All animal research was approved by the ParaTechs 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. All mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Vasectomized male CD1 mice (>2 months old) were used to induce pseudopregnancy. Female CD1 mice between 2 and 5 months old were used as sperm recipients. Male C3H mice (>2 months old) with proven fertility were used as sperm donors. The vivarium was maintained at 20–22 °C with an average relative humidity of 35–50 % under a 12:12 h light:dark (light from 8 am to 8 pm) cycle. Mice were housed in standardized ventilated microisolation caging (Innovive, San Diego, CA, USA). Mice had access to Teklad Global 19 % protein extruded rodent diet #2919 (Harlan Laboratories, Madison, WI, USA) and bottled sterile water (Innovive) ad libitum.
Sperm isolation and preparation
Human tubal fluid medium (HTF) (Irvine Scientific, Santa Ana, CA, USA, Cat #90126) was prepared by adding 4 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA, Cat #A3311), and filter sterilizing. The HTF media was equilibrated prior to use for at least 30 min at 37 °C in the presence of 5 % CO2. Fresh sperm was collected from the cauda epididymis and vas deferens of C3H males. The sperm was capacitated in 500 µl freshly prepared HTF media under embryo culture grade mineral oil (Sigma-Aldrich, Cat #M8410) at 37 °C in the presence of 5 % CO2 for 45 min.
Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG, Cat# AFP8456A) were obtained from the National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, (Torrance, CA, USA) and diluted in sterile phosphate buffered saline (PBS) (Butler-Schein, Dublin, OH, USA, Cat #002477) to 1000 IU/ml, and stored at −80 °C until use. Female CD1 mice for this study were either naïve (chosen by outward appearance to be in estrus) or were injected with hormones for induction of estrus or superovulation. For naïve females, estrus cycle indicators were observed as previously described (Behringer et al. 2014). Superovulated females were prepared by intraperitoneal (i.p.) injection with 5 IU PMSG and hCG administered ~47 h apart at various time schedules relative to the vivarium light–dark cycle. To induce a more natural ovulation condition in CD1 females, the hormones were reduced to 1 IU per i.p. injection.
After optimization of the procedure, a total of 40 µl of fresh sperm was transferred to the uterine horns of female CD1 mice. The transfer was performed without the use of anesthesia or analgesia and took <1 min per mouse. Sperm transfer was carried out using a commercially available device originally developed for nonsurgical embryo transfer in mice, the NSET™ device (ParaTechs, Lexington, KY, USA). The NSET device, a tapered Teflon catheter connected to a specially designed hub, was attached to a Rainin P20 pipette (Mettler-Toledo, Columbus, OH, USA). The device was loaded with 20 µl sperm from the edge of the sperm sample, avoiding clumps. The un-anesthetized recipient female was placed on a wire bar cage top and allowed to grasp the surface with its forefeet. The mouse was held at the base of the tail using a thumb and forefinger, and the rear angled slightly upward, while lightly pressing the base of the hind legs with two fingers from the same hand. The holding technique has been described previously for nonsurgical embryo transfer (Green et al. 2009) and a video of the procedure is available (www.paratechs.com/nset/). Either the small or large speculum included with the device was used to hold open the vagina. If the larger diameter speculum was used, the cervix could be visually located with the aid of a direct light source. The NSET tip was inserted through the cervix and into the uterus. Once the NSET hub contacted the speculum, the sperm was expelled by pressing the pipette plunger to the first stop. The procedure was then repeated to deliver a total sperm volume of 40 μl. The speculum was removed, and the mouse was immediately paired with a vasectomized male overnight. No post-procedure monitoring was required.