Steve Johnston

The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z

BackgroundSex-determining systems have evolved independently in vertebrates. Placental mammals and marsupials have an XY system, birds have a ZW system. Reptiles and amphibians have different systems, including temperature-dependent sex determination, and XY and ZW systems that differ in origin from birds and placental mammals. Monotremes diverged early in mammalian evolution, just after the mammalian clade diverged from the sauropsid clade. Our previous studies showed that male platypus has five X and five Y chromosomes, no SRY, and DMRT1 on an X chromosome. In order to investigate monotreme sex chromosome evolution, we performed a comparative study of platypus and echidna by chromosome painting and comparative gene mapping.ResultsChromosome painting reveals a meiotic chain of nine sex chromosomes in the male echidna and establishes their order in the chain. Two of those differ from those in the platypus, three of the platypus sex chromosomes differ from those of the echidna and the order of several chromosomes is rearranged. Comparative gene mapping shows that, in addition to bird autosome regions, regions of bird Z chromosomes are homologous to regions in four platypus X chromosomes, that is, X1, X2, X3, X5, and in chromosome Y1.ConclusionMonotreme sex chromosomes are easiest to explain on the hypothesis that autosomes were added sequentially to the translocation chain, with the final additions after platypus and echidna divergence. Genome sequencing and contig anchoring show no homology yet between platypus and therian Xs; thus, monotremes have a unique XY sex chromosome system that shares some homology with the avian Z.

DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide.

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41 °C and 45 °C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24h), or acute (1h) exposure to each treatment followed by incubation at 37 °C over a period of 24h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.

Elevated dietary intake of Zn-methionate is associated with increased sperm DNA fragmentation in the boar

Boars fed on ration of 200 ppm Zn methionate showed a significant increase (P < 0.001) in sperm DNA fragmentation when compared to animals fed on non-supplemented and rations containing 150 ppm Zn methionate. There was a positive correlation (R2 = 0.207; P = 0.002) between % sperm DNA fragmentation (SDF) and the concentration of Zn in spermatozoa. Increased Zn in the diet also resulted in a non-proportional increase in Zn concentration in the testis and spermatozoa but not in the epididymis; Zn in sperm accumulated at levels up to 50 times higher than that found in the seminal plasma and 10-13 times that found in the epididymis and testis, respectively. These results show that supplementation of dietary Zn at a concentration of 200 ppm had an adverse effect on boar sperm DNA quality and may be related to the ability of spermatozoa to accumulate Zn during spermiogenesis.

Effects of cryopreservation on mitochondrial function and heterogeneity, lipid raft stability and phosphatidylserine translocation in koala (Phascolarctos cinereus) spermatozoa

Koala sperm mitochondria were examined by cryomicroscopy using the fluorescent probe JC-1, which distinguishes high (red) and low (green) mitochondrial membrane potential (MMP). At normal body temperature, approximately 70% of live and untreated spermatozoa exhibited high MMP whereas <3% of live untreated spermatozoa exhibited low potential. A third class, in which single midpieces contained mixed mitochondrial populations, was also detected. Heterogeneity was noted in the level of MMP between individual koalas, individual spermatozoa and even between mitochondrial gyres within single midpieces. MMP of the live sperm population was not significantly affected by glycerol but was suppressed by freezing and thawing treatments. After thawing, MMP declined significantly during rewarming, especially as the temperature increased from 5 to 35 degrees C. The distribution of the ganglioside GM1 was examined using fluorescent-labelled cholera toxin B. In fresh, untreated koala spermatozoa GM1 was detected on the head and midpiece, but not on the principal piece. No significant redistribution of GM1 was observed after chilling and cryotreatment. Phosphatidylserine translocation across the plasma membrane was examined using fluorescent-labelled annexin V. Few fresh spermatozoa exhibited phosphatidylserine translocation (approximately 1%); this was not increased by chilling or cryopreservation, thus implying that cryotreatment had little effect on plasma membrane lipid asymmetry.

The effect of environmental variables on the activity patterns of the southern hairy-nosed wombat (Lasiorhinus latifrons) in captivity: onset, duration and cessation of activity

The influence of scotoperiod, ambient temperature and humidity on the activity of captive wombats was examined to differentiate environmental influence from that pertaining to food availability. The activity of 12 wombats (Lasiorhinus latifrons) housed at Rockhampton Zoo was remotely monitored by digital video-surveillance for one year, with environmental variables simultaneously recorded via dataloggers. Mean proportion of total daily time spent active was 18.2 ± 1.8%. Mean daily time spent active varied seasonally according to changes in temperature and humidity. Mean daily activity was greater during winter (301 ± 17 min) and spring (295 ± 16 min), than summer (234 ± 16 min) and autumn (238 ± 15 min) with differences due to a decrease in the number and length of activity bouts in summer and autumn; presumably these results reflect a need to maintain heat balance associated with exposure to elevated temperatures. Activity was positively correlated with temperature during winter (r = 0.60) and spring (r = 0.52) but negatively correlated during summer (r = –0.58) and autumn (r = –0.49), suggesting the existence of a thermoneutral zone where activity is maximised. We conclude that activity in captive wombats is influenced by environmental variables.

Sperm chromatin in beef bulls in tropical environments

Sperm chromatin status was assessed in 565 Zebu and Zebu crossbred beef bulls in extensive tropical environments using the sperm chromatin structure assay (SCSA). The SCSA involved exposure of sperm to acid hydrolysis for 0.5 or 5.0 minutes, followed by flow cytometry to ascertain relative amounts of double-stranded (normal) and single-stranded (denatured) DNA, which was used to generate a DNA fragmentation index (%DFI). With conventional SCSA (0.5-minute SCSA), 513 bulls (91%) had <15 %DFI, 24 bulls (4%) had 15 to 27 %DFI, and 28 bulls (5%) had >27 %DFI. In 5.0-minute SCSA, 432 bulls (76%) had <15 %DFI, 68 bulls (12%) had 15 to 27 %DFI and 65 bulls (12%) had >27 %DFI. For most bulls, the SCSA was repeatable on two to four occasions; however, because most bulls had <15 %DFI, repeatability of the SCSA will need to be determined in a larger number of bulls in the 15 to 27 %DFI and >27 %DFI categories. The %DFI was negatively correlated with several bull semen parameters and the strongest negative correlation was with normal sperm. There was a strong positive correlation between %DFI and sperm head abnormalities. Based on these findings, most Zebu beef bulls in extensive tropical environments had relatively stable sperm chromatin. Based on the apparent negative correlations with conventional semen parameters, we inferred that the SCSA measured a unique feature of sperm quality, which has also been suggested for other species. Further studies on the relationships between sperm chromatin stability and fertility are required in beef bulls before chromatin status can be used as an additional predictor of the siring capacity of individual bulls in extensive multiple-sire herds.

Koala habitat use and population density: using field data to test the assumptions of ecological models

In principle, conservation planning relies on long-term data; in reality, conservation decisions are apt to be based upon limited data and short-range goals. For the koala (Phascolarctos cinereus), frequently reliance is made on the assumption that indirect signs can be used to indicate behavioural preferences, such as diet choice. We examined the relationship between the use of trees by koalas and the presence of scats beneath those trees. Tree use was associated with scat presence on 49% of occasions when koalas were radio-tracked in both central Queensland (n = 10 koalas) and south-east Queensland (n = 5 koalas), increasing to 77% of occasions when trees were rechecked the following day. Koala densities were correlated with scat abundance at sites with koala density between ~0.2 and 0.6 koalas per hectare. Our results confirm that scat searches are imprecise indicators of tree use by koalas, but demonstrate that these searches can be used, with caveats, to estimate koala population densities. We discuss how errors in estimating or applying predictive model parameters can bias estimates of occupancy and show how a failure to validate adequately the assumptions used in modelling and mapping can undermine the power of the products to direct rational conservation and management efforts.

Anesthesia of captive African wild dogs (Lycaon pictus) using a medetomidine-ketamine-atropine combination.

Abstract Seven captive male African wild dogs (Lycaon pictus) weighing 25–32 kg each, were anesthetized by i.m. injection via hand syringe with a combination of 1.5 mg/kg ketamine, 40 μg/kg medetomidine, and 0.05 mg/kg atropine. Following endotracheal intubation, each animal was connected to a bain closed-circuit system that delivered 1.5% isoflurane and 2 L/min oxygen. Atipamezole (0.1 mg/kg i.v.; 0.1 mg/kg i.m.) was given at the end of each procedure (60 min following injection of medetomidine/ketamine/atropine). Time to sternal recumbency was 5–8 min. Times to standing after atipamezole administration were 8–20 min. This anesthetic regimen was repeated on three separate occasions (September 2000, February 2002, and October 2002) on all males to perform electroejaculation procedures. Each procedure was <80 min from injection to standing. Dogs showed excellent muscle relaxation during the procedures. Arterial blood samples were collected at 10-min intervals for blood gases in one procedure (September 2000). Separate venous samples were taken from each dog during each procedure for hematology and biochemistry. These values were within the normal range for this species. Arterial hemoglobin oxygen saturation (SpO2) and heart rate (HR) were monitored continuously in addition to other anesthesia monitoring procedures (body temperature, respiratory rate [RR], capillary refill time, blink response, pupil position, deep pain perception reflex). All dogs maintained relatively stable SpO2 profiles during monitoring, with a mean (± SD) SpO2 of 92% ± 5.4%. All other physiological variables (HR, RR, body temperature, blood pressure) were within normal limits. Following each procedure, normal behavior was noted in all dogs. All the dogs were reunited into the pack at completion of their anesthetic procedures. An injectable medetomidine–ketamine–atropine combination with maintenance by gaseous isoflurane and oxygen provides an inexpensive, reliable anesthetic for captive African wild dogs.