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

Summary

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

Abstract

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.

Introduction

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 reported^1,2. 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.

Protocol

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

1. Preparation
   1. Make FERT medium (heparin- and hypotaurine-free). Prepare FERT-TALP medium supplemented with 25 m....

Results

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 representati.......

Discussion

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 transpo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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