Van den Berghe, Femke (2018) A conservation management toolkit: developing assisted breeding and behavioural management tools for the African wild dog (Lycaon pictus). PhD thesis, James Cook University.

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Abstract

The African wild dog (AWD; Lycaon pictus) is endangered with the current population estimated at 6,600 animals, scattered over several subpopulations in Southern and Eastern Africa. They show a complex social structure including a separate male and female hierarchy and a cooperative breeding system where subdominants usually do not breed but help in raising the pups. To maintain a viable captive population and genetic diversity, animals are often translocated between institutions to form a new breeding pack. Similarly, a metapopulation management plan has been introduced in South Africa, involving the reintroduction of AWDs in small protected areas and regular translocations of individuals between subpopulations. However, due to their complex social structure, new pack formations can often lead to aggression between animals resulting in injury or even mortality.

Sperm freezing, and development of artificial insemination (AI) techniques, can aid species management and conservation of the AWD. The use of semen cryopreservation and AI could overcome problems of intra-pack aggression associated with new pack formations by supplementing genetic diversity without disrupting existing pack structure; and thereby facilitating captive breeding and metapopulation management. In addition, transporting spermatozoa instead of live animals reduces the risk of disease transmission and has ecological and economic benefits. Sperm from free-roaming males could be used to increase genetic diversity in captivity, avoiding the removal of animals from the wild. Lastly, establishing a sperm bank of genetically valuable animals will provide a genetic back-up of the remaining population, providing a buffer against possible threats. Therefore, the aim of this thesis was to develop assisted breeding and behavioural management techniques to enable the application of AI in this species, through the following objectives: (i) determine the effect of social rank on subordinate male fertility (Chapter 2); (ii) develop a sperm freezing protocol (Chapter 3); (iii) determine if Dog Appeasing Pheromone (DAP) can reduce stress and aggression associated with temporary pack separation (Chapter 4); and (iv) validate the use of behaviour and faecal steroid hormone profiles as a non-invasive way to time the fertile period in AWD females for timed AI (Chapter 5).

The study included n=15 males from 5 packs housed in zoological institutions in the US (ABQ, Albuquerque BioPark, Albuquerque, NM; TOP, Topeka Zoo, Topeka, KS; BRK, Brookfield Zoo, Chicago, IL; BIN, Binder Park Zoo, Battle Creek, MI; and OKC, Oklahoma City Zoo, Oklahoma City, OK) and n=13 males and n=3 females from 3 packs housed at Harnas Wildlife Foundation, Gobabis, Namibia (BRU, Brutus pack; PLA, Platform pack; SAN, San pack). Males were immobilised during the pre-breeding season (n=12; ABQ, BRK, BIN, TOP) and breeding season (n=24; ABQ, BRK, BIN, OKC, BRU, PLA, SAN) and male fertility parameters including hormones (faecal androgen - fAM and glucocorticoid metabolite - fGCM concentrations), prostate and testes volume, preputial gland size, semen collection success, and multiple measures of sperm quality were recorded (objective 1). Sperm samples of sufficient quality collected in the breeding season were split and frozen using 2 canine freezing protocols: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution (objective 2). In addition, males from US packs were treated topically either with DAP (n=11; 4 packs) or placebo solution (n=12, 4 packs), applied at the end of anaesthesia prior to reintroduction into the pack (objective 3). Behavioural interactions as well as fGCM and fAM were examined from 3 days before (objective 1) until 4-6 days after immobilisation (objective 3). Moreover, behavioural interactions, faecal progesterone (fPM) and estrogen (fEM) metabolite concentrations were examined for n=3 alpha females during their periovulatory period. Furthermore, each female was immobilised on 2 or 3 occasions at this time to evaluate vulvar size, and serum progesterone and oestrogen levels as well as perform vaginal cytology, vaginoscopy, and ovarian ultrasound (objective 4).

Semen could be collected successfully from all alpha males but from only half the subordinate males in the pre-breeding season, with higher urine contamination in lower ranking animals. Fertility parameters did not differ between social ranks, except for a lower sperm progressive motility and normal morphology in subordinates. In the breeding season, preputial gland size increased with social rank, but no difference between ranks was observed in any other parameter, including sperm quality (objective 1). Eight ejaculates of sufficient quality were frozen in the breeding season. Sperm motility persisted for up to 8h after thawing for Protocol 2, while it dropped to nearly 0% after 2h incubation for Protocol 1. In addition, viability and acrosome integrity of spermatozoa were higher for Protocol 2 throughout post-thaw incubation (objective 2). The application of DAP to males at the end of anaesthesia and prior to reintroduction, did not alter the rise in fGCM levels after intervention. However, fAM increased in placebo but was prevented in DAP treated animals. On the day of reintroduction, DAP treated packs tended to show lower rates of contact-dominance and active-submission behaviour, but higher rates of non-contact dominance behaviour (objective 3). In females, late oestrus (fertile period) could be clearly distinguished from early oestrus by an increase in frequency of certain types of behavioural interactions between the alpha male and female (affiliative behaviour, sexual follow, male initiating behaviour, ride-up, and copulation). One female exhibited an anovulatory cycle while the remaining 2 showed a significant 2.5- to 3-fold increase in fPM levels and similar declining fEM levels (after a rise in pro-oestrus) compared to baseline. However, vaginal cytology and vaginoscopy results deviated from patterns seen in domestic dogs, and blood progesterone showed lower levels. Moreover, ovarian structures were difficult to visualise by ultrasound. As such, without frequent sampling, these invasive diagnostic techniques are unsuitable to determine the fertile phase in wild canids such as the African wild dog (objective 4).

In conclusion, reproductive suppression of subordinate males appears to be behaviourally mediated, given that AWD males of all social ranks produce semen of similar quality during the breeding season, making them suitable candidates for sperm banking (objective 1). In addition, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it appropriate for sperm banking and artificial insemination initiatives (objective 2). The observed effects of DAP on AWD hormones and behaviour could decrease the risk of agonistic interactions, making it a useful tool to help manage temporary pack separation, needed when performing semen freezing and AI (objective 3). Lastly, distinctive behaviours during late oestrus, together with an increase in faecal progesterone and decline in faecal oestrogen in AWD females, could potentially be used to determine the timing of the fertile period (objective 4). These results combined facilitate sperm banking and the application of AI in the African wild dog, thereby assisting management and conservation of the species.

Item ID:	54780
Item Type:	Thesis (PhD)
Keywords:	African wild dog, artificial insemination, behaviour, biobanking conservation, cortisol, cryopreservation, endocrinology, Equex STM, faeces, Lycaon pictus, physiology, prostate gland, semen, sperm bank, sperm, testis, testosterone, wildlife
Related URLs:	https://researchonline.jcu.edu.au/54249/ https://researchonline.jcu.edu.au/52999/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 2: Van den Berghe, F., Paris, M.C.J., Sarnyai, Z., Briggs, M.B., Millar, R., Ganswindt, A., and Paris, D.B.B.P. (2018) Social dominance does not affect semen quality in African wild dogs (Lycaon pictus). Reproduction, Fertility and Development, 30 (1). p. 188. Chapter 3: Van den Berghe, Femke, Paris, Monique Christina Johanna, Briggs, Michael Brent, Farstad, Wenche Kristin, and Paris, Damien Boyd Bertrand Paul (2018) A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog(Lycaon pictus). Cryobiology, 80. pp. 18-25.
Date Deposited:	26 Jul 2018 05:17
FoR Codes:	06 BIOLOGICAL SCIENCES > 0608 Zoology > 060803 Animal Developmental and Reproductive Biology @ 50% 05 ENVIRONMENTAL SCIENCES > 0502 Environmental Science and Management > 050202 Conservation and Biodiversity @ 50%
SEO Codes:	96 ENVIRONMENT > 9608 Flora, Fauna and Biodiversity > 960899 Flora, Fauna and Biodiversity of Environments not elsewhere classified @ 50% 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 25% 97 EXPANDING KNOWLEDGE > 970107 Expanding Knowledge in the Agricultural and Veterinary Sciences @ 25%
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