Artificial insemination and parthenogenesis in the whitespotted bamboo shark Chiloscyllium plagiosum

Abstract

Non-lethal methods for semen collection from elasmobranchs to better understand species reproduction has accompanied the development of artificial insemination. Ejaculates (n = 82) collected from whitespotted bamboo sharks Chiloscyllium plagiosum (n = 19) were assessed and cold-stored raw or extended at 4 °C. Females (n = 20) were inseminated with fresh or 24-48 h cold-stored raw or extended semen and paternity of offspring determined with microsatellite markers. Insemination of females with fresh semen (n = 10) resulted in 80 hatchlings and 27.6% fertility. Insemination of females with semen cold-stored 24 h (n = 4) and 48 h (n = 1) semen resulted in 17 hatchlings and fertilization rates of 28.1% and 7.1% respectively. Two females inseminated with fresh or cold-stored semen laid eggs that hatched from fertilization and parthenogenesis within the same clutch. Parthenogenesis rate for inseminated females was 0.71%. Results demonstrate artificial insemination with cold-stored semen can provide a strategy for transport of male genetics nationally and internationally, precluding the need to transport sharks. Production of parthenotes in the same clutch as sexually fertilized eggs highlights the prevalence of parthenogenesis in whitespotted bamboo sharks and poses important considerations for population management.

Figure 1

Whitespotted bamboo shark Chiloscyllium plagiosum spermatozuegmata and spermatozoa micrographs illustrating the morphology and acrosome location for aggregated and individual spermatozoa. Scanning electron micrograph of a spermatozeugma fragment (a) revealing sperm with heads closely aligned and embedded in a matrix core. Matching phase contrast and fluorescent micrographs of a spermatozeugma with radially aligned heads (b, d) and individual spermatozoan (c, e) with acrosomes highlighted by fluorescein isothiocyanate conjugated Arachis hypogaea agglutinin (PNA-FITC) stain. Whitespotted bamboo shark spermatozoa (c) possess a helical head with an acrosome (e), a helical midpiece and a flagellum with a transient cytoplasmic sleeve (arrow) at the junction of the midpiece and tail that was shed after acquiring motility.

Figure 2

The effect of cold storage on whitespotted bamboo shark Chiloscyllium plagiosum ejaculates. Plasma membrane integrity (a) and motility (b) of ejaculates cold-stored raw or extended with elasmobranch modified Hank’s balanced salt solution (E-HBSS) or artificial seawater (ASW) and initial mean (dotted line) and standard error (grey rectangle) of ejaculates. Treatment bars are capped with a circle (estimated marginal mean) and error bars (95% confidence interval). *Indicates significant difference among treatments at each time point (p < 0.05).

Figure 3

Oviposition dates for whitespotted bamboo shark Chiloscyllium plagiosum females after artificial insemination. Egg cases are usually laid in pairs, one egg from each side of the paired reproductive tract. Egg cases classified as fertile were assigned to categories: hatched, parthenote or embryonic death. Egg cases classified as infertile had ova without evidence of embryonic development. Empty or wind egg cases did not contain ova.

Figure 4

Fertility, expressed as a percentage of egg cases laid, for five females that stored sperm for more than 3 months from artificial insemination and laid pairs of fertile eggs. Fertility (smoothed loess local regression line) with 95% confidence interval (grey) for females (n = 5) beginning 6 days after and ending 116 days after artificial insemination. Individual female fertility is distinguished by marker color.