The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas.

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (± SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.

Seasonal reproduction in wild and captive male koala (Phascolarctos cinereus) populations in south-east Queensland.

The effects of breeding season (late spring to early autumn) on south-east Queensland male koala fertility were examined to improve the efficacy of the AI procedure and to determine the practicality of using free-range animals as semen donors for a genome resource bank. Seasonal changes in male koala reproductive function were assessed in a wild free-range population (n = 14; obtained every 6 weeks from January to November 2005), a necropsied healthy wild population (n = 84; obtained monthly from September 2004 to August 2005) and a captive population (n = 7; obtained monthly from October 2005 to October 2006). Reproductive parameters investigated included bodyweight, coat score, sternal gland area and activity, testosterone secretion, reproductive anatomy volume and semen quality (before and after cryopreservation). Collectively, these findings show that reproduction in male koalas from south-east Queensland changes seasonally and that winter appears to be the optimal season in which to collect semen samples by electroejaculation. While it was possible to repeatedly collect semen from free-range koalas for future genetic management via potential storage in a genome resource bank, the survival of these spermatozoa after cryopreservation was poor and will require further improvement.

Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage.

In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with 'true' DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

Individual variability in post-thaw sperm survival in a captive koala population.

This study documented the extent of individual animal variation with respect to two proven methods of sperm cryopreservation in a captive population of 22 koalas. Semen samples were collected by electroejaculation, diluted in Tris-citrate glucose and equilibrated to 4 degrees C before being further diluted and frozen in media containing a final concentration of either 14% (v/v) glycerol or 12.5% (v/v) dimethylacetamide (DMA). There were significant differences in post-thaw survival of spermatozoa from different animals that were independent of pre-freeze semen quality. Glycerol proved to be a better cryoprotectant than DMA in terms of maintaining motility, plasma membrane integrity and high mitochondrial membrane potential; however, there was no difference between cryoprotectants with regards to their ability to prevent chromatin relaxation. While a positive correlation was observed between motility and mitochondrial membrane potential, both before and after cryopreservation, the slopes of the pre-freeze regression lines of these relationships were significantly altered following cryopreservation, suggesting that the efficiency of energy generation by the mitochondria was lowered by the freeze-thaw process. Based on a cluster analysis of the post-thaw semen viability parameters, the koalas in this study could be divided into two distinct groups; Cluster 1 had significantly higher sperm viability compared to the other cluster, regardless of the cryoprotectant used. The unpredictability of assessing post-thaw survival from pre-freeze koala semen parameters requires further investigation but is likely to be associated with variation in ejaculate composition or inherent genetic differences between animals.

The effect of cryoprotectant on kangaroo sperm ultrastructure and mitochondrial function.

This study examined the effect of cryoprotectants (20% DMSO, a 10% DMSO/10% glycerol mixture, 20% glycerol and 1M sucrose solution) on kangaroo sperm structure and function, along with the effect of varying concentrations of glycerol on sperm mitochondrial function. Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 min at 35 degrees C in each cryoprotectant and the plasma membrane integrity (PMI) and motility assessed using light microscopy. The same samples were fixed for TEM and the ultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerol on the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubated with JC-1 in Tris-citrate media at 35 degrees C for 20 min in a range of glycerol concentrations (0%, 5%, 10%, 15% and 20%) and the mitochondrial membrane potential (MMP) and plasma membrane integrity determined. As expected, incubation of spermatozoa in 20% glycerol for 10 min resulted in a significant reduction in motility, PMI and ultrastructural integrity. Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility or PMI but a significant loss of structural integrity when compared to the control spermatozoa (0% cryoprotectant). However, 20% DMSO was overall less damaging to sperm ultrastructure than glycerol, a combination of 10% glycerol and 10% DMSO, and sucrose. While all glycerol concentrations had an adverse effect on mitochondrial function, the statistical models presented for the relationship between MMP and glycerol predicted that spermatozoa, when added to 20% glycerol, would lose half of their initial MMP immediately at 35 degrees C and MMP would halve after 19.4 min at 4 degrees C. Models for the relationship between PMI and glycerol predicted that spermatozoa would lose half of their initial PMI after 1.8 min at 35 degrees C and PMI would halve after 21.1 min at 4 degrees C. These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermatozoa then it is best administered at 4 degrees C and that mitochondrial function is more sensitive to glycerol than PMI. Future research should be directed at investigating strategies that reduce exposure of spermatozoa to glycerol during processing and that test the cryoprotective properties of 20% DMSO for kangaroo spermatozoa.

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol.

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA-10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7+/-1.9% and 22.7+/-5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10-20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.

Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa.

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

Control of the koala (Phascolarctos cinereus) anterior pituitary-gonadal axis with analogues of GnRH.

The aim of the present study was to determine whether analogues of gonadotrophin-releasing hormone (GnRH) could be used to both induce an acute testosterone response and suppress anterior pituitary function in male koalas, and induce a luteal phase in female koalas. Experiment 1 characterised the steroidogenic response of male koalas to administration of 30 microg (4.3 microg kg(-1)) natural-sequence GnRH. Intra-muscular injection of natural-sequence GnRH induced the release of LH and testosterone with peak concentrations at 30 min (3.7 +/- 1.9 ng mL(-1)) and 2 h (5.4 +/- 0.5 ng mL(-1)), respectively. In Experiment 2, a single injection of the GnRH antagonist acyline (100 microg (14.3 microg kg(-1)) or 500 microg (71.4 microg kg(-1))) did not influence the testosterone response to subsequent injections of natural-sequence GnRH. In Experiment 3, 4 microg (~0.67 microg kg(-1)) of the GnRH agonist buserelin induced a luteal phase in five female koalas based on a LH surge, secretion of progestogen, and a normal-length oestrous cycle. The findings have shown that (1) natural-sequence GnRH can be used to test gonadotroph cell function and determine the testosterone-secreting capacity of male koalas, (2) the GnRH antagonist, acyline, at the dose rates used, does not suppress the pituitary-testis axis in male koalas, and (3) the GnRH agonist, buserelin, induces a normal luteal phase in female koalas.

Successful artificial insemination in the koala (Phascolarctos cinereus) using extended and extended-chilled semen collected by electroejaculation.

Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, and makes available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for artificial insemination is complicated because koalas are induced ovulators, and it is thought that ovulating factors are present in the semen, so that semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of this study were designed to determine whether artificial insemination using undiluted, extended, and extended-chilled semen collected by electroejaculation was capable of inducing a luteal phase and/or the production of pouch young. In Experiment 1, 1 ml undiluted electroejaculated semen, 2 ml diluted (1:1) semen, and 1 ml diluted (1:1) semen resulted in seven of nine, six of nine, and six of nine koalas showing a luteal phase, respectively; four pouch young were produced in each treatment. A second artificial insemination experiment was conducted in which 2 ml diluted (1:1) semen was administered in three groups of nine koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, and the final group received semen that had been chilled for 72 h. In the first group, five females had a luteal phase, but none became pregnant. In group 2, two of the five females that had a luteal phase gave birth, whereas in group 3, four of the six females that had a luteal phase produced pouch young. In addition, experiment 3 was conducted to determine whether it was possible to produce pouch young by naturally mating koalas that were in the latter stages of their behavioral estrus; this information is important to the logistics of transporting koala semen for artificial insemination by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on Day 8 of estrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on Day 10 of estrus produced pouch young. The majority of females (21 of 22) in experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended, or extended-chilled semen to produce koala offspring up to Day 8 of estrus at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The pouch young produced in this study represent the first marsupials born following artificial insemination of extended-chilled semen and bring the total number of koalas produced by artificial insemination to 31.

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.