 10.1210/en.2015-1618. PMID: 26677880; PMCID: PMC4733112. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733112/
Sepulveda-Rincon LP, Islam N, Marsters P, Campbell BK, Beaujean N, Maalouf WE (2017) Embryo cell allocation patterns are not altered by biopsy but can be linked with further development. Reproduction 154(6):807-814. doi: 10.1530/REP-17-0514. PMID: 28971891; PMCID: PMC5747100. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747100/
Steele KH, Hester JM, Stone BJ, Carrico KM, Spear BT, Fath-Goodin A (2013) Nonsurgical embryo transfer device compared with surgery for embryo transfer in mice. J Am Assoc Lab Anim Sci 52(1):17-21. PMID: 23562028; PMCID: PMC3548196. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548196/
Stone BJ (2020) Nonsurgical Embryo Transfer Protocol for Use with the NSET™ Device. Methods Mol Biol 2066:107-111. doi: 10.1007/978-1-4939-9837-1_9. PMID: 31512211. https://link.springer.com/protocol/10.1007%2F978-1-4939-9837-1_9
Sullivan-Pyke C, Mani S, Rhon-Calderon EA, Ord T, Coutifaris C, Bartolomei MS, Mainigi M. (2020) Timing of exposure to gonadotropins has differential effects on the conceptus: evidence from a mouse model†. Biol Reprod. 103(4):854-865. doi: 10.1093/biolre/ioaa109. PMID: 32584398; PMCID: PMC8179426. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179426/
Tian X, Anthony K, Neuberger T, Diaz FJ (2014) Preconception zinc deficiency disrupts postimplantation fetal and placental development in mice. Biol Reprod 90(4):83. doi: 10.1095/biolreprod.113.113910. PMID: 24599289; PMCID: PMC4076385. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076385/
Valachova B, Cubinkova I, Brezovakova V, Hanes J, Jadhav S (2019) Rederivation of transgenic rodent models expressing disease modified tau protein - a report. SJLAS 45(5):1-8. doi.org/10.23675/sjlas.v45i0.915. http://www.sjlas.org/index.php/SJLAS/article/view/915/
Weinerman R, Feng R, Ord TS, Schultz RM, Bartolomei MS, Coutifaris C, Mainigi M. (2016) Morphokinetic Evaluation of Embryo Development in a Mouse Model: Functional and Molecular Correlates. Biol Reprod. 94(4):84. doi: 10.1095/biolreprod.115.134080. PMID:
26911427; PMCID: PMC6322449. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322449/
Weinerman R, Ord T, Bartolomei MS, Coutifaris C, Mainigi M (2017) The superovulated environment, independent of embryo vitrification, results in low birthweight in a mouse model. Biol Reprod. 97(1):133-142. doi: 10.1093/biolre/iox067. PMID: 28859279; PMCID: PMC6248797. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248797/
Yoon J, Juhn KM, Yoon SH, Ko Y, Lim JH (2017) Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation. Clin Exp Reprod Med 44(1):15-21. doi: 10.5653/cerm.2017.44.1.15. PMID: 28428939; PMCID: PMC5395547. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395547/
Zhang S, Mesalam A, Lee KL, Song SH, Khan I, Yuan Y, Wenfa LV, Kong IK (2019) Effect of Predator Stress on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer. J Am Assoc Lab Anim Sci. 58(3):304-310. doi: 10.30802/AALAS-JAALAS-18-000085. PMID: 30971328; PMCID: PMC6526485. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526485/
Avella MA, Baibakov B, Dean J (2014) A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans. J Cell Biol 205(6):801-9. doi: 10.1083/jcb.201404025. PMID:24934154; PMCID: PMC4068139. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068139/
Lough-Stevens M, Ghione CR, Urness M, Hobbs A, Sweeney CM, Dean MD (2021) Biology of Reproduction, 2021, 104(3),684–694, Male-derived copulatory plugs enhance implantation success in female Mus musculus,https://doi.org/10.1093/biolre/ioaa228
Stone BJ, Steele KH, Fath-Goodin A (2015) A rapid and effective nonsurgical artificial insemination protocol using the NSET™ device for sperm transfer in mice without anesthesia. Transgenic Res24(4):775-81. doi: 10.1007/s11248-015-9887-3. PMID:26065409; PMCID: PMC4504984.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504984/
Chen J, Zhang H, Zhou Z, Yang Z, Ding Y, Zhou Z, Zhong E, Arulanandam B, Baseman J, Zhong G (2014) Chlamydial induction of hydrosalpinx in 11 strains of mice reveals multiple host mechanisms for preventing upper genital tract pathology. PLoS One 9(4):e95076. doi: 10.1371/journal.pone.0095076. PMID: 24736397; PMCID: PMC3988139. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988139/
Coers J, Gondek DC, Olive AJ, Rohlfing A, Taylor GA, Starnbach MN (2011) Compensatory
T cell responses in IRG-deficient mice prevent sustained Chlamydia trachomatis infections. PLoS Pathog 7(6):e1001346. doi: 10.1371/journal.ppat.1001346. PMID: 21731484; PMCID: PMC3121881. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121881/
Davila SJ, Olive AJ, Starnbach MN (2014) Integrin α4β1 is necessary for CD4+ T cell-mediated protection against genital Chlamydia trachomatis infection. J Immunol 192(9):4284-93. doi:
10.4049/jimmunol.1303238. PMID: 24659687; PMCID: PMC3995848. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995848/
Fischer A, Harrison KS, Ramirez Y, Auer D, Chowdhury SR, Prusty BK, Sauer F, Dimond Z, Kisker C, Hefty PS, Rudel T (2017) Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense. Elife 6:e21465. doi: 10.7554/eLife.21465. PMID: 28347402; PMCID: PMC5370187. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5370187/
Gondek DC, Olive AJ, Stary G, Starnbach MN (2012) CD4+ T cells are necessary and sufficient to confer protection against Chlamydia trachomatis infection in the murine upper genital tract. J Immunol 189(5):2441-9. doi: 10.4049/jimmunol.1103032. PMID: 22855710; PMCID:
Helble JD, Starnbach MN. (2019) A Chlamydia trachomatis Strain Expressing Ovalbumin
Stimulates an Antigen-Specific CD4+ T Cell Response in Mice. Infect Immun. 87(7):e00837-18. doi: 10.1128/IAI.00837-18. PMID: 30988057; PMCID: PMC6589065. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589065/
Helble JD, Reinhold-Larsson NV, Starnbach MN. (2019) Early Colonization of the Upper
Genital Tract by Chlamydia muridarum Is Associated with Enhanced Inflammation Later in Infection. Infect Immun. 87(9):e00405-19. doi: 10.1128/IAI.00405-19. PMID: 31285254; PMCID: PMC6704611. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704611/
Helble JD, Gonzalez RJ, von Andrian UH, Starnbach MN. (2020) Gamma Interferon Is Required for Chlamydia Clearance but Is Dispensable for T Cell Homing to the Genital Tract. mBio. 11(2):e00191-20. doi: 10.1128/mBio.00191-20. PMID: 32184237; PMCID: PMC7078466. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078466/
Helble JD, Mann AO, Starnbach MN. (2020) Antigen-specific memory and naïve CD4+ T cells following secondary Chlamydia trachomatis infection. PLoS One. 15(10):e0240670. doi: 10.1371/journal.pone.0240670. PMID: 33091023; PMCID: PMC7580951. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580951/
Karunakaran KP, Yu H, Jiang X, Chan Q, MoonKM, Foster LJ, Brunham RC (2015) Outer membrane proteins preferentially load MHC class II peptides: implications for a Chlamydia trachomatis T cell vaccine. Vaccine 33(18):2159-66. doi: 10.1016/j.vaccine.2015.02.055. PMID: 25738816; PMCID: PMC4390527. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390527/
LaBrie SD, Dimond ZE, Harrison KS, Baid S, Wickstrum J, Suchland RJ, Hefty PS (2019) Transposon mutagenesis in Chlamydia trachomatis identifies CT339 as a ComEC homolog important for DNA uptake and lateral gene transfer. MBio 10(4):e01343-19. doi: 10.1128/mBio.01343-19. PMID: 31387908; PMCID: PMC6686042. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686042/
Nguyen NDNT, Olsen AW, Lorenzen E, Andersen P, Hvid M, Follmann F, Dietrich J. (2020) Parenteral vaccination protects against transcervical infection with Chlamydia trachomatis and generate tissue-resident T cells post-challenge. NPJ Vaccines. 5(1):7. doi:
10.1038/s41541-020-0157-x. PMID: 31993218; PMCID: PMC6978417. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978417/
Nogueira CV, Zhang X, Giovannone N, Sennott EL, Starnbach MN (2015) Protective immunity against Chlamydia trachomatis can engage both CD4+ and CD8+ T cells and bridge the respiratory and genital mucosae. J Immunol 194(5):2319-29. doi: 10.4049/jimmunol.1402675. PMID: 25637024; PMCID: PMC4340718. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340718/
Olive AJ, Gondek DC, Starnbach MN (2011) CXCR and CCR5 are both required for T cell-mediated protection against C. trachomatis infection in the murine genital mucosa. Mucosal Immunol 4(2):208-16. doi: 10.1038/mi.2010.58. PMID: 20844481; PMCID: PMC3010299. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010299/
Olive AJ, Haff MG, Emanuele MJ, Sack LM, Barker JR, Elledge SJ, Starnbach MN. (2014) Chlamydia trachomatis-induced alterations in the host cell proteome are required for intracellular growth. Cell Host Microbe. 15(1):113-24. doi:
10.1016/j.chom.2013.12.009. PMID: 24439903; PMCID: PMC3932326. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932326/
Pal S, Tifrea DF, Zhong G, de la Maza LM. (2018) Transcervical Inoculation with Chlamydia trachomatis Induces Infertility in HLA-DR4 Transgenic and Wild-Type Mice. Infect Immun. 86(1):e00722-17. doi: 10.1128/IAI.00722-17. PMID: 29038126; PMCID: PMC5736824. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736824/
Ramsey KH, Schripsema JH, Smith BJ, Wang Y, Jham BC, O'Hagan KP, Thomson NR, Murthy AK, Skilton RJ, Chu P, Clarke IN (2014) Plasmid CDS5 influences infectivity and virulence in a mouse model of Chlamydia trachomatis urogenital infection. Infect Immun 82(8):3341-9. doi: 10.1128/IAI.01795-14. PMID: 24866804; PMCID: PMC4136204. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136204/
Shao L, Zhang T, Liu Q, Wang J, Zhong G. (2017) Chlamydia muridarum with Mutations in
Chromosomal Genes tc0237 and/or tc0668 Is Deficient in Colonizing the Mouse Gastrointestinal Tract. Infect Immun. 85(8):e00321-17. doi: 10.1128/IAI.00321-17. PMID: 28584162; PMCID: PMC5520443. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520443/
Stary G, Olive A, Radovic-Moreno AF, Gondek D, Alvarez D, Basto PA, Perro M, Vrbanac VD, Tager AM, Shi J, Yethon JA, Farokhzad OC, Langer R, Starnbach MN, von Andrian UH (2015) VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science 348(6241):aaa8205. doi: 10.1126/science.aaa8205. PMID: 26089520; PMCID: PMC4605428. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605428/
Tang L, Zhang H, Lei L, Gong S, Zhou Z, Baseman J, Zhong G (2013) Oviduct infection and hydrosalpinx in DBA1/j mice is induced by intracervical but not intravaginal inoculation with Chlamydia muridarum. PLoS One 8(8):e71649. doi: 10.1371/journal.pone.0071649. PMID: 23940777; PMCID: PMC3734308. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734308/
Tang L, Yang Z, Zhang H, Zhou Z, Arulanandam B, Baseman J, Zhong G (2014) Induction of protective immunity against Chlamydia muridarum intracervical infection in DBA/1j mice. Vaccine 32(12):1407-13. doi: 10.1016/j.vaccine.2013.10.018. PMID: 24188757; PMCID: PMC3943569. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943569/
Vicetti Miguel RD, Quispe Calla NE, Pavelko SD, Cherpes TL (2016) Intravaginal Chlamydia trachomatis challenge infection elicits TH1 and TH17 immune responses in mice that promote pathogen clearance and genital tract damage. PLoS One 11(9):e0162445. doi: 10.1371/journal.pone.0162445. PMID: 27606424; PMCID: PMC5015975. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015975/
Vicetti Miguel RD, Quispe Calla NE, Dixon D, Foster RA, Gambotto A, Pavelko SD, Hall-Stoodley L, Cherpes TL (2017) IL-4-secreting eosinophils promote endometrial stromal cell proliferation and prevent Chlamydia-induced upper genital tract damage. Proc Natl Acad Sci U S A
114(33):E6892-E6901. doi: 10.1073/pnas.1621253114. PMID: 28765368; PMCID: PMC5565408. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565408/
Yu H, Karunakaran KP, Jiang X, Brunham RC (2016) Subunit vaccines for the prevention of mucosal infection with Chlamydia trachomatis. Expert Rev Vaccines 15(8):977-88. doi: 10.1586/14760584.2016.1161510. PMID: 26938202; PMCID: PMC4981183. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981183/
Zhang Y, Shao L, Li X, Zhong G (2017) Uterotubal junction prevents chlamydial ascension via innate immunity. PLoS One 12(8):e0183189. doi: 10.1371/journal.pone.0183189. PMID: 28797102; PMCID: PMC5552320.
Zhang T, Huo Z, Ma J, He C, Zhong G (2019) The plasmid-encoded pGP3 promotes Chlamydia evasion of acidic barriers in both stomach and vagina. Infect Immun 87(5):e00844-18. doi: 10.1128/IAI.00844-18. PMID: 30858342; PMCID: PMC6479032. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479032/
Armstrong GM, Maybin JA, Murray AA, Nicol M, Walker C, Saunders PTK, Rossi AG, Critchley HOD (2017) Endometrial apoptosis and neutrophil infiltration during menstruation exhibits spatial and temporal dynamics that are recapitulated in a mouse model. Sci Rep 7(1):17416. Doi: 10.1038/s41598-017-17565-x. PMID: 29234102; PMCID: PMC5727295. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727295/
Barrette VF, Adams MA, Croy BA (2012) Endometrial decidualization does not trigger the blood pressure decline of normal early pregnancy in mice. Biol Reprod 86(3):66. Doi:
10.1095/biolreprod.111.096958. PMID: 22156477; PMCID: PMC3380067. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380067/
Cousins FL, Murray A, Esnal A, Gibson DA, Critchley HO, Saunders PT (2014) Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation. PloS One. 9(1):e86378. Doi: 10.1371/journal.pone.0086378. PMID: 24466063; PMCID: PMC3899239. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899239/
Li S, Garcia M, Gewiss RL, Winuthayanon W (2017) Crucial role of estrogen for the mammalian female in regulating semen coagulation and liquefaction in vivo. PloS Genet 13(4):e1006743. Doi: 10.1371/journal.pgen.1006743. PMID: 28414719; PMCID: PMC5411094. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411094/
Lab AnimalSciences 2014 by Dr. Barbara Stone, Director of NSET Technology, ParaTechsCorporation, C.E. Credits: CE. https://www.labroots.com/webinar/the-future-of-mouse-embryo-transfer-achieving-the-3rs-with-the-nset-device
Moreno-Moya JM, Ramírez L, Vilella F, Martínez S, Quinonero A, Noguera I, Pellicer A, and Simon C (2014) Complete method to obtain, culture, and transfer mouse blastocysts nonsurgically to study implantation and development. Fertility and Sterility Forum. https://pubmed.ncbi.nlm.nih.gov/24355048/
Stone B, ParaTechs Corporation. mNSET (Non-Surgical Embryo Transfer) Device for Mice 60010 Full Demonstration and QuickProcedure Video (2019) https://youtu.be/eQ4LuKNXQtw
Stone B, ParaTechs Corporation. mNSET (Non-Surgical Embryo Transfer) Device for Mice 60010 Quick Procedure DemonstrationVideo (2019) https://youtu.be/ItFo8zacPnw
Animal Welfare Review:
Ormandy EH, Dale J, Griffin G (2011) Genetic engineering of animals: ethical issues, including welfare concerns. Can Vet J52(5):544-50. PMID: 22043080; PMCID: PMC3078015. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078015/
CARD-IP Mouse Sperm and Embryo Cryopreservation presentation by ParaTechs’ Director of NSET Technology, Dr. Barbara Stone, October24 – 28, 2016; Institut Pasteur, Paris, France. This one-week course presented a combination of lectures and intensive hands-on sessions to learn the most up-to-date CARD methods in mouse cryopreservation. Dr. Stone led the portion of the course, “Embryo Transfer and Artificial Insemination in Mice using the NSET Device”. The aim of this course was to introduce the newest CARD methods to researchers and technicians involved in mouse archiving and/or managing transgenic facilities and who are willing to implement these new methods in their work. These techniques were taught directly by the team that devised them. https://www.pasteur.fr/en/education/programs-and-courses/pasteur-workshops-old. View the history of the CARD courses here: https://www.mouse-ivf-training.com/archives/category/overseas.
University of Veterinary Medicine Vienna, Cryo & Embryo Transfer Course, September 5-9, 2016. The Institute of Laboratory Animal Science and Biomodels Austria at the University of Veterinary Medicine Vienna offer a comprehensive course on cryopreservation, embryo transfer and other methods of assisted reproduction in mice. The course was intended to give technicians and scientists state-of-the-art background knowledge and hands-on training in the methods routinely used at the University of Veterinary Medicine Vienna.
National Center for Biological Sciences (NCBS), August 16-22, 2016, Tata Institute of Fundamental Research, Bengaluru INDIA. ParaTechs’ Dr. Barbara Stone, Director of NSET Technology, gave a lecture presentation and hands-on workshop as a course instructor for the NSET Device Technology during the NCBS Animal Care and Resource Center Mouse Cryobiology / IVF Workshop.
During the 2016 District 5 AALAS Meeting, sponsored by the Southern Ohio Branch AALAS, Kendra Steele, Ph.D. presented advances in 3Rs practices for assisted reproductive techniques in rodents, “Using the NSET (device) for embryo transfer and artificial insemination in mice and rats”. The conference was held in Covington, Kentucky USA, May 11-13, 2016. [PDF]
During the 12th Transgenic Technology Meeting in Edinburgh Scotland, October 6-8, 2014, Dr. Barbara Stone presented the poster, “A Rapid and Effective Nonsurgical A.I. Protocol using the NSET Device for Sperm Transfer in Unanesthetized Mice.” For more information regarding the TT2014 meeting please visit the ISTT website.
Stone B, (2014) Embryo Transfer in Mice using the NSET Device. Presentation and Hands-on Workshop during the 2014 CARD-RPCI Mouse Sperm and Embryo Cryopreservation Course held at Roswell Park Cancer Institute, Buffalo, NY, USA, Sept 15-19, 2014.
Stone B, (2014) The Future of Mouse Embryo Transfer: Achieving the 3Rs with the NSET Device. Abstract presented during the 2014 District 5 AALAS Meeting held May 14-16, 2014, Lexington, Kentucky, USA. [PDF]
Steele K, Stone B, Hester J, Fath-Goodin A. (2014) Non-surgical Embryo Transfer in Mice Is An Easy, Effective, and Ethical Replacement For Surgery. Poster presented at the 2014 District 5 AALAS Meeting held May 14-16, 2014, Lexington, Kentucky, USA. [PNG]
Stone B. (2014) A Non-surgical Uterine Transfer Technique for Mouse Embryos after Cryopreservation, In Vitro Fertilization, ES-cell Injection, and Sperm during Artificial Insemination. Poster presented at the 2014 District 5 AALAS Meeting held May 14-16, 2014, Lexington, Kentucky, USA. [PDF]
Stone B, (2014) The Future of Mouse Embryo Transfer: Achieving the 3Rs with the NSET Device. Industry Track Presentation presented during the February 2014 Laboratory Animal Science BioConference Live [PDF]
Steele K, Stone B, Hester J, Fath-Goodin A. (2013) Non-surgical Embryo Transfer in Mice Is An Easy, Effective, and Ethical Replacement For Surgery. Poster presented at: The 64TH AALAS National Meeting October 27-31, 2013 in Baltimore, MD. [PNG]
Stone B. (2013) Successful Use of the NSETTM Device for Non-surgical Uterine Transfer of Embryos or Sperm. Poster presented at: The 64TH AALAS National Meeting October 27-31, 2013 in Baltimore, MD and also during the Kentucky Innovation & Entrepreneurship Conference August 29, 2013. [PDF]
Steele K, Hester J, Stone B, Spear B, Fath-Goodin A. (2012) Non-surgical embryo transfer with the NSETTM device is a 3Rs refinement technique that reduces stress in CD-1 mice. Poster presented at: The 12th FELASA SECAL Congress. 2013 June 10-13; Barcelona Spain [PDF]
Stone B. (2013) Successful use of the NSET device for non-surgical transfer of blastocysts after in vitro fertilization, cryopreservation, or ES-cell injection and sperm transfer for artificial insemination. Poster presented at: The 11th Meeting of the International Society of Transgenic Technologies. 2013 February 25-27; Guangzhou China. [PDF]
Steele K, Hester J, Stone B, Spear B, Fath-Goodin A. (2012) Non-surgical embryo transfer with the NSETTM device is a 3Rs refinement technique that reduces stress in CD-1 mice. Poster presented at: The 14th annual NIH SBIR/STTR Conference. 2012 May 30-June 1; Louisville KY, USA. Also presented at AALAS National Meeting. 2012 November 6-8; Minneapolis MN, USA [PDF]
Damiani P, Coffee R, Boutin S, Vitale J, Grass D, Soerensen U. (2011) Production of Germfree Chimeric Mice using Non-surgical Embryo Transfer from Embryos Shipped Overnight in a Portable Incubator System. Poster presented at AALAS National Meeting. 2011 October 2-6. San Diego CA, USA.
Williams M. (2011). Rederivation of a Colony using non-surgical embryo transfer. Presentation given at 2011 ANZLAA 2011 Annual Conference. 2011 Sept. 14-16. Hobart Tasmania.
ParaTechs Corporation is a privately held Biotechnology company formed in 2004 and is founded on intellectual property from the University of Kentucky, USA.