Facultad de Medicina Veterinaria y Zootecnia of the Universidad Nacional Autónoma de Mexico (Veterinarian Medicine School of the National University of Mexico)
Abstract
The low reproduction rate of the captive dolphins in the aquariums of Mexico City have led to the studies of assistant reproduction techniques (ART). The cryopreservation of semen is one of the most important stages of the research of the ART. The research has been carried out in the Aragón Acuario, the property of CONVIMAR SA de CV, situated in Mexico City on the altitude of 2240 meters above the sea level, with a male dolphin, 15 years old, Tursiops truncatus , 10 years of which in captivity.
The method of the research involves sample collection once a week, semen collected by masturbation using positive reinforcement. Once collected semen is analyzed for macroscopic traits (volume, color, pH) and microscopic (concentration, motility percentage, status, acrosome integrity and membrane permeability). The process of cryopreservation begins the centrifugation of the sample (10 minutes, 600G) to eliminate seminal plasma (supernatant), which permits to increase longevity and viability of the sperm (Howard, 1993). The pellet of sperm is resuspended with Triladyl® (Minitüb, Germany), made of one part of egg yolk and three parts of distillated water to obtain the final concentration of 800 x 106 sperms/ml. One milliliter of resuspended semen is mixed with 0.5% (v/v) of Equex® STM paste (Nova Chemical Sales Inc., Scituate, MA), which is extender use to increase the survival and longevity of the semen of different species. The straws of 0.5 ml were filled and frozen with the velocity of 0.18-0.20 °C/min until it reached 5°C and then the straws were gradually frozen until -80°C in nitrogen vapors and finally until - 196 °C in liquid nitrogen. The procedure is controlled by the application of digital thermocouple (Lutron modelo TM-914C). The thaw process is undertaken after 15 days in a hot water tub (36°C, 1 minute), and then motility percentage, status, membrane permeability, and acrosome integrity are evaluated.
The Results The results of the semen collection turned out to be optims according to the reported for the species (Robeck and O'Brein 2004; Robeck et al., 2005) (concentration1580 ± 1125.61 x 106 cel/ml; % motility 95.2 ± 3.27; status 4.5 ± 0.35; % acrosome damage 1.2 ± 0.4; % mortality 2.7 ± 1.39). The facility of manual semen freezing in the Aragon aquarium (where there is no specialized laboratory) is considered to be the right one. However it resulted in obtaining lower rates of individual semen freezing-thawing then reported so far (% motility 20 ± 6.12; status 2.2 ± 0.57; % acrosome damage 22.4 ± 4.33; % mortality 71 ± 7.4). These results were improved by a couple of percent while using EQUEX® STM paste, before filling the straws (% motility 28 ± 12.54; status 3 ± 0.79; % acrosome damage 17.6 ± 2.5; % mortality 69 ± 7.4).
The preliminarily of these results and scarcity of samples analyzed so far does not permit any statistic conclusions, however, the obtain data allows us to consider the paste to have a protective effect on spermatic cells during the freezing process as it was proved for other species (Peña and Linde-Forsberg, 2003; Rota et al., 1997; Robeck and O'Brien, 2004). The results of the research obtain so far are satisfactory enough to consider the continuation of the investigation.
References
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