Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The overall goal of this technique in to assess the cryo preserved dolphin sperm functionality by the heterologous in vitro fertilization using cryo preserved dolphin spermatozoa and zona intact bovine oocytes. In this video, we will show how to collect, evaluate, and cryo preserve dolphin spermatozoa. Then we will assess sperm interaction and penetration up to zona pellucida.

Male pronuclear formation and hybrid embryo cleavage. One critical issue in the development of assisted reproductive technologies is the evaluation of sperm function which is best indicated by its ability to fertilize. Homologous IVF techniques are commonly used for the evaluation of sperm function but obtaining dolphin oocyte is very difficult.

The main advantage of heterologous in vitro fertilization is its simplicity. Bovine oocytes are readily obtainable and can be processed in vitro by well tested protocols. We would also demonstrate how to cryopreserve dolphin spermatozoa using liquid nitrogen vapors.

A simple technique that does not require highly specialized freezing equipment. For this procedure, the male dolphin should be previously trained for six months using positive reinforcement. Let the animal lie in dorsal recumbency near the edge of the pool.

There perform various tactile stimulations by gently pressing on the cranial area of the genital groove to elicit voluntary extrusion of the penis. After achieving a complete erection, direct the penis tip to a sterile container. Maintain the ejaculate at 37 degree Celsius until the analysis.

As needed repeat the tactile stimulation for successive ejaculate collections using a new polypropylene glass each time. Immediately after the collection evaluate the ejaculates keeping them at 37 degree Celsius throughout. Determine the volume, color, pH, and osmolarity of the ejaculates.

Then from a 10 microliter aliquot, determine the spermatozoa concentration in a counting chamber. Place the ejaculate on to one slide on a heated microscope stage at 37 degree Celsius. Using a computer assisted analysis system evaluate the sperm's motility.

If necessary, dilute the sperm with fert medium for easier viewing. Next using a 20 microliter aliquot of ejaculate evaluate the sperm's viability and morphology by conventional staining with Eosin Nigrosin and Spermac. For cryopreservation, first transfer the sperm sample to a centrifuge tube.

Then pellet the sperm at 250 G for five minutes. Now over the course of five minutes slowly make a 1:1 dilution of the sperm sample with trace egg yolk based buffer containing 1.5%glycerol. Then chill the sample at 5%Celsius for 1.5 hours.

An hour later repeat the 1:1 dilution with the same buffer and then maintain the sperm suspension at five degree Celsius for 10 minutes. Next load the suspension into 0.25 milliliter straws and heat seal the straws. Load the straws into a rack and suspend the rack 4.5 centimeters above liquid nitrogen for 10 minutes.

Then plunge the straws into the liquid nitrogen for long-term storage. For this protocol, use bovine ovaries kept warm saline collected within the last three hours. Begin by washing the ovaries three times with saline.

Always keeping them at 37 degree Celsius. Next aspirate all the two millimeter to eight millimeter follicles using an 18 gauge needle and syringe. Transfer to a 50 milliliter tube and let oocytes settle for 15 minutes.

Then remove the supernatant and re suspend the follicles in PBS. Recover the COCs under a stereo microscope in 90 millimeter dishes. Select grade one and two oocytes which have homogenous cytoplasm and are surrounded by at least three cumulous cell layers.

Transfer these oocytes to PBS in a 35 millimeter dish. Next wash the COCs three times in PBS and once in maturation medium. Then load the COCs into four well dishes at 50 COCs and 500 milliliters of maturation medium per well then incubate the COCs for 24 hours.

Once the oocytes have expanding cumulous cells they are mature. Then wash them twice in fert medium and transfer them to four well dishes in groups of 50 with 250 milliliters of fert medium per well. Now return the COCs to their incubator and prepare the sperm for fertilization.

Fill a frozen straw containing dolphin spermatozoa and a straw containing bovine spermatozoa in a water bath at 37 degree Celsius for 50 seconds. Then evaluate their motility subjectively using a phase contrast microscope. Next transfer the sperm sample to a density gradient tube and centrifuge it for 10 minutes at 250 G.Carefully remove the supernatant.

And add back three milliliters of the washing solution. Then re suspend the sperm with gentle mixing. Now centrifuge the tube again and remove most of the supernatant leaving the sperm in 300 microliters.

Now count the sperm and adjust the concentration to two million per milliliter diluted with fert medium. Add 250 microliters of the suspension to each well of oocytes to have a final sperm concentration of one million per milliliter. Under a stereo microscope observe the expanded matured oocytes surrounded by either the motile bovine or dolphin spermatozoa which are smaller than bovine spermatozoa.

Coincubate the gametes for 18 hours. To evaluate the sperm interaction with the zona pellucida, remove 20 oocytes from the dish at 2.5 hours post coincubation. Remove the loosely attached spermatozoa by vigorously pipetting through a narrow bore Pasteur pipette.

Next fix and stain the oocytes with hooks to 3342. Transfer the stained oocytes individually into two microliter droplets of mounting medium and count the number of spermatozoa attached to the oocytes using a phase contrast microscope. Starting at 18 hours of coincubation transfer the 10 presumptive zygotes to a 15 milliliter centrifuge tube containing two milliliters of PBS and vortex gently for three minutes to remove the cumulous cells.

Wash twice in PBS fix and stain as previously described. Additionally at 18 hours of co incubation collect 50 zygotes for culturing. Vortex them in PBS for three minutes and then wash them twice in PBS.

After washing select zygotes with homogenous cytoplasm for culturing. Transfer them in groups of 25 into 25 microliter droplets of synthetic oviductal fluid supplemented with 5%fetal calf serum. Then culture the presumptive zygotes for 48 hours and evaluate their cleavage rate using a stereo microscope.

Later further evaluate their genetic makeup by PCR according to the text protocol. The spermatozoa of frozen thawed dolphin ejaculates were about 85%motile and 70%progressively motile. These numbers are representative of high quality cryo preserved spermatozoa.

Dolphin spermatozoa were attached to bovine zona pellucida after 2.5 hours of coincubation at a higher percentage than bovine spermatozoa. The dolphin spermatozoa were able to penetrate the zona intact bovine oocytes leading to pronuclear formation and cleavage. In the heterologous in vitro fertilization group no polyspermy was observed.

Pronuclear formation reached the highest percentage at 24 hours. Pronuclear formation was quicker in the homologous group. The cleavage rate at 48 hours was lower in heterologous fertilizations than in homologous fertilizations.

PCR confirmed the presence of dolphin genetic material in the hybrid embryos. Primers to the dolphin 18 s ribosome gene resulting in 190 base pair product were tested. Lanes one to 30 are dolphin bovine hybrids.

Controls were dolphin DNA in the S lanes. And two cell bovine embryos in the C lanes. The protocol presented for autologous in vitro fertilization has shown that bottlenose dolphin are able to fertilize intact bovine oocytes resulting in hybrid embryos.

Further development and optimization of this technique will facilitate the evaluation of dolphin sperm functionality. Cryopreservation of dolphin spermatozoa using liquid nitrogen vapors is an inexpensive rapid and simple method that yields good profound motility parameters and allows for successful heterologous in vitro fertilization. The observation that dolphin sperm are capable of fertilizing bovine oocytes opens a new space for research on the reproduction of the dolphin and other marine animals.

Also raises questions about the mechanisms of fertilization that remains largely unanswered.