Abstract

Semen collection in captive bottlenose dolphins is facilitated by their natural propensity for masturbation.¹ Manual manipulation under operant conditioning to obtain semen from bottlenose dolphins was first described some time ago² and is now common practice. However, information about the characteristics of the semen, such as motility, viability, density and volume is limited.^3,4,5 There is also a lack of information on the characteristics of semen from dolphins of different ages / sexual maturity status. To be able safely and reliably to obtain high quality semen is a key component in assisted reproductive technologies (ART), such as artificial insemination (AI). Therefore, thorough understanding of normal ejaculate characteristics for the species of interest, is required for the development of ART.⁶

As part of a study that aims to ascertain specific information about reproductive development in male Tursiops aduncus, semen was collected at the same time as ultrasonographic examination of the testes and evaluation of serum testosterone. Semen was collected weekly from three sexually mature dolphins (M1, M2 and M3) and every two months in an immature dolphin (M4). Ejaculates were collected from each individual in succession. The end of each collection 'session' was determined by the absence of semen in spite of apparent effort, the presence of urine in the ejaculate, or behavioral signs indicating lack of cooperation by the subject. Semen was collected into wide-mouth, screw closure, polypropylene bottles (Nalgene® Labware, USA). After collection, samples were transferred into 1.5ml Eppendorf tubes and stored at 36°C for transportation and evaluation in the laboratory within an hour of collection.

Semen samples were assessed for motility (% total motility, % forward motility and rate forward motility), viability, density and volume. Motility was assessed on a warm stage mounted on a microscope, under a bright field at magnification x400. Samples were stained with eosin-nigrosin to assess viability. Prior to ascertaining sperm density using new Neubaur-improved double net ruling haemocytometers (Paul Marienfeld, Germany), samples were diluted at ratios determined during the initial motility assessment. Volume was measured with micropipettes or graduated glass cylinders (10ml or 25ml). pH of the first, last and some intermediate ejaculates in the series was measured using 4-patch pH test strips (Neutralit pH range 5.0-10.0, pH unit 0.5, Merck, Germany).

The number and volume of ejaculates collected per session from each subject varied (Table 1). The older the male, the higher was the number of consecutive ejaculates in a single collection session. The first ejaculate was nearly always higher in volume than subsequent ejaculates in a series. Overall, the older males produced ejaculates of higher volume. pH appeared to be higher in semen of the younger subjects (Table 1).

Table 1. Ejaculate characteristics: Ejaculate number, volume and pH. Between October 2002 and February 2005, 2012 ejaculates were collected from the four subjects in 367 sessions.

Table 2. Ejaculate quality: Spermatozoa total motility, viability and density.

*Sum of sperm count divided by sum of semen volume of the session

Generally, the ejaculate quality of all the mature males was good, with maximum motility and viability >90% and density >700x10⁶/ml (Table 2). The older the male, the higher was the ejaculate quality. There were greater variations in motility and viability in the younger male (M3). The first ejaculate was poorer in motility and viability compared with the subsequent 2-5 ejaculates in a series. High density samples (>700 x10⁶/ml) most often occurred in the first 5 ejaculates in a series, but have been recorded in ejaculates 10, 11, 12 or 14 (M1).

A lack of spermatozoa occurred only once in the semen from M1 and M2, but more frequently in M3. Spermatozoa have not been found in M4. No seasonal pattern in sperm density has been observed in any subject.

This study found differences in ejaculate characteristics and quality between three mature subjects of different ages, with older males having higher values in all the semen parameters investigated. It may be speculated that for ejaculate characteristics to reach these levels is a process that requires several years. Continued monitoring of M3 and M4 will provide more information about this. Young male Tursiops aduncus (< 10y) can produce high quality ejaculates and should be monitored and considered for early inclusion in controlled breeding programmes / AI using fresh or cryopreserved semen.

Acknowledgments

The authors are grateful to Gary Wong, Harriet Chiu and the trainers of Ocean Park's Marine Mammal Department for their invaluable contributions in dolphin training, husbandry and semen collection, and the Clinical Laboratory staff for their support, assistance and the author's training in semen evaluation.

This project is funded by the Research Grants Council of Hong Kong (Grant ref: PolyU5287/01).

References

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