Aspects of the reproduction of male and female African penguins (Spheniscus demersus) with special reference to sperm biology and cryopreservation

Abstract

In the marine environment, penguins have been described as curators and serve a critical role in ecological balance. The African penguin (Spheniscus demersus) has undergone a rapid population decline, mainly due to disturbances in their natural habitat. The African penguin was up-listed from vulnerable to endangered on the IUCN Red List for Threatened Species in 2010 and thus urgent conservation action is required. Integral to long-term conservation action of any species is a basic knowledge of its reproductive biology, which is currently lacking for African penguins. The main aim of this investigation was to evaluate techniques for the collection of semen in African penguin and to determine sperm quality in order to cryopreserve sperm for in vitro fertilization (IVF) purposes of captive and wild populations. Semen was collected once a week during two breeding seasons from two captive African penguins. Ejaculates (n=51) were obtained over two breeding seasons (Jan-Feb and Jun-Oct) and evaluated for semen volume, sperm concentration, sperm vitality, sperm motility and sperm morphology. In addition twelve (six females and six males, n=4 were breeding pairs) captive African penguins were monitored for hormone (estradiol, testosterone, progesterone) levels prior to and after the egg-laying period. The testes were asymmetrical in adults, with the right testis on average shorter in length, width and volume respectively (16.80 ± 4.37 mm; 7.93 ± 2.63 mm; 0.75 mL) compared to the left testis (25.39 ± 5.85 mm; 11.48 ± 4.09 mm; 2.46 mL). The ovaries displayed variation in shape and size among the penguins, the adult ovary has a mean length of 25.72 ± 5.37 mm and a mean width of 9.02 ± 3.87 mm. The Follicles ranged from white small follicles with a diameter of <0.01 mm to mature, yellow large follicles which had a maximum diameter of 22 mm. The testis and ovary histological features such as structure, weight and size, can give a clear indication of breeding status in African penguin. Estradiol levels showed a biphasic pattern in three of the four breeding females, whereas no clear pattern could be followed in other hormones investigated. Semen volume ranged from 0.01 to 0.1 ml, sperm concentration from 802.6 to 7808.8 x106 /ml and total number of sperm per sample ranged from 3.42 to 740.18 x106. The percentage total motility was between 40.1 and 87.1%. The recorded velocities was for curvilinear velocity (VCL 81.5 ± 10.2 μm/s), straight-line velocity (VSL 42.72 ± 7.3 μm/s) and average path velocity (VAP 59.4 ± 8.2 μm/s), and kinematics at straightness of track (STR 71.4 ± 8.9 %), linearity of track (LIN 52.4 ± 8.1 %), amplitude of lateral head displacement (ALH 2.3 ± 0.2 μm) and beat cross frequency (BCF 16.8 ± 3.8 Hz). Sperm quality and semen parameters were similar across all samples collected over breeding seasons. In comparison to fresh semen, percentage total motility of thawed semen decreased to 16.8% after two hours in liquid nitrogen. Since spermatozoa differ notably in their morphology within species, phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy were used to examine structural abnormalities. The sperm morphology is almost identical and largely resembles that of non-passerine birds in terms of the filiform head, small acrosome and mid-piece containing 13 spherical mitochondria, arranged around the proximal and distal centrioles in a single helix. The ultrastructure of the sperm principal piece revealed the typical 9+2 microtubular arrangement without any outer dense fibres. An unusual feature was the occurrence of multiple axonemes contained in one plasmalemma in 4% of spermatozoa. Multiple axonemes found in penguin flagella could be an apomorphism that distinguish them from other bird spermatozoa. This research represents a critical step in the conservation and long-term survival of the African penguin by evaluating techniques for the collection and determination of sperm quality in order to cryopreserve sperm for IVF.