3.3.3 Artificial insemination using frozen sperm†
Takao Sasado, Yasuko Kota, Makoto Furutani-Seiki, Hisato Kondoh
JST/SORST Kondoh Research Group, Kinki-chihou Hatsumei Center, Yoshida-Kawaracho 14, Sakyo-ku, Kyoto 606-8305, Japan
188.8.131.52 An overview†
The procedure of artificial insemination using frozen sperm comprises the following three steps:
(1) Collection of mature unfertilized eggs
(2) Thawing frozen sperm
(3) Insemination using the thawed sperm
In our routine described below, we observed a 92% fertilization rate and a 68% hatching rate of fertilized eggs, in 70 trials using sperm of heterozygous mutant carrier male Medaka of Kyoto-Cab background and unfertilized eggs of Kyoto-Cab or Kyoto-Kaga. Long storage of sperm in liquid nitrogen has not affected the fertilization rate over the period of our 3-year experience.
Starting from 100 unfertilized eggs, at least 50 fish can be obtained using a capillary full of frozen sperm. The procedure can be scaled up or down as appropriate, combining sperm from several capillaries or using a clipped part of capillary. (A capillary can be clipped using chilled nippers.)
There are three possible ways to collect mature unfertilized eggs: [A] squeezing the female abdomen); [B] making a section in abdomen and isolating the ovaries (Yamamoto, 1975); and [C] by mating females with sterile males (Naruse et al., 1985). The first two procedures are described here.
(1) Balanced salt solution (BSS) (Iwamatsu, 1983; Ando and Wakamatsu, 1995). Adjust pH to 7.4~8.0 (Iwamatsu, 1984). Prepare as described in the previous chapter.
(2) 0.3% artificial sea salt in reverse osmosis water
(3) 0.03% artificial sea salt and 0.5 ppm methylene blue in reverse osmosis water for embryo culture.
(1) For collection of unfertilized eggs
• Fish net
• 6 cm plastic culture dish
• Two fine tweezers (Dumont No.5)
• Dissecting scissors or spring scissors
• Disposable polyethylene Pasteur pipette (e.g. LP ITALIANA SPA, Pasteur pipette, #135030)
• Stereoscopic microscope
The following are used for squeezing out the eggs
• Cotton wool (5X5cm)
• Squeeze bottle containing BSS, used to moisten cotton wool
• Tweezers to hold eggs using sides of long tips (e.g., KFI, Tokyo Japan, K-3 GG).
(2) For sperm handling
• A Dewar flask (1 L) containing liquid nitrogen
• A dry ice block covered with aluminum foil in a Styrofoam box (inner size 27X17X17 cm)
• Leather gloves
• A clamp to hold cryo-tubes: a Lorna non-perforating towel clamp for surgical operation (e.g., FRIGZ Medico Japan, DJF28-0811089) is convenient
• Blunt forceps to pick up cryo-tubes from liquid nitrogen
• Fine tweezers
• 0.5 mL plastic centrifuge tubes
(3) For insemination
• 24-well plates
• Capillary dispensers: accessories for commercial glass capillaries (minicaps disposable microcapillaries, 10 microL, Hirschmann laborgeraete #9000110)
• Inverted phase contrast microscope
• Slide glass
 Preparation of egg-laying females.
Rear a sufficient number of well-fed females that are laying eggs. Separate the females from males the day before collection of unfertilized eggs, after females have laid the day’s eggs. Eight or more mature unfertilized eggs are usually collected from such a female.
 Collection of mature unfertilized eggs from females.
Mature unfertilized eggs are ovulated into the ovarian lumen one hour before onset of the lighting cycle (14 hours light and 10 hours dark) (Iwamatsu, 1978). Eggs collected (by any of the methods) within 4 hours after start of a lighting cycle can be stored in BSS at room temperature for at least 3 hours without reduction in fertilization or hatching rates. Care must be taken in handling unfertilized eggs, because in soft chorion they are fragile and tend to be activated by mechanical stimulation.
[A] Collection of mature unfertilized eggs by squeezing.
(1) Anesthetize the female fish by keeping it in ice-cold water for ten seconds.
(2) Place it with its back down in the center of the cotton wool piece moistened with BSS, and hold the fish using thumb and one finger so that it is sandwitched between layers of the cotton wool. Squeeze out the mature unfertilized eggs using the fingers to gently press posteriorly the area of ovarian lumen (area between the stiff dorsal musculature and soft belly). (If guts are eviscerated or if immature oocytes are squeezed out, this indicates too much pressure by the fingers which can damage the female.)
(3) Hold eggs delivered onto cotton wool on the side of tweezers tips, and transfer them into BSS. Do not try to hold unfertilized eggs between tips of the tweezers, as they are too fragile to be held.
(4) Release the female into 0.3% artificial sea salt.
(5) Repeat the process until the desired number of eggs is collected.
(6) Keep females in 0.3% sea salt water overnight for recovery, and release them into ordinary fish water with males. Severely damaged fish should be killed by anesthesizing with cold. In our experience, around 80% of squeezed females will fully recover within two weeks and will be reusable.
[B] Collection of mature unfertilized eggs by abdominal section (Yamamoto, 1939).
(1) Under anesthesia in ice-cold water for 10 seconds, kill the female fish by quickly piercing the brain and cutting the spinal cord using the dissecting scissors.
(2) Then cut the abdominal wall from anus to heart along the midline with the scissors. Push the dorsal abdomen with the thumb and one finger, and pull out the gut using dissecting scissors.
(3) Cut the peritoneum from the anterior dorsal side to the posterior to expose the ovary, cut out the oviduct, and place the ovary in BSS in a culture dish.
(4) Hold and break the dorsal ovary epithelium using two pairs of tweezers and collect the mature unfertilized eggs located in the ovarian lumen.
(5) Repeat this procedure until a sufficient number of eggs are collected.
NB: This is a time-consuming procedure involving ovary isolation, the sorting of mature eggs from among oocytes, and sacrificing fish.
 Sorting of mature unfertilized eggs.
(1) Collect only mature unfertilized eggs, eliminating activated eggs or oocytes.
(2) Remove attaching filaments, either by spooling them with two pairs of tweezers or cutting with fine scissors.
 Place ca. 100 eggs in BSS in a well of a 24-well plate. Remove excess BSS so that eggs are just beneath the liquid surface.
 Hold a 0.5 mL tube upright with the lid open. Tube body is used as a capillary holder, while the lid is used as a well for sperm dilution. Pour 90 microL of BSS into the well of the lid.
 Handling of frozen sperm capillaries.
Minimize temperature increase of frozen sperm specimens. Use pre-chilled equipment while wearing leather gloves. Cryo-tubes are handled on dry ice block covered with aluminum foil in a Styrofoam box.
(1) Take sperm capillary-containing cryo-tubes from storage boxes, and place them in liquid nitrogen in a Dewar flask.
(2) Pick up a cryo-tube from the liquid nitrogen, hold the tube with a clamp (Lorna clamp) on the dry ice, and open the cap.
(3) Pick up a capillary using fine tweezers, and place it in the body of the 0.5 mL tube. (If capillaries are frozen together, tweezers can be used to separate them.)
(4) Close cap of the cryo-tube, and return it to the liquid nitrogen.
 Insemination using thawed sperm.
(1) Connect the capillary to a capillary dispenser, and when 10 microL of sperm suspension has thawed in the ambient temperature, place it in 90 microL of BSS in the lid of the 0.5 mL tube, and rinse the capillary with BSS to collect all sperm.
(2) Remove BSS from the well of unfertilized eggs using a Pipetman. Mix 100 microL of sperm suspension well with a Pasteur pipette, pour it on the eggs, and mix the egg-sperm mixture gently with the pipette.
(3) Incubate the egg-sperm mixture in ambient temperature for 15 minutes, gently mixing it every few minutes.
(4) Sample a small amount of sperm suspension on a glass slide and check the motility and fraction of the moving sperm under a phase-contrast microscope.
 Collection of fertilized eggs and second round insemination of eggs remaining unfertilized.
(1) Recover the liquid in the well containing sperm back to the tube lid, add ca. 2 mL BSS to eggs in the well, and transfer the eggs and BSS to a 6 cm culture dish.
(2) Sort fertilized and unfertilized eggs under a stereoscopic microscope. Normally, more than 80% of eggs will be fertilized within 15 min.
(3) Rinse the fertilized eggs a few times with BSS.
(4) Unfertilized eggs should be returned to the well of the 24-well plate, added with the recovered sperm suspension, and incubated further until a large majority of them are fertilized.
(If fertilization is inefficient due to low-motility of the sperm, add newly thawed sperm to the recovered sperm suspension.)
 Replace the egg medium from BSS with 0.03% sea salt water at blastula stage. Discard abnormal embryos at the time the medium is changed and as they are recognized during the following days.
N.B. Use a new set of materials for each set of inseminations to avoid contamination. Tweezers and other metallic items can be cleaned using 70% methanol.
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