Experimental Summary Examples
Example 1:
Only normal husbandry of mice is planned. Breeding procedures: One male and one female (8
weeks of age or older) are put together at the end of the afternoon. Detection of a vaginal plug in the
morning is considered E0.5. Mice are weaned at 20-21 days of age. At that time, tail snips of 2-3 mm are
taken for genotyping the mice and an earpunch is used to mark individuals to distinguish them from their
cagemates. Tail snips are necessary so that we can have enough DNA to do southern blots to confirm
the PCR genotyping results as needed. Only four weaned mice are housed in one cage.
To prepare mouse embryos needed for this study, mice will be mated overnight and examined the
following day for vaginal plugs. When the embryos are to be harvested, the mother will be euthanized by
anesthesia as described in Q21. The embryos will be rapidly dissected in cold PBS and placed
immediately in fixative for embedding or in lysis buffer for extraction of nucleic acids or protein. For cell
growth in vitro, tissue is rapidly dissected in cold, sterile PBS and trypsinized before plating on gelatincoated tissue culture dishes. When there are many embryos, each will be stored individually in PBS on
ice until the uterine wall and decidua can be dissected away. Rnf4 null embryos die before E15, so
embryos will be harvested at earlier stages. If any embryos are dissected between E15 and birth, they will
be decapitated.
Example 2:
Housing
Mice will be housed in ventilated, solid bottomed Innovive ™ disposable caging. All animals are fed
irradiated feed, and provided acidified water and autoclaved bedding. All animals will be checked daily. All
animals will be group-housed if possible, and provided with either Enviro-Dri or Mouse Igloos®.
Occasionally, male mice may be housed individually to prevent the occurrence of double litters or if
fighting occurs. Also occasionally female mice may be singly house if pregnant and waiting for delivery of
a litter in a harem mating scheme or if there are no other age and genetically compatible animals in that
colony. Individually housed mice will also be provided with either Enviro-Dri or Mouse Igloos®.
Colony Animal Procedures
Breeding will either be monogamous or harem breeding. Weaning will usually be at 3 weeks of age, but in
some strains or knockouts with slower growth rates, it will be delayed until 4 weeks. Small weanlings may
be supplied with supplementary nutrition, such as Transgenic Dough™ or Clear H20™ gel cups.
Genotyping by tail sampling will occur prior to three weeks of age by collection of <0.5 cm of distal tail,
using a sharp scissors that has been sterilized in a glass bead sterilizer. For very small mice, genotyping
may be delayed until up to 28 days, although this is not common. Other methods of genotyping that may
be used include use of tissue acquired from ear punching for identification. Identification methods for
available pups include ear punching, ear tagging, and tattooing, depending on PI request. If a PI requests
a mouse older than 28 days to be genotyped and cannot use a 2mm ear punch then the mouse will be
anesthetized with isoflurane gas 1-5% given to effect via anesthetic machine. Induction in a chamber,
followed by maintenance with a nosecone. Anesthetic depth is assessed by lack of response to toe pinch.
After tail biopsy the mouse will be provided ketoprofen 5 mg/kg subcutaneously for 3 days post-sampling.
If special procedures are needed to maintain a colony (e.g., different requirements for a specific type of
transgenic mouse) they will be added via an amendment. Occasionally toe-clipping will be used to both
identify neonatal animals and provide tissue for genotyping. In this case, animals <7 days old may have
the distal phalanx of one toe removed by sharp dissection (scissors or scalpel blade) on the hind feet
only. This method will be used only when it is necessary to genotype very young animals because of
genetically-based neonatal lethality. At this age, identification of individual pups is problematic. We have
tried tail and toe tattooing, which are suggested alternatives to toe-clipping, and have been unable to
unequivocally identify altricial rodents.
On occasion at PI request, mice will be weighed, the weight recorded, and the data provided to
investigators.
Some investigators request that we specify the date of copulation (DPC). For these requests, 1 or 2
females will be paired with males. Then the following morning females will be checked for copulation plug.
Occasionally, investigators request that we feed research diets (e.g., high fat, doxycycline-containing,
high iron) prior to transfer of animals out of the colony. The justification for the use of these diets is
provided in the approved “parent” protocol. In any case, all diets fed will be either autoclaved or irradiated
so as not to compromise the disease-free status of our colony. Animals be be weighed for diet related
requests or for conformation of pregnancy in timed mated mice. Weighing could be done as frequent as
daily. In addition, PIs may request that we provide them with blood samples for phenotyping purposes. In
such cases, we will follow the methods described in the section below describing survival blood draws.
Survival blood draw volumes will not exceed 1% of total blood volume. In addition, PIs sometimes request
that we euthanize animals and provide tissues. All euthanasia will be by methods as described below in Q
21.
Survival Blood Draws
Survival bleeding:
Used for phenotyping, or glucose readings
Frequency
no more often than monthly
Volume
<1% body weight in blood volume
(250 µl for a 25 gram mouse)
Routes
maxillary (requires no anesthesia)
Rederivations
For rederivation purposes, females will be superovulated, which involves IP injection of hormones
(pregnant mare serum gonadotropin 5 IU/mouse in 0.1xml, and human chorionic gonadotropin 5
IU/mouse in 0.1 ml) 46-48 hours apart, and then exposing treated females to males. Females will be
euthanized 1-2 days later for isolation of the reproductive tracts.
The Transgenic animal facility provides us with the hormomes. The PMSG is a non-pharmaceutical-grade
compound from the National Hormone and Peptide Program (based in NIH); is used because of the
unavailability of pharmaceutical-grade equivalents. These compounds will be used as described in the All
–Campus Policy and SOP on the use of non-pharmaceutical grade materials.
(http://www.rarc.wisc.edu/policy/2010-037.html).
Some preparations of hCG may be available in pharmaceutical grade, but because of increased
performance in fertility and hence, a decrease in the number of animals needed, an alternative nonpharmaceutical-grade formulation is used. The UW Transgenic Animal Facility tested numerous hCG
compounds and found the one from the National Hormone and Peptide Program to give the best
hormonal response in multiple strains of mice, thus limiting the number of females needed per
experiment. Additionally, pharmaceutical-grade compounds that are available are not at the appropriate
concentration for mice and may give us unpredictable results. Use of the compound is in accordance with
the ACAPAC policy. PMSG and hCG is diluted using sterile saline in an eppendorf tube and then kept
frozen in a -20 freezer.
Example 3:
The division of sperm into X and Y aliquots using flow cytometry is very detrimental to the viability of
sperm cells. Therefore a single, timed laparoscopic AI is required to ensure sperm cells are at the site of
fertilization prior to ovulation. Estrus synchronization protocols are required to ensure the proper timing of
laparoscopic AI. All drugs administered for these procedures will be of pharmaceutical grade.
Weaned sows can be administered an FDA approved drug called OvuGel. OvuGel is a GnRH agonist
(triptorelin acetate, 100 μg/mL) deposited (2 mL) into the sow vagina at 96 h after weaning. The agonist
will induce an LH surge resulting in synchronized ovulation.
Gilts with a known estrus will be administered for up to 14 d with an oral dose (6.8 mL) of Matrix
(altrenogest). Doses would start any day from 11 to 15 of their estrous cycle. In order to specifically
control insemination timing, a chorionic gonadotrophin (Chorulon, 500 IU) will be injected subcutaneous
with an 18 gauge, 1.5” needle at 96 h after the last dose of Matrix. An alternative to Chorulon is OvuGel
given at 96 h after the last dose of Matrix. OvuGel is not yet approved for gilts.
All animals will be scanned transrectally using a real time ultrasound (RTU) and 7.5 MHz linear
transducer customized to a fixed angled PVC holder starting 72 h after weaning for sows or 24 h after the
last Matrix dose in gilts to determine ovarian status, follicle size and time of ovulation. The technician
performing the technique will wear gloves and lubricate the probe with obstetrical lubricant or mineral oil.
A finger will be gently inserted into the anus prior to the insertion of the adaptor. The adaptor with the
probe will be quickly but gently inserted with a rotating motion in the rectum. The lubricated probe will be
gently pushed cranially by firmly holding on to the adaptor outside the animal. The transducer will be
advanced while looking for reference points such as the bladder until the ovary is recognized. Scanning
will be conducted a minimum of 2 times per day over the expected estrous days until ovulation has been
confirmed.
Females laparoscopically inseminated will be checked for pregnancy between day 18-24 with a RTU and
3.5-5.0 MHz probe. Females will be housed in a gestation crate to minimize forward and lateral
movement. External observation requires obstetrical lubricant or mineral oil to effectively transmit and
receive ultrasound waves through the skin. The gel is placed on transducer surface and the transducer is
firmly applied to the animal’s abdomen.
Females that have been synchronized for estrus and have not ovulated will be prepared for a
laparoscopic AI. Water and feed will be withheld for 12 h prior to anesthesia. On the day of surgery the
females will be anesthetized, prepped for surgery and laparoscopically inseminated using a total of 10
million sperm per uterine side. The insemination will occur in the oviduct and uterotubal junction or just the
uterotubal junction at 6-12 h prior to ovulation. Females confirmed pregnant via RTU will either be allowed
to carry to term or taken to a local slaughter plant so fetuses can be retrieved and counted. Surgical
embryo recovery is also an option to quantify fertilization, embryo development and quality an accessory
sperm binding.
Weaned sows will be given OvuGel 96 h after weaning to induce a time ovulation. After detection of
standing estrus as determined according to SRTC SOP protocols, the sow will be inseminated using a
deep uterine catheter. Immediately prior to insemination the vulva of the sow will be cleaned with a
sponge and warm soapy water and wiped dry. The outer catheter of the deep uterine catheter will be
inserted in the vagina at 45 degree angle and pushed into the lumen of the cervix until resistance is met.
Next the inner uterine catheter will be fed through the lumen of outer cervical catheter and pushed
through the cervical pads and into the uterine lumen. The catheter will continue to be pushed through the
uterus until resistance is encountered. A bottle of diluted semen is connected to the inner uterine catheter,
squeezed and the semen is deposited in the uterus. After all the semen has been deposited in the uterus
the inner catheter is slowly removed from uterus and both catheters are removed from the sow.
For this protocol the following phrases are considered to be the same: Flushing; Embryo
Retrieval; Recovery of Preimplantation embryos. Some females (gilts or sows) that have been
inseminated via laparoscopic AI or deep uterine insemination will have embryos recovered from either the
2 to 8 cell or morulla/blastocyt stage of embryos. Regardless of stage of development the surgery
procedures are identical. Females will be withheld feed and water for at least 12 h prior to anesthesia. On
the day of surgery the females will be anesthetized, and prepped for surgery as described later in this
protocol.