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.

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.

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.

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.

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.

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.

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.

Semen collection, ejaculate characteristics and in vitro manipulation of spermatozoa from six species of captive flying-fox (Pteropus spp.).

Seminal characteristics are described in six Pteropus species including the critically endangered P. rodricensis. Spermic ejaculates (~40µL) were collected using electro-ejaculation on 406 of 413 attempts. All flying-fox species had mean percentages of acrosome- and plasma-membrane (PM)-intact spermatozoa of >66% and >73%, respectively; the predominant sperm abnormalities found across all species were damaged, folded or missing acrosomes, bent midpieces and coiled tails. Seminal pH ranged from a low of 7.5 in P. giganteus to a high of 8.2 in P. alecto with the other species in between. Electro-ejaculates recovered in short succession from P. alecto revealed no differences in sperm quality, allowing spermatozoa to be utilised for multi-treatment experiments that evaluated the effects of transportation, incubation temperature and in vitro physico-chemical environments on acrosome and PM integrity. Pteropus alecto spermatozoa were successfully held at ~27°C and 37°C for up to 6h before a reduction in PM integrity (P=0.003) was observed. Acrosome and PM integrity decreased (P<0.000) when P. alecto spermatozoa were incubated at 37°C for 30min in a Tris-citrate buffer of pH 9.0 but remained stable at pH 5.0 to 8.0. Pteropus alecto mean (± s.e.m.) seminal osmolality was 307.0±2.5mOsmkg(-1); nevertheless, spermatozoa were tolerant of media ranging from 160 to 1190mOsmkg(-1) but exposure to media of ≤160mOsmkg(-1) resulted in increased acrosome damage (P<0.000).

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.

Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay.

The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n=3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1h (T1) and 24h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r=0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.

Spermatozoa of Sminthopsis murina (Mammalia: Metatheria) exhibit an unusually high degree of chromatin stability in the absence of disulphide bonding in protamine 1.

Although all but a single genus (Planigale) of the metatheria so far examined contain no cysteine residues in protamine 1, we report a remarkable level of chromatin stability in the spermatozoa of the common dunnart, Sminthopsis murina. S. murina cauda epididymal spermatozoa and somatic epithelial cells were exposed to a combination of graded treatments to lyse sperm protein and induce sperm DNA damage via standard freeze-thaw protocols and post-thaw incubation at 37°C for 48h, exposure to sodium nitroprusside (SNP) and the enzyme AluI restriction endonuclease. Sperm DNA fragmentation was assessed using the comet assay and sperm chromatin dispersal test. Although S. murina somatic cells showed DNA fragmentation following protein lysis and after treatment with all the protocols specifically designed to induce chromatin damage, sperm DNA fragmentation was only observed following moderate to severe proteolytic exposure and treatment with the restriction endonuclease; there was also an increase in the baseline halo of spermatozoa treated with an aggressive reducing agent, but no corresponding evidence of fragmented DNA, suggesting that cysteine residues may be functioning to conform tertiary and/or quaternary chromatin structure. Given that the protamine 1 of S. murina contains no cysteine, we suggest that the source of these residues is possibly the histone fraction of the chromatin and that the high level of stability is potentially related to prolonged sperm survival in the female's reproductive tract.

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.

Orchitis and Epididymitis in Koalas (Phascolarctos cinereus) Infected With Chlamydia pecorum.

Although Chlamydia causes disease of the urethra and prostate of male koalas, its impact on the testis and epididymis has not been examined. This study describes chronic-active and granulomatous orchitis and epididymitis with interstitial fibrosis associated with infection by Chlamydia pecorum in 2 of 18 adult male koalas being euthanized at a koala hospital, 8 of which also had chlamydial prostatitis. By immunohistochemistry and transmission electron microscopy, chlamydial inclusions were demonstrated within Sertoli cells directly associated with mild inflammation surrounding intact seminiferous and epididymal tubules, marked pyogranulomatous inflammation around disrupted tubules, replacement of tubules by interstitial fibrosis, and aspermia. The presence of C. pecorum but not Chlamydia pneumoniae was detected by quantitative polymerase chain reaction of formalin-fixed tissues of the left and right testes and right epididymis in 1 animal. This is the first report of orchitis and epididymitis in a koala infected with C. pecorum.

Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples.

Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.