Thermoregulation of the bovine scrotum 2: simulated acute and chronic heat waves reduces the scrotal thermoregulatory capability of Wagyu bulls.

The objective of this study was to investigate the effect of acute and chronic heat load events on scrotal temperature (ST), body temperature (BT) and bull behaviour, and to examine the interrelationship between these parameters; the underlying hypothesis was that adverse heat treatments delivered in a temperature controlled environment will lead to thermoregulatory dysfunction of the bull scrotum. Six sexually mature Wagyu bulls were used in this study with data loggers surgically implanted into the abdominal cavity and scrotum. Body temperate and ST were recorded at 30-min intervals for the duration of the study. There were two housing locations used throughout the study, outdoor pens and climate control rooms. The study was designed as a four-phase crossover design with two heat treatments: (1) a 5-day acute challenge, and (2) a 14-day chronic challenge. The study was also blocked by phase to control for systematic change between phases with a thermoneutral (TN) phase in outdoor pens between each heat challenge. Observations within the climate rooms were conducted at 1-h intervals and data on panting scores (PS), respiration rate (RR), posture (standing or lying) and general behaviours (feeding, drinking, ruminating) recorded. Ambient temperature (AT, °C) and relative humidity (RH, %) were obtained at 10-min intervals and used to calculate the temperature humidity index (THI). Multiple models were conducted using a linear mixed effects model that contained different permutations of date and time factors and interactions as well as inclusion of an autoregressive parameter. The strongest model based on Akaike's information criterion (AIC) was selected and further analysed. Ambient conditions during heat treatments were consistent with heat load and bulls showed typical physiological symptoms of the same. Maximum ST for acute and chronic treatments occurred once AT had exceeded 34 °C for at least 3 h (acute 35.59 °C at 1500 h; chronic 35.18 °C at 1400 h), whereas during TN conditions, maximum ST was at 2100 h. All phases showed variation in ST throughout the day. There were strong cross correlations between ST and RR during the heat treatments (acute r = 0.918, P < 0.0001; chronic r = 0.916, P < 0.0001), but not during TN (r = 0.411, P < 0.05). Our results confirmed that the ST of the bulls used in this study was not held at a constant temperature and that there was a possible connection between ST and RR. We have shown that during a period of heat load, the thermoregulatory mechanisms thought responsible for maintaining bovine ST appear to breakdown.

Plasma and acrosomal membrane lipid content of saltwater crocodile spermatozoa.

This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is documented. The mean (±s.d.) ratio of unsaturated to saturated membrane fatty acids within the plasma membrane was 2.57±0.50, and was determined to be higher than a similar analysis of the lipids found in the acrosomal membrane (0.70±0.10). The saltwater crocodile sperm plasma membrane also contained remarkably high levels of cholesterol (mean (±s.d.) 40.7±4.5 nmol per 106 sperm cells) compared with the spermatozoa of other amniote species that have so far been documented. We suggest that this high cholesterol content could be conferring stability to the crocodile sperm membrane, allowing it to tolerate extreme osmotic fluxes and rapid changes in temperature. Our descriptive analysis now provides those interested in reptile and comparative sperm physiology an improved baseline database for interpreting biochemical changes associated with preservation pathology (e.g. cold shock and cryoinjury), epididymal sperm maturation and capacitation/acrosome reaction.

Body temperature of free-ranging koalas (Phascolarctos cinereus) in south-east Queensland.

The distribution of the koala (Phascolarctos cinereus) in Queensland is predicted to contract as a result of climate change, driven by the frequency, intensity and duration of heatwaves and drought. However, little is known about the physiological responses of this species to environmental extremes under field conditions. This study aimed to establish the efficacy of surgically implanted thermal radio transmitters and data loggers to measure the body temperature of free-ranging koalas across a range of environmental conditions and ambient temperatures. Five free-ranging koalas in southeast Queensland were implanted with thermal transmitters and data loggers waxed together as a single package. Body temperatures were recorded for variable periods ranging from 3 to 12 months. Diurnal rhythms in body temperature were detected irrespective of season. The long-term diurnal body temperature peak for all koalas occurred between 16:00 and 17:00 h and body temperature was 36.7-36.9 °C, the long-term nadir occurred between 07:00 and 08:00 h and body temperature was 35.4-35.7 °C. Koala body temperatures as low as 34.2 °C and as high as 39.0 °C were recorded. Thermolability became apparent when ambient temperatures were outside the deduced thermal neutral zone for koalas (14.5-24.5 °C): heat was accumulated during the day and dissipated during the cool of the night. While this study is the first to report on body temperature of free-ranging koalas in their normal behavioural context, further investigations are necessary to determine the physiological boundaries of the thermal niche for this species, in order to better equip models that will more accurately predict the impacts of climate change on koalas.

Protamine composition of koala and wombat spermatozoa provides new insights into DNA stability following cryopreservation.

To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynamics over 24h of post-thaw incubation. Following validation of a wombat sperm chromatin dispersion test, wombat DNA showed a rate of SDF that was 6-fold higher than for koala spermatozoa (P=0.038). Finally, we examined whether expected differences in chromatin compactness, associated with protamine sequence, had an effect on restriction site accessibility of sperm DNA. Thawed spermatozoa were exposed to Alu I and EcoR1 endonuclease restriction enzymes and the SDF dynamics were observed. Koala spermatozoa exposed to Alu I showed a greater rate of SDF (P=0.01), whereas wombat spermatozoa exposed to EcoR1 showed a greater rate of SDF (P=0.032). We conclude that restriction sites in these species are differentially present or exposed and potentially account for differences in SDF dynamics. Although differences in the arginine composition of protamine may explain relative differences in SDF following cryopreservation, they do not support the hypothesis that increased arginine composition increases DNA stability; rather, increased arginine composition in the wombat may reduce post-thaw chromatin swelling.

Recording body temperature in koalas (Phascolarctos cinereus): a comparison of techniques.

OBJECTIVE: Compare the use of four techniques to measure body temperature in koalas: intraperitoneal (thermal data logger and temperature sensitive radio transmitter), rectal (certified thermometer), tympanic (infrared thermometer), and hind foot (infrared camera). METHODS: The body temperature data collected concurrently from the intraperitoneal loggers were used as the benchmark in the analyses. RESULTS: The rectal, foot and tympanic methods consistently recorded lower body temperature when compared with the benchmark. There was a strong positive relationship (R2 = 0.79) between logger and rectal measurements, but no significant relationship between logger and foot or logger and tympanic measurements. CONCLUSION: Rectal measurements can be used to record internal body temperature, with the caveat that such measurements will generally register a temperature approximately 0.25°C lower than the actual intraperitoneal temperature.

Behavioral and endocrine responses to season and social dynamics of captive male southern hairy-nosed wombats (Lasiorhinus latifrons).

Although southern hairy-nosed wombats (SHN wombats; Lasiorhinus latifrons) rarely breed in captivity, further knowledge of their reproductive physiology and behavior is likely to improve their breeding potential. This study examined the effect of seasonal variation and changes in social dynamics on the physiology and behavior of a captive population of male SHN wombats (n = 6). Seasonal changes in urinary testosterone metabolites (UTM), urinary cortisol metabolites (UCM), qualitative estimates of spermatorrhoea (QS), aggressive behavior and reproductive behavior were measured over an 11-month period. While there was no effect of month on QS (GLM ANOVA, P = 0.27), reproductive behavior (GLM ANOVA, P = 0.19) or aggressive behavior (Tukey pairwise comparisons), the secretion of UTM (GLM ANOVA, P = 0.051) was only marginally affected by season, compared to that reported for wild male SHN wombats. Mean UCM concentrations of July and August 2016 were significantly higher than those between October 2015 and January 2016 (Tukey pairwise comparisons). To examine social dynamics, two trials of animal positioning exchange with the enclosure system were implemented and behavioral data were examined for each trial over a six week period; UTM, UCM and general behaviors (n = 27) were measured for each trial. Neither UTM nor UCM concentration varied significantly (P ≥ 0.45) before and after the exchanges. "Scratching" decreased at the group level following the animal exchange in both trials, suggesting reduction in self-grooming may be a behavioral response to novel stimuli. UCM and UTM concentrations were both positively correlated with "standing still" and "body rub" behaviors. This may be evidence of a hormonal control of a "freezing behavioral response" to external stimuli and marking behavior, respectively. As there was no evidence that changing the social dynamics affected reproductive or agonistic behavior or hormone concentrations, it was concluded that captive male wombats in this study showed reduced reproductive seasonality compared to wild wombats and that animal exchange resulted in a behavioral response to novel stimuli but was not sufficient to affect testosterone or cortisol secretion, within the context of our study.

Measurement of testosterone and cortisol metabolites and luteinising hormone in captive southern hairy-nosed wombat (Lasiorhinus latifrons) urine.

This study reports the validation and use of enzyme immunoassays (EIA) to measure changes in plasma and urinary luteinizing hormone, testosterone metabolites (UTM) and cortisol metabolites (UCM) in captive southern hairy-nosed wombats (Lasiorhinus latifrons). GnRH agonist and ACTH agonist challenges were conducted to validate urinary testosterone (male wombat only) and cortisol (male and female wombats) EIAs. Following intra-muscular injection of 8-12µg buserelin (n=4 males), there was a significant increase in both plasma (P<0.001) and urinary testosterone concentrations (P<0.001) 60min and 21h after administration, respectively. Plasma LH levels were elevated (p<0.05) at 20min but there was no significant increase found in urinary LH concentrations after injection. Intra-muscular injection of Synacthen® Depot (250µg) (n=3 males, 3 females) resulted in a significant increase (p<0.05) in plasma cortisol secretion 15min and in urinary cortisol concentrations 3h post injection, respectively. Sex-related differences in cortisol secretion were also reported in this study. These findings indicate that (1) urinary LH might not be an appropriate index for describing the reproductive status in captive male L. latifrons, and (2) the UTM and UCM assays appear to be suitable for the assessment of the testicular steroidogenic capacity and the adrenocortical activity in captive southern hairy-nosed wombats, respectively.

Measurement of bovine body and scrotal temperature using implanted temperature sensitive radio transmitters, data loggers and infrared thermography.

Synchronous and continuous measurement of body (BT) and scrotal temperature (ST) without adverse welfare or behavioural interference is essential for understanding thermoregulation of the bull testis. This study compared three technologies for their efficacy for long-term measurement of the relationship between BT and ST by means of (1) temperature sensitive radio transmitters (RT), (2) data loggers (DL) and (3) infrared imaging (IRI). After an initial pilot study on two bulls to establish a surgical protocol, RTs and DLs were implanted into the flank and mid-scrotum of six Wagyu bulls for between 29 and 49 days. RT frequencies were scanned every 15 min, whilst DLs logged every 30 min. Infrared imaging of the body (flank) and scrotum of each bull was recorded hourly for one 24-h period and compared to RT and DL data. After a series of subsequent heat stress studies, bulls were castrated and testicular tissue samples processed for evidence of histopathology. Radio transmitters were less reliable than DLs; RTs lost >11 % of data, whilst 11 of the 12 DLs had 0 % data loss. IRI was only interpretable in 35.8 % of images recorded. Pearson correlations between DL and RT were strong for both BT (r > 0.94, P < 0.001) and ST (r > 0.80, P < 0.001). Surgery produced temporary minor inflammation and scrotal hematoma in two animals post-surgery. Whilst scar tissue was observed at all surgical sutured sites when bulls were castrated, there was no evidence of testicular adhesion and normal active spermatogenesis was observed in six of the eight implanted testicles. There was no significant correlation of IRI with either DL or RT. We conclude that DLs provided to be a reliable continuous source of data for synchronous measurement of BT and ST.

Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1 (fast freeze) or -6.0°Cmin-1 (slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.

Thermoregulation of the bovine scrotum 1: measurements of free-range animals in a paddock and pen.

The bull's scrotum and scrotal cord vasculature has traditionally been regarded as a thermoregulatory device for maintaining optimal testicular temperature for normal spermatogenesis. This assumption has mostly been derived from discrete measurements using thermocouples with limited data correlating continuous scrotal temperature (ST) to body temperature (BT). From mid-summer to early autumn, four Wagyu bulls (9-18 months) were surgically implanted with two data loggers (DL) logging at 30 min intervals: one on the right hand side flank and the other was attached to the visceral vaginal tunic of the mid-testis. Bulls were firstly housed in a paddock (PK) for 13 days and then moved to individual pens (IP), again for 13 days. Repeated measures analysis modelled the long-term and diurnal trends in BT and ST. While both day and time of day (TOD) were significant effects for ST at both housing locations (P < 0.005), only TOD showed significance for BT at both locations (P < 0.0001). Significant effects were seen between bulls with ST (F = 167.2, P < 0.001) but not BT (F = 0.03, P = 0.863), suggestive of variation in individual bull thermoregulatory capacity. Dual peaks were observed in ST at 0500 and 2130 h when housed in PK but not IP, suggesting ST may be influenced by external stimuli such as postural or behavioural changes. Reporting concurrent and continuous BT and ST will allow further investigation into factors influencing bovine ST and should be useful in selecting bulls with high degrees of thermoregulation capacity.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus).

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen-thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r=0.90; P=0.037). Results of the SCDt immediately following thawing and after 5h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE: Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

First report of a spermatic granuloma and varicocele in a marsupial: A Koala (Phascolarctos cinereus) Case Study.

This study reports the first documented clinical case of a spermatic granuloma and varicocele in a marsupial. Initial clinical presentation included gross morphological changes in the left scrotal cord, epididymis and testis. Ultrasonography of the scrotum and spermatic cord, and gross and histopathological examination after hemicastration, confirmed the condition as a spermatic granuloma affecting the left caput epididymis, with a varicocele in the left proximal spermatic cord, which was causing azoospermia and infertility. Semen quality and serum testosterone secretion following a GnRH challenge was assessed prior to, and following surgery. After hemi-castration, an increase in androgen secretion to within normal reference ranges for the koala was observed with a subsequent increase in semen production and sperm quality resulting in the sire of a pouch young, 12months later.

Surgical implantation of temperature-sensitive transmitters and data-loggers to record body temperature in koalas (Phascolarctos cinereus).

BACKGROUND: Under predicted climate change scenarios, koala distribution in Australia is expected to be adversely affected. Recent studies have attempted to identify suitable habitat, based on models of bioclimatic regions, but to more accurately reflect the thermal tolerance and behavioural adaptations of the various regional populations, the koala's response to periods of heat stress will need to be investigated at the individual animal level. OBJECTIVE: To explore the safety and suitability of temperature-sensitive intra-abdominal implants for monitoring core body temperature in the koala. METHODS: A temperature-sensitive radio transmitter and thermal iButton data-logger, waxed together as a package, were surgically implanted into the abdominal cavity of four captive koalas. In one animal the implant was tethered and in the other three, it was left free-floating. RESULTS: After 3 months, the implants were removed and all four koalas recovered without complications. The tethering of the package in the one koala resulted in minor inflammation and adhesion, so this practice was subsequently abandoned. The free-floating deployments were complication-free and revealed a diurnal body temperature rhythm, with daily ranges of 0.4-2.8°C. The minimum recorded body temperature was 34.2°C and the maximum was 37.7°C. The difference in the readings obtained from the transmitters and iButtons never exceeded 0.3°C. CONCLUSIONS: The suitability of the surgical approach was confirmed, from both the animal welfare and data collection points of view.

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa.

Assisted reproductive technologies are of great importance for increasing the genetic diversity in captive animals. The use of bovine or murine oocytes in heterologous IVF provides advantages compared to homologous IVF in nondomestic animals, such as the accessibility to oocytes and the availability of well-developed in vitro maturation systems. The aim of this study was to determine the heterologous IVF parameters using cryopreserved dolphin spermatozoa and zona-intact bovine or murine oocytes and to examine the nuclear chromatin status of the dolphin spermatozoa. All the processes involved in the fertilization including embryo cleavage were observed by confocal microscopy and hybrid embryo formation was confirmed by polymerase chain reaction. Heterologous bovine IVF showed no polyspermy, lower percentages of pronuclear formation, and a lower cleavage rate compared to homologous IVF group (34.8% vs. 89.3%). Heterologous murine IVF showed a lower cleavage rate than homologous IVF (9.6% vs. 77.1%). With respect to dolphin sperm chromatin, it was more stable, i.e. more resistant to EDTA-SDS decondensation than the bovine sperm chromatin. This study revealed the stability of the dolphin sperm chromatin and the ability of the dolphin spermatozoa to penetrate zona-intact bovine and murine oocytes, leading to hybrid embryo formation.

Use of the gonadotrophin-releasing hormone antagonist azaline B to control the oestrous cycle in the koala (Phascolarctos cinereus).

The present study examined the effectiveness of the gonadotrophin-releasing hormone (GnRH) antagonist azaline B to suppress plasma LH and 17ß-oestradiol concentrations in koalas and its potential application for oestrous synchronisation. In Experiment 1, single subcutaneous injections of azaline B successfully blocked the LH response to exogenous mammalian (m) GnRH in a dose-dependent manner; specifically, 0 mg (n = 4) did not suppress the LH response, 1 mg azaline B (n = 6) suppressed the LH response for 24 h (P < 0.05), 3.3 mg azaline B (n = 8) suppressed the LH response significantly in all animals only for 3 h (P < 0.05), although in half the animals LH remained suppressed for up to 3 days, and 10 mg azaline B (n = 4) suppressed the LH response for 7 days (P < 0.05). In Experiment 2, daily 1 mg, s.c., injections of azaline B over a 10-day period during seasonal anoestrus (June-July; n = 6) suppressed (P < 0.01) the LH response to mGnRH consecutively over the 10-day treatment period and, 4 days after cessation of treatment, the LH response had not recovered. Experiment 3 was designed to test the efficacy of daily 1 mg, s.c., azaline B over 10 days to suppress plasma LH and 17ß-oestradiol concentrations and ultimately synchronise timed return to oestrus during the breeding season. Although azaline B treatment did not suppress basal LH or 17ß-oestradiol, oestrus was delayed in all treated females by 24.2 days, but with high variability (range 9-39 days). Overall, the present study demonstrates that the GnRH antagonist azaline B is able to inhibit the LH response in koalas to exogenous mGnRH and successfully delay the return to oestrus. However, although azaline B clearly disrupts folliculogenesis, it has not been able to effectively synchronise return to oestrus in the koala.

The use of a synthetic progesterone, levonorgestrel (LNG), to control the oestrous cycle in the koala.

This study investigated the efficacy of a synthetic progestogen, levonorgestrel (LNG), to control koala ovarian activity for the purposes of oestrous synchronisation. Captive koalas were administered either saline control or a 70-mg LNG implant on Day 2 of oestrus. Urogenital cytology, oestrous behaviour and plasma oestradiol-17ß and LH concentrations were monitored over a 6-week period. After LNG implant removal females were monitored to determine if the return to oestrus was synchronised. LNG-treated koalas immediately ceased displaying oestrous behaviour, showed no evidence of cornified epithelial cells in smears of urogenital cytology and exhibited low plasma oestradiol-17ß concentrations throughout the implantation period. In contrast, oestradiol-17ß levels in control koalas showed evidence of continued cyclic activity associated with behavioural oestrus and increased cornified epithelial cells in urogenital smears on Days 33 to 35 after saline injection. After implant removal, LNG-treated koalas exhibited oestrus at 13, 14, 17 and 30 days after implant removal. Plasma LH concentrations varied throughout the study period with no significant time (P = 0.49) or treatment (P = 0.13) effect. Overall results from this study suggest that LNG implants in koalas can inhibit oestrous behaviour and reduce circulating oestradiol-17ß levels before oestrus, most likely by preventing development of the pre-ovulatory follicle. However, there was no evidence of LH suppression by the LNG implants. Removal of LNG implants resulted in the synchronous return to oestrus in three of the four treated koalas. Further studies on a larger population are required to validate these findings.

Plasma prolactin concentrations during lactation, pouch young development and the return to behavioural oestrus in captive koalas (Phascolarctos cinereus).

Plasma prolactin (PRL) concentrations in captive koalas during lactation were determined by serial blood sampling. PRL concentrations were low (1.3 ± 0.1 ng mL-1; n = 5) during early lactation until pouch young (PY) began to emerge from the pouch (around Day 130) before significantly (P < 0.05) increasing between Day 161 and Day 175 (5.3 ± 1.0 ng mL-1). A significant (P < 0.001) peak in PRL (7.7 ± 0.6 ng mL-1) coincided with maturing young between Day 189 and Day 231. All females failed to exhibit any signs of oestrous behaviour until Day 268.8 ± 8.5 (n = 4), some 102 ± 19 days before PY were weaned following achieving target weights of 2.5-2.7 kg. Throughout lactation, plasma LH concentrations were relatively high (range 4.9-8.7 ng mL-1) and LH responses to exogenous gonadotrophin-releasing hormone were observed in all koalas at all times during lactation.