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RESEARCH ARTICLE
Contents Vol 21(7)

Assessment of semen quality, sperm cryopreservation and heterologous IVF in the critically endangered Iberian lynx (Lynx pardinus)

Natalia Gañán A , Raquel González A , J. Julián Garde B , Fernando Martínez C , Astrid Vargas C , Montserrat Gomendio A D and Eduardo R. S. Roldan A E F
+ Author Affiliations
- Author Affiliations

A Grupo de Ecología y Biología de la Reproducción, Museo Nacional de Ciencias Naturales, CSIC, 28006 Madrid, Spain.

B Instituto de Investigación en Recursos Cinegéticos, CSIC-UCLM-JCCM, 02071 Albacete, Spain.

C Programa de Conservación Ex-Situ del Lince Ibérico, Centro de Cría en Cautividad ‘El Acebuche’, Parque Nacional de Doñana, Matalascañas, 21760 Huelva, Spain.

D Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UK.

E Royal Veterinary College, London NW1 0TU, UK.

F Corresponding author. Email: roldane@mncn.csic.es

Reproduction, Fertility and Development 21(7) 848-859 https://doi.org/10.1071/RD08226
Submitted: 10 October 2008  Accepted: 17 May 2009   Published: 17 July 2009

Abstract

Semen traits and factors affecting sperm cryopreservation were assessed in the Iberian lynx (Lynx pardinus), a species regarded as the most endangered felid in the world. For cryopreservation, semen was washed, resuspended in a Tes–Tris-based diluent (TEST) or a Tris-based diluent (Biladyl), both with 20% egg yolk and 4% glycerol, loaded into straws, cooled to 5°C using an automated programmable system and frozen on nitrogen vapour. Heterologous IVF of in vitro-matured domestic cat oocytes was used to test the fertilising ability of cryopreserved spermatozoa. Electroejaculates from five males were obtained. Characterisation of the electroejaculates revealed mean (± s.e.m.) values of 3.3 ± 0.6 × 106 total spermatozoa, 73.6 ± 4.6% motile spermatozoa, 23.7 ± 4.0% morphologically normal spermatozoa and 40.7 ± 2.3% spermatozoa with intact acrosomes. After thawing a higher percentage of motile spermatozoa was seen in TEST than in Biladyl (34.0 ± 6.2% v. 7.5 ± 4.8%, respectively; P < 0.05); however, there were no differences in the percentage of intact acrosomes between the two diluents. Iberian lynx spermatozoa fertilised domestic cat oocytes in vitro, with higher fertilisation rates observed for spermatozoa cryopreserved in TEST than in Biladyl, although the difference did not reach statistical significance (20.5 ± 4.5% v. 11.5 ± 6.8%, respectively). There were positive significant relations between the fertilisation rates and both the percentage of normal spermatozoa and the percentage of spermatozoa with an intact acrosome before cryopreservation (P = 0.04). This first report of the collection and cryopreservation of Iberian lynx semen and analysis of fertilising ability is an important step in the development of assisted reproductive techniques for this critically endangered felid species.

Additional keywords: assisted reproductive technologies, felid, genome resource bank, teratospermia.


Acknowledgements

The authors thank David Wildt, JoGayle Howard, Rachel M. Santymire and Katharine Pelican for initial support in setting up the techniques for semen collection and evaluation. Juana Bergara, Luis Díez Klink, José Rodríguez and David Rodríguez from the ‘El Acebuche’ captive breeding centre helped with various tasks. Ana del Olmo, Lucía Arregui and Adrián Sestelo collaborated with fieldwork in 2005 and 2006. Eva Martín Becerra, Sandra Prol Rollán, Begoña de la Cruz are thanked for their collaboration in the collection and analyses of samples. The photograph in Fig. 1a was taken by Ana del Olmo. Borja Heredia and Miguel Aymerich (Ministry of the Environment, Spain) and Miguel Angel Simón (Junta de Andalucia, Spain), are gratefully acknowledged for their support. The authors thank the Consejería de Medio Ambiente, Junta de Andalucía, for allowing us to collect Iberian lynx biomaterial for this project. Animal protection societies (ANAA, GATA, SPAP, CIACAM) and veterinary clinics (Ventas, Gattos, Mediterraneo, Arco Iris, Alonso Cano, Puerta de Toledo, Capacés, Ramón y Cajal, Retiro, Ayala y Alberto Alcocer, Castellana, Pío XII and Zurbano), all in Madrid, Spain, provided us with samples from domestic cats. N.G. received financial support from the Spanish Research Council (CSIC-I3P program) and the BBVA Foundation. R.G. received financial support from the Spanish Research Council (CSIC-I3P program). E.R.S.R. is the recipient of a Royal Society Wolfson Research Merit Award. Funding for this work was obtained from the Spanish Ministry of the Environment, the Spanish Research Council and the BBVA Foundation. Novauto (Ford Motor Co., Madrid, Spain) generously donated a vehicle for fieldwork.


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