Pteropus spp. (flying-foxes) worldwide are in desperate need of conservation. Over 60% of species are threatened and 10% are on the verge of extinction. Assisted reproductive technologies (ART), such as artificial insemination (AI), have the potential to play a primary role in the conservation of endangered Pteropus spp., through the genetic and reproductive management of captive colonies. The development of AI technology in Pteropus spp. has been hampered by an inability to preserve spermatozoa and by the atypical reproductive biology of the female.
The primary aims of this thesis were to utilise ART in order to: 1) improve semen collection and preservation protocols, 2) evaluate male seasonality, its relationship with semen and sperm quality, and its modification for reproductive management of captive populations, and 3) successfully manipulate the female oestrous cycle to control the timing of insemination to maximise fertilisation success following AI.
The successful collection of 406 ejaculates from six species of Pteropus, has demonstrated that electro-ejaculation is a safe and reliable method of collecting semen from these species. This success has enabled ejaculate characteristics to be described in four Pteropus species for the first time: P. giganteus, P. hypomelanus, P. vampyrus and the critically endangered P. rodricensis. Multi-treatment investigations into sperm preservation are now possible after the finding that up to three ejaculates, recovered in quick succession, from individual P. alecto did not differ. Furthermore, P. alecto spermatozoa were successfully stored at ambient temperature (~27oC) and 36.5oC for up to 6h before losses (P<0.05) to acrosomal and plasma membrane integrity were observed.
The acrosomal and plasma membrane fatty acid composition of five Pteropus spp. spermatozoa were investigated. The presence of polyunsaturated fatty acids (PUFAs) in sperm membranes limits the effects of cold shock; however, the predominant membrane fatty acids of Pteropus spp. were saturated fatty acids and not PUFAs, such as docosapentaenoic acid (DPA; 22:5) and docosahexaenoic acid (DHA; 22:6). In fact, DPA was not detected in any of the five Pteropus spp. examined, with DHA only detected above trace levels in P. vampyrus. This overall lack of PUFAs may explain why Pteropus spp. spermatozoa are prone to damage during cooling procedures.
Male reproductive seasonality and the effect of contraceptive implants were monitored in captive P. alecto (six controls; five treated with 4.7 mg deslorelin) for 15 months. Seasonal changes in testicular volume, body weight and testosterone secretion were observed in untreated males; however, there was no corresponding change in sperm quality, and seminal vesicle gland (SVG) secretions remained present within ejaculates. Testosterone concentration, in treated males, reached basal levels by 32d and remained at a nadir. Sperm motility was reduced at 1mo, SVG secretions were absent at 4mo and aspermic ejaculates were first recorded 5mo post-deslorelin treatment. The return of motile spermatozoa in one individual coincided with a return of SVG secretions 13mo post-treatment. This indicates that deslorelin is an effective reversible contraceptive in P. alecto and that P. alecto males are capable of producing motile, membrane-intact spermatozoa and SVG secretions throughout the year.
Synchronising AI with oestrus and ovulation is important to maximise fertilisation. Pteropus spp. do not show systematic increases in systemic sex steroids associated with oestrus, nor is there overt behavioural oestrus, suggesting exogenous hormonal stimulation may be required to circumvent the need to detect natural oestrus when timing AI. Two doses of pregnant mare serum gonadotropin (PMSG; 15IU, n=4; 30IU, n=4) were examined in adult female P. alecto. Both doses of PMSG induced ovulation, however single ovulations occurred in 75% of the 15IU group, while evidence of a bilateral ovarian response in all 30IU females, indicated over-stimulation of the reproductive tract.
The ability of exogenous hormones, PMSG and oestradiol benzoate (OB), to induce oestrus was investigated using daily ethological observations of male-female interactions. Experiment 1 involved three female groups: i) ovariohysterectomised (OHX), ii) intact-untreated and, iii) intact PMSG-treated (25IU; 0d). Males differentiated PMSG-treated, from untreated or OHX groups. Female receptivity was indicated by increased behaviours that regulate or pace the rate of copulatory stimulation received, accompanied by low levels of antagonism in response to male approaches. Experiment 2 involved administration of OB (days 0, 1 and 2) using three groups of OHX females: i) no dose (controls), ii) low dose (0.1mg) and, iii) high dose (1.0mg); vaginal cytology was also examined (days 1, 3 and 5). Oestradiol did not alter vaginal cytology and superficial epithelial cells remained dominant. Spermatozoa were present every day in vaginal smears of each group. While male behaviour indicated some capacity to differentiate between females on the basis of their hormonal status, mating patterns were apparently indiscriminate.
This body of work has contributed to a greater understanding of Pteropus species reproductive knowledge and will enable the continued development of ART and facilitate technology transfer between species to ensure the successful development of AI protocols for endangered Pteropus species.