Effects of Age and Captive Environment on Sperm Quality Metrics in Critically Endangered Mississippi

Gopher Frogs

a. b.

Ashley Watt*1, Ruth Marcec-Greaves2, Kristin Hinkson3, Sinlan Poo3, Beth Roberts3, Trevor Pitcher1

University of Windsor1. Detroit Zoological Society2. Memphis Zoo3. Dallas Zoo3. Email: watta@uwindsor.ca

• Animals in captivity often experience lower fertilization success than their wild counterparts.

• One of the limiting factors that leads to low fertilization success is the quality and quantity of sperm produced by males in captivity.

• Ageing, or senescence – a decline in performance or function with age – can be expressed by a decline in sperm quality in later years1.

• Senescence has been the focus of many reproductive studies to understand the potential deleterious effects of age on sperm quality and the potential downstream consequences on fertilization success; however, only a handful of studies have evaluated the effects of age on sperm quality in frogs2,3,.

• Study the effects of age on sperm quality and fertilization success in the critically endangered Mississippi gopher frog (Lithobates sevosus).

• Evaluate sperm quality metrics between males in different age categories (1 – 2 years old, 3 – 4 years old, and 8 – 9 years old).

• Identify an optimal breeding age to improve reproductive outcomes.

• Explore how the captive environment can influence sperm quality, and whether it may act independently of age or together.

a. Males (N = 30) at the Detroit Zoo (N = 11; captive-bred and wild-origin), the Memphis Zoo (N = 4, captive-bred and wild-origin), and the Dallas Zoo (N = 15, wild-origin) were hormonally induced (GnRH + HCG or GnRH) to collect spermic urine.

b. Spermic urine was collected to assess sperm quality (i) motility, (ii) concentration, (iii) morphology and abnormalities.

c. Fertilization and hatching success were compared between old males ( 8 – 9 years old) and young males (1 – 2 years old) housed at the Detroit Zoo using a split-clutch in-vitro fertilization design.

c.

Figure 1. Log-transformed means (± 1 SE) for a) sperm total length (μm), b) head length (μm) across males of different ages (years) and zoos (Dallas, Detroit, Memphis) in the Mississippi gopher frog (Lithobates sevosus).

This research was funded by the Natural Sciences and Engineering Research Council of Canada. We would like to thank the support of Bradley Lawrence (Reptile and Amphibian Supervisor; Dallas Zoo), Steve Reichling (Director of Conservation and Research; Memphis Zoo), Mark Vassallo (Enrichment Technician; Detroit Zoo), Nizarina Guxholli and Mariah Demelo (Laboratory Assistants; University of Windsor).

• Age did not influence sperm motility or sperm concentration

• Older males (8 – 9 years old) had longer sperm than younger males (1 - 2 years old and 3 - 4 years old) and fewer abnormalities than younger males (1 – 2 years old).

• Older males (8 – 9 years old) had higher hatching success than younger males (1 – 2 years old).

• Male Mississippi gopher frogs maintain their reproductive capacity across their lifespan.

• Captive environment (Detroit Zoo, Memphis Zoo, Dallas Zoo) had a significant effect on all sperm quality and quantity metrics.

• Sperm quality was not influenced by hormone protocol or origin.

(1) Saino, N., Ambrosini, R., Martinelli, R., Møller, A. P. (2002). Mate fidelity, senescence in breeding performance and reproductive trade-offs in the barn swallow. Animal Ecology, 71(2), 309-319. (2) Roth, T. L., Szymanski, D. C., Keyster, E. D. (2010). Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas). Theriogenology, 73, 501-511. (3) Hettyey, A., Vági, B., Penn, D. J., Hoi, H., Wagner, R. H. (2012). Post-meiotic intra-testicular sperm senescence in a wild vertebrate. PLoS ONE, 7(12), e50820.

Captive Environment: