Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.
The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.
Assisted reproductive technologies are poorly developed in wild animals, including marine mammals. The lack of sensitive methods to assess sperm fertilizing success contributes to the slow development of reproductive technologies in species such as dolphin. It was not until recently that the basic seminal parameters of the bottlenose dolphin (Tursiops truncatus) were reported1,2. However, variables such as motility and morphology, although widely used, give limited information on reproductive efficiency. The best indicator of sperm quality is the evaluation of the fertilizing potential.
Recently, our group used a method to assess dolphin sperm fertilizing potential by assessing male pronuclear formation and/or hybrid embryo formation after heterologous IVF using zona intact bovine oocytes3. The use of dolphin-bovine heterologous IVF has important advantages over homologous IVF, as it overcomes the difficulty of obtaining dolphin oocytes and facilitates the use of well-tested in vitro bovine oocyte maturation systems. In order to avoid species specificity, heterologous fertilization is generally performed in the absence of ZP. Although it allows for the evaluation of the ability of the acrosome-reacted spermatozoa to fuse with the vitelline membrane, it impairs the evaluation of other features related to fertilization. The procedure described uses zona intact oocytes and permits the evaluation of the following parameters: sperm zona binding and attachment, penetration, polyspermy, pronuclear formation, and hybrid embryo cleavage.
Here, we present several protocols for sperm collection, basic sperm analysis, spermatozoa freezing as well as the evaluation of the dolphin sperm functionality by assessing male pronuclear and/or hybrid embryo formation after heterologous IVF using zona intact bovine oocytes.
Ethics statement: All the experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). All the experiments were performed in accordance to the Guide for the Care and Use of Laboratory Animals, as adopted by the Society for the Study of Reproduction, and to the Animal Welfare Act for the care of Marine Mammals.
1. Dolphin Sperm Collection and Cryopreservation
2. Heterologous In Vitro Fertilization Using Zona Intact Bovine Oocytes
3. PCR
All the results, tables, and figures (1 and 2) presented here were reproduced with permission3.
Dolphin spermatozoa have high motility after freezing and thawing
The frozen-thawed dolphin ejaculates showed percentages (% ± SD) of 84.5 ± 5.3 total motile and 69.1 ± 5.1 progressive motile spermatozoa. In motility terms, these numbers are representative of high-quality cryopreserved spermatozoa.
Dolphin spermatozoa are capable of penetrating zona intact bovine oocytes and producing hybrid embryos
Figure 1A and Table 1 show dolphin spermatozoa attached to the bovine ZP after 2.5 h of co-incubation. Dolphin spermatozoa were attached at a higher percentage than bovine spermatozoa (Figure 1B and Table 1). Indeed, fluorescence microscopy shows that dolphin spermatozoa are able to penetrate zona intact bovine spermatozoa (Figure 1C), leading to pronuclear formation and cleavage (Figure 1E and Table 1). This was confirmed by confocal microscopy (Figure 1D and Figure 1F). No polyspermy was observed in oocytes from the heterologous IVF group after 12 h of co-incubation (Table 1). Pronuclear formation in the heterologous IVF reached the highest percentage at 24 h, a longer time than in the homologous group, which occurred at 18 h of co-incubation (Table 1). The cleavage rate at 48 h after incubation was lower in the heterologous than the homologous IVF (Table 1). A spontaneous parthenogenetic activation rate of 8.0% was observed in bovine matured unfertilized oocytes at 48 h (Table 1).
Finally, the PCR results confirmed the presence of dolphin genetic material in the hybrid embryos (Figure 3) by evaluating the presence of a dolphin reference gene that encodes for the ribosomal 18 S protein (Figure 2).
Figure 1: Dolphin spermatozoa attached to the bovine zona pellucida (ZP). Attached dolphin (A) and bovine (B) spermatozoa after 2.5 h of co-incubation with zona intact bovine oocytes. Gametes were stained with Hoechst and visualized under phase-contrast microscope (40X magnification). A dolphin spermatozoa in the periviteline space of a bovine oocyte (C and D) and a decondensing dolphin sperm head in the bovine oocyte cytoplasm (E). Images were captured under a confocal microscope and correspond to the equatorial plane of the presumptive zygote (Hoechst staining; 40X magnification). Two pronuclei (F) observed under a phase contrast microscope after 24 h of heterologous IVF consisting of the co-incubation of dolphin spermatozoa with zona intact bovine oocytes (Hoechst staining; 40X magnification; Scale bar = 25 µm). The figure was reproduced from reference3 with permission. Please click here to view a larger version of this figure.
Figure 2: Representative electrophoresis (2% agarose gel ethidium bromide stained) showing the PCR amplification results for the 18 S dolphin gene. Lanes 1 to 30 show presumptive dolphin/bovine hybrid embryos; lanes S1 and S2 feature 4 ng/mL and 0.4 ng/mL dolphin sperm DNA, respectively; and lanes C1 to C5 feature two-cell bovine embryos and H2O (water) (the blank control). The arrow indicates the 190-bp band corresponding to the amplified 18 S rDNA. The figure was reproduced from reference3 with permission. Please click here to view a larger version of this figure.
Figure 3: Representative images of hybrid embryos. Representative image of a two-cell hybrid embryo (A) at 48 h of incubation (Hoechst staining; 40X magnification). Unstained hybrid embryos at 48 h of incubation, at different stages of division, visualized under the stereomicroscope (B). Please click here to view a larger version of this figure.
Table 1: Rates of attachment, polyspermy, pronuclear formation, and cleavage following homologous and heterologous co-incubation with bovine oocytes and bovine or dolphin spermatozoa, respectively. Variables were analyzed with ANOVA (Kruskal-Wallis and Mann-Whitney test). The values are expressed as the mean ± SEM (range) of twelve replicates; n = the total number of oocytes or presumptive zygotes examined. The table was reproduced from reference3 with permission. Please click here to view a larger version of this figure.
For many different mammalian species, there are diverse advantages to using frozen-thawed sperm. These include the ability to transfer valuable genetic material, the potential for worldwide distribution, the low risk of contamination, and the capability to preserve male gametes for decades. The use of cryopreserved sperm is essential for the bottlenose dolphin, because this species is protected under Appendix II of CITES, which limits the transport and exchange of animals between different aquatic parks. Dolphins transport is an activity that creates logistical problems and dangerous risks. However, refraining from transporting them also has consequences, such as the risk of introducing consanguinity to a captive population of animals. One solution is the cryopreservation of sperm. Some authors have cryopreserved dolphin sperm5,7,8,9, with very encouraging results. Moreover, in 2005, the first birth of a dolphin calf conceived by artificial insemination using cryopreserved sperm was reported10. Some years later, this was done using sex-sorted spermatozoa11.
Dolphin sperm have been cryopreserved using different techniques, from sophisticated methods, such as a programmable freezer5, to common methods, such as dry ice12 or liquid nitrogen vapors2,9,13. The main advantage of using dry ice or liquid nitrogen vapors is the possibility to cryopreserve at the same location where the sperm is extracted, without the use of highly specialized equipment. Sperm frozen in straws on polystyrene in liquid nitrogen vapors is a simple, quick, and inexpensive method13. In practice, when this methodology has been used on dolphins, sperm motility was 65%. However, it is important to consider how difficult it is to establish a standardized method: the technique may be influenced by external factors, such as temperature, humidity, polystyrene size, etc. Therefore, further studies are necessary to optimize and standardize the procedure in order to improve the success of using sperm frozen with the liquid nitrogen vapor method.
In order to optimize a freezing protocol, it is important to develop a reliable method for the functional assessment of the spermatozoa. IVF is a good means of testing the sperm potential for fertilization. Ideally, dolphin oocytes should be used for IVF, but the difficult and laborious procedures needed to obtain oocytes from females represent an important constraint. Therefore, the possibility of using heterologous IVF opens a new means to test fertilization efficiency in the dolphin. Oocytes from other species can be used for heterologous dolphin IVF. The results show that heterologous IVF between bovine oocytes and dolphin spermatozoa can be performed. Frozen and thawed dolphin spermatozoa can penetrate the zona intact bovine oocyte and generate a hybrid embryo.
The presented combination of protocols for collecting, freeze-thawing, and performing heterologous IVF with bovine oocytes have been successfully demonstrated and represents an initial step for the optimization of cryopreservation and the evaluation of dolphin sperm. It is important to note that there are still many unknowns in these procedures. However, the ability to cryopreserve dolphin semen makes it accessible and allows it to be kept in laboratories. This will facilitate scientific studies and can lead to a better understanding of the physiology, composition, and behavior of spermatozoa.
The evaluation of sperm by heterologous IVF can ease the optimization of the freezing protocols, but it may also be used in the future to test and select males for fertility efficiency. One important consideration is the relationship between real fertilization numbers and heterologous IVF values. In addition, and due to the novelty of heterologous IVF between these two phylogenetically distant species, bovine and dolphin, new biological hypotheses can be built. Altogether, these protocols open a new space for research on the reproduction of the dolphin and other marine mammals.
The authors have nothing to disclose.
his work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2015-70140-R to D. Rizos, AGL2015-66145R to A. Gutierrez-Adan and J. F. Pérez-Gutiérrez) and Seneca Foundation of Murcia (Grant 20040/GERM/16 to F. García-Vázquez)
FERT-TALP medium | Merck | |
TCM-199 | Sigma | M-4530 |
Hoechst 33342 | Sigma | B-2261 |
4-well dishes | Nunc | 176740 |
Density gradient BoviPure | Nidacon International | BP-100 |
Washing solution Boviwash | Nidacon International | BW-100 |
Magnesium chloride | Promega | A35 1H |
Sterile water | Mili Q sintesis A10 Millipore | A35 1H |
Buffer Tris Borate EDTA | Sigma | T4415 |
MB agarose | Biotools | 20.012 |
5X GoTaq flexi buffer | Mili Q sintesis A10 Millipore | M 890 A |
MB agarose | Biotools | 20.012 |
Taq polymerase | Promega | |
SafeView | NBS Biologicals Ltd. | M 890 A |
Makler counting chamber | Sefi Medical | |
Thoma chamber | Hecht-Assistant | |
pHmeter MicropH 2000 | Crison Instruments | |
Osmometer Advanced micro osmometer 3300 | Norwood | |
Computer assisted sperm analysis system | Projectes y Serveis R+D | |
Stereomicroscope MZ 95 | Leica | |
Epifluorescent optics Eclipse Te300 | Nikon | |
Confocal assistant 4.02 software | Bio-Rad | 3D analysis software |
Confocal laser scanning microscopy | Bio-Rad | |
Micropippetes (P2. P20, P200, P1000) | Gilson | |
Microcentrifuge tubes | VWR | |
UV iluminator | Bio-Rad | |
PCR Thermal cycler Primus 96 Plus | MWG AG Biotech |