Associate Professor John Clulow

For oviparous animals such as amphibians, the presence or absence of conspecifics can influence site selection, with each life history stage potentially influencing the decision-making process in a different manner. In the present study, we tested the effect of conspecific life history stage on oviposition site selection in the sandpaper frog, Lechriodus fletcheri. We quantified preferences for spawning in pools that were unused by conspecifics, as well as pools with conspecific spawn or tadpoles present, or both. The study included two ecological contexts: a system of naturally occurring breeding pools and a field experiment with 40 artificial pools that controlled for habitat variation. Mothers preferred to oviposit in pools that already contained conspecific spawn, despite the likelihood of intense resource competition upon offspring hatching. This could be due to the potential benefits obtained by providing offspring access to a significant nutrient supply upon hatching via cannibalism of conspecific tadpoles and might be an important adaptation for completing tadpole development in highly ephemeral and resource-limited pools prior to desiccation. In contrast, mothers avoided ovipositing in pools with conspecific tadpoles, a probable adaptation to avoid their own offspring from becoming victims of cannibalism themselves prior to hatching. Such nuances in the effect of conspecific presence on offspring survival highlight the often complex decision-making process that amphibians need to make when selecting oviposition sites, as well as the influence cannibalism can have on the evolution of reproductive behaviour.

Amphibians are under threat from many drivers resulting in declining populations. Restoration and creation of habitat is a method used to reverse amphibian declines. The green and golden bell frog (Litoria aurea) is distributed in southeastern Australia, and is threatened by the fungal pathogen Batrachochytrium dendobatidis (chytrid), an introduced fish (the plague minnow, Gambusia holbrooki), and habitat loss. There have been numerous wetland restoration attempts to combat population declines in this species, which have been largely unsuccessful in producing persisting populations. Here we present a robust model for the creation of breeding habitat for the L. aurea population on Kooragang Island, New South Wales, which is based off thorough review of the literature and past pilot studies and experiments. We describe in detail the habitat, land use history, and wetland habitat design formulation and construction so that the context of the habitat creation is understood and so construction can be repeatable and the design can be further refined. The habitat features passive controls for chytrid and G. holbrooki, and contains the most optimum breeding habitat for L. aurea based upon current knowledge. This is the first attempt in our knowledge to create wetlands in an open system that have the potential to passively manage chytrid.

The ¿Compassionate Conservation¿ movement is gaining momentum through its promotion of ¿ethical¿ conservation practices based on self-proclaimed principles of ¿first-do-no-harm¿ and ¿individuals matter¿. We argue that the tenets of ¿Compassionate Conservation¿ are ideological - that is, they are not scientifically proven to improve conservation outcomes, yet are critical of the current methods that do. In this paper we envision a future with ¿Compassionate Conservation¿ and predict how this might affect global biodiversity conservation. Taken literally, ¿Compassionate Conservation¿ will deny current conservation practices such as captive breeding, introduced species control, biocontrol, conservation fencing, translocation, contraception, disease control and genetic introgression. Five mainstream conservation practices are used to illustrate the far-reaching and dire consequences for global biodiversity if governed by ¿Compassionate Conservation¿. We acknowledge the important role of animal welfare science in conservation practices but argue that ¿Compassionate Conservation¿ aligns more closely with animal liberation principles protecting individuals over populations. Ultimately we fear that a world of ¿Compassionate Conservation¿ could stymie the global conservation efforts required to meet international biodiversity targets derived from evidenced based practice, such as the Aichi targets developed by the Convention on Biological Diversity and adopted by the International Union for the Conservation of Nature and the United Nations.

Compassionate conservation focuses on 4 tenets: first, do no harm; individuals matter; inclusivity of individual animals; and peaceful coexistence between humans and animals. Recently, compassionate conservation has been promoted as an alternative to conventional conservation philosophy. We believe examples presented by compassionate conservationists are deliberately or arbitrarily chosen to focus on mammals; inherently not compassionate; and offer ineffective conservation solutions. Compassionate conservation arbitrarily focuses on charismatic species, notably large predators and megaherbivores. The philosophy is not compassionate when it leaves invasive predators in the environment to cause harm to vastly more individuals of native species or uses the fear of harm by apex predators to terrorize mesopredators. Hindering the control of exotic species (megafauna, predators) in situ will not improve the conservation condition of the majority of biodiversity. The positions taken by so-called compassionate conservationists on particular species and on conservation actions could be extended to hinder other forms of conservation, including translocations, conservation fencing, and fertility control. Animal welfare is incredibly important to conservation, but ironically compassionate conservation does not offer the best welfare outcomes to animals and is often ineffective in achieving conservation goals. Consequently, compassionate conservation may threaten public and governmental support for conservation because of the limited understanding of conservation problems by the general public.

Invasive fish threaten many native freshwater fauna. However, it can be difficult to determine how invasive fish impact animals with complex life cycles as interaction may be driven by either predation of aquatic larvae or avoidance of fish-occupied waterbodies by the terrestrial adult stage. Mosquitofish (Gambusia spp.) are highly successful and aggressive invaders that negatively impact numerous aquatic fauna. One species potentially threatened by Gambusia holbrooki is the green and golden bell frog (Litoria aurea). However, G.¿holbrooki's role in this frog's decline was unclear due to declines driven by the chytrid fungal disease and the continued co-existence of these fish and frogs in multiple locations. To clarify the extent to which Gambusia is impacting L.¿aurea, we conducted 3¿years of field surveys across a deltaic wetland system in south-east Australia. We measured the presence and abundance of aquatic taxa including G.¿holbrooki, and L.¿aurea frogs and tadpoles, along with habitat parameters at the landscape and microhabitat scale. Generalized linear models were used to explore patterns in the abundance and distributions of L.¿aurea and G.¿holbrooki. We¿found strong negative associations between G.¿holbrooki and tadpoles of most species, including L.¿aurea, but no apparent avoidance of G.¿holbrooki by adult frogs. Native invertebrate predators (Odonata and Coleoptera) were also absent from G.¿holbrooki-occupied ponds. Due to the apparent naivety of adult frogs toward G.¿holbrooki, the separation of G.¿holbrooki and tadpoles, plus the abundance of alternative predators in G.¿holbrooki-free ponds, we conclude that the impact of G.¿holbrooki on L.¿aurea recruitment is likely substantial and warrants management action.

Ontogenetic changes in disease susceptibility have been demonstrated in many vertebrate taxa, as immature immune systems and limited prior exposure to pathogens can place less developed juveniles at a greater disease risk. By causing the disease chytridiomycosis, Batrachochytrium dendrobatidis (Bd) infection has led to the decline of many amphibian species. Despite increasing knowledge on how Bd varies in its effects among species, little is known on the interaction between susceptibility and development within host species. We compared the ontogenetic susceptibility of post-metamorphic green and golden bell frogs Litoria aurea to chytridiomycosis by simultaneously measuring three host-pathogen responses as indicators of the development of the fungus¿infection load, survival rate, and host immunocompetence¿following Bd exposure in three life stages (recently metamorphosed juveniles, subadults, adults) over 95¿days. Frogs exposed to Bd as recently metamorphosed juveniles acquired higher infection loads and experienced lower immune function and lower survivorship than subadults and adults, indicating an ontogenetic decline in chytridiomycosis susceptibility. By corresponding with an intrinsic developmental maturation in immunocompetence seen in uninfected frogs, we suggest these developmental changes in host susceptibility in L. aurea may be immune mediated. Consequently, the physiological relationship between ontogeny and immunity may affect host population structure and demography through variation in life stage survival, and understanding this can shape management targets for effective amphibian conservation.

Conspecific attraction plays an important role in habitat selection of several taxa and can affect and determine distribution patterns of populations. The behaviour is largely studied and widespread among birds, but in amphibians, its occurrence seems limited to breeding habitats of adults and gregarious tadpoles. The Australian green and golden bell frogs (Litoria aurea) have suffered considerable shrinking of their original distribution in south-eastern Australia since the 1970s. Currently, with only about 40 populations remaining, the species is considered nationally threatened. In natural conditions, these frogs are aggregated in the landscape and do not seem to occupy all suitable ponds within the occurrence area. To date, studies focusing on the frogs¿ habitat have failed in finding a general habitat feature that explains current or past occupancy. This led us to the hypothesis that social cues may play a key role in habitat selection in this species. Using two choice experiments, we tested the preference of juvenile green and golden bell frogs for habitats containing cues of conspecifics of similar size versus habitats without conspecific cues. Tested frogs did not show a preference for habitats containing only scent from conspecifics but did prefer habitats where conspecifics were present. Our results show that conspecific attraction is a determining factor in juvenile green and golden bell frog habitat selection. To our knowledge, this is the first time the behaviour is shown to occur in juvenile frogs in the habitat selection context. From a conservation management point of view, the behaviour may help to explain the failure of reintroductions to areas where the frogs have been extinct, and the non-occupation of suitable created habitats in areas where they still inhabit and develop appropriated management strategies.

Although amphibians are one of the most threatened animal groups, little published evidence exists on effective management programs. In order for conservation initiatives to be successful, an understanding of habitat use patterns is required to identify important environmental features. However, habitat use may differ between the different sexes and age classes due to different behavioural and resource requirements. For this study, we compared microhabitat use during the active breeding season among the sexes and age classes in the endangered green and golden bell frog Litoria aurea, a species which has had several failed management programs. We found aquatic vegetation was selected for by every L. aurea class, and should be the focus of future management plans for this species. Females were the only class to select for terrestrial vegetation more than availability. Increasing the amount of terrestrial vegetation around ponds may help encourage female occupancy, and possibly improve management outcomes, as they are typically a limiting resource. Although large rock piles have been used in past L. aurea habitat management, they were selected for by adults and juveniles, but not metamorphs. Therefore, large rocks may not be necessary for captive breeding portions of management initiatives, which typically only involve tadpoles and metamorphs prior to release. The results indicate that the most appropriate management plans should contain a habitat mosaic of various microhabitats, such as a large proportion of aquatic and terrestrial vegetation with patches of bare ground and a small proportion of rocks for basking and shelter. Recognizing differences in microhabitat use patterns between individuals in a population and implementing them into management strategies should be a pivotal step in any conservation plan.

A bias in conservation research has meant that population viability analysis has focused primarily on mammals and birds with slow life histories. The global amphibian decline has demonstrated the capacity for fast life-history species to experience decline. However, little is known about the viability of remnant populations of these species as patterns of decline cannot be inferred from other species with different life-history strategies. Population viability analysis was performed on the threatened frog, Litoria aurea, which exhibits high temporal variability in population size due to its fast life-history traits. Projections of population size from the viability model were highly variable, and removing parametric uncertainty only slightly improved overall model certainty, thus demonstrating the limits of population viability analysis for predicting abundance in fast life-history species. Sensitivity analysis identified recruitment of adults, female survival, male survival and rate of maturity as having the most impact on population viability. This population viability model provides a starting point to incorporate future research findings and better elucidate the causes of local extinction in this species. This study also reinforces the importance of egg-juvenile survival for amphibian populations, but also exemplifies the variability of amphibian viability analyses for identifying important parameters. As a case study for amphibian conservation, this analysis shows the utility of population viability analyses for fast life-history species, even with incomplete knowledge of all life-history stages.

Biodiversity loss caused by invasive species is particularly problematic in freshwater ecosystems, which are among the world's most threatened habitats. Invasive fish such as the eastern mosquitofish, Gambusia holbrooki, have been implicated in the decline of amphibians, which suffer high extinction rates globally. Although G. holbrooki is one of the most studied freshwater fish, its diet shows wide geographic variation and its impact on amphibian populations remains unclear. Stable isotopes C and N were used in 10 urban ponds in Sydney, Australia to compare the diet of G. holbrooki in January, April and May 2013 using a stable isotope mixing model. Gambusia holbrooki was carnivorous and fed on invertebrates (24¿39%), tadpoles (25¿32%) and conspecifics (20¿45%). In contrast to previous studies, primary producers were a negligible part of Gambusia holbrooki diet (<10%). Its diet in late autumn comprised a high proportion of conspecifics (up to 45%) owing to the depletion of other food sources before winter (metamorphosis of larvae). This study provides evidence of high rates of predation on native tadpoles and invertebrates by a highly invasive fish. This knowledge should be incorporated into amphibian releases through head-starting tadpoles or using soft releases where tadpoles are placed in predator-free enclosures until larvae are large enough to avoid predation. Considering the dire conservation status of amphibians globally and the growing interest for invertebrates, it is suggested that stable isotopes are valuable to identify threats from predation in order to target conservation practice toward suitable priorities. Copyright © 2016 John Wiley & Sons, Ltd. 13 15

Social aggregations are widespread among animal groups. They are relatively common in amphibian larvae, likely conferring protection against predators, advantages for microhabitat selection, foraging efficiency, and thermoregulatory efficiency. Group formation involves selection of individuals to group with by the other members, and several tadpoles are reported to recognise and prefer to aggregate with siblings or familiar individuals. In Australia, tadpoles of the endangered green and golden bell frog, Litoria aurea, are attracted to conspecifics and form schools. We conducted two choice experiments for captive breed tadpoles of this species to test their grouping preferences. Tadpoles preferred to aggregate with conspecifics to heterospecifics of a sympatric species; however, when conspecifics were absent they preferred to aggregate with the heterospecifcs than to remain alone. Tadpoles also preferred unfamiliar kin to unfamiliar non-kin conspecifics, but had no preferences between unfamiliar and familiar siblings. Once widespread in southeast Australia, the green and golden bell frog has suffered considerable declines and local extinctions in recent decades. Susceptibility to chytridiomycosis is likely the major threat for most remaining fragmented populations and the major challenge for reintroduction programs. The strong gregarious behaviour of this species may affect disease dynamics, especially chytridiomicosis that continues to threaten remaining wild populations.

The chytrid fungus Batrachochytrium dendrobatidis has been implicated in the decline and extinction of amphibian populations worldwide, but management options are limited. Recent studies show that sodium chloride (NaCl) has fungicidal properties that reduce the mortality rates of infected hosts in captivity. We investigated whether similar results can be obtained by adding salt to water bodies in the field. We increased the salinity of 8 water bodies to 2 or 4 ppt and left an additional 4 water bodies with close to 0 ppt and monitored salinity for 18 months. Captively bred tadpoles of green and golden bell frog (Litoria aurea) were released into each water body and their development, levels of B. dendrobatidis infection, and survival were monitored at 1, 4, and 12 months. The effect of salt on the abundance of nontarget organisms was also investigated in before and after style analyses. Salinities remained constant over time with little intervention. Hosts in water bodies with 4 ppt salt had a significantly lower prevalence of chytrid infection and higher survival, following metamorphosis, than hosts in 0 ppt salt. Tadpoles in the 4 ppt group were smaller in length after 1 month in the release site than those in the 0 and 2 ppt groups, but after metamorphosis body size in all water bodies was similar . In water bodies with 4 ppt salt, the abundance of dwarf tree frogs (Litoria fallax), dragonfly larvae, and damselfly larvae was lower than in water bodies with 0 and 2 ppt salt, which could have knock-on effects for community structure. Based on our results, salt may be an effective field-based B. dendrobatidis mitigation tool for lentic amphibians that could contribute to the conservation of numerous susceptible species. However, as in all conservation efforts, these benefits need to be weighed against negative effects on both target and nontarget organisms.

Surveillance of pathogens can lead to significant advances towards making effective decisions in research and management for species threatened by disease. Batrachochytrium dendrobatidis has been a major contributing factor to the global decline of amphibians. Knowledge of the distribution of B. dendrobatidis can contribute to understanding patterns of species decline and prioritizing action. Therefore, we surveyed four spatially distinct populations of a B. dendrobatidis susceptible species, the green and golden bell frog (Litoria aurea), for evidence of infection in the population. Three mainland populations were infected at a prevalence of 3.5¿28.3 %, with median infection loads of 0.28¿627.18 genomic equivalents (GE). Conversely, we did not detect infection in an island population 3 km from the mainland; the isolation and infrequent visitation of the island suggests that the pathogen has not arrived. Management actions for B. dendrobatidis and conservation of susceptible frog species are heavily dependent on the presence and absence of the pathogen in the population. Prevention of the accidental introduction of B. dendrobatidis and safe guarding genetic diversity of L. aurea is necessary to preserve unique diversity of the island population, whereas containment and control of the pathogen can be directed towards mainland populations. Knowledge of disease dynamics also provides a context to understand the ecology of remaining populations as variation in the physiology or habitat of the mainland populations have facilitated persistence of these populations alongside B. dendrobatidis. Other islands should be a priority target in disease surveillance, to discover refuges that can assist conservation.

With the incidence of emerging infectious diseases on the rise, it is becoming increasingly important to identify refuge areas that protect hosts from pathogens and therefore prevent population declines. For the chytrid fungus Batrachochytrium dendrobatidis, temperature and humidity refuge areas for amphibian hosts exist but are difficult to manipulate. Other environmental features that may affect the outcome of infection include water quality, drying regimes, abundance of alternate hosts and isolation from other hosts. We identified relationships between water bodies with these features and infection levels in the free-living hosts inhabiting them. Where significant relationships were identified, we used a series of controlled experiments to test for causation. Infection loads were negatively correlated with the salt concentration of the aquatic habitat and the degree of water level fluctuation and positively correlated with fish abundance. However, only the relationship with salt was confirmed experimentally. Free-living hosts inhabiting water bodies with mean salinities of up to 3.5 ppt had lower infection loads than those exposed to less salt. The experiment confirmed that exposure to sodium chloride concentrations >2 ppt significantly reduced host infection loads compared to no exposure (0 ppt). These results suggest that the exposure of amphibians to salt concentrations found naturally in lentic habitats may be responsible for the persistence of some susceptible species in the presence of B. dendrobatidis. By manipulating the salinity of water bodies, it may be possible to create refuges for declining amphibians, thus allowing them to be reintroduced to their former ranges.

Although human-modified habitats often result in a loss of biodiversity, some have been found to serve as habitat refuges for threatened species. Given the globally declining status of amphibians, understanding why some species are found in heavily modified environments is of considerable interest. We used the endangered green and golden bell frog (. Litoria aurea) as a model to investigate the factors influencing their distribution toward industrial areas within a landscape. The number of permanent waterbodies within a kilometer of surveyed sites was the best predictor of L. aurea occupancy, abundance and reproduction. It appears that industrial activities, such as dredging and waste disposal inadvertently created refuge habitat for L. aurea to fortuitously persist in a heavily modified landscape. Future conservation plans should mimic the positive effects of industrialization, such as increasing the number of permanent waterbodies, especially in areas containing ephemeral or isolated waterbodies and threatened with drought. Our findings also suggest that despite amphibians being relatively small animals, some species may require a larger landscape than anticipated. Recognizing life history traits, in combination with a landscape-based approach toward species with perceived limited motility, may result in more successful conservation outcomes. Identifying why threatened species persist in heavily disturbed landscapes, such as industrial sites, can provide direction toward future conservation efforts to prevent and reverse their decline.

Conspecific attraction can prevent occupancy of restored or created habitats by limiting dispersal to unoccupied areas. This may cause problems for threatened taxa where habitat restoration and creation programmes are implemented as part of species recovery plans. Studies on birds have found that the introduction of artificial communication cues such as calling can increase occupancy of restored habitat. The endangered green and golden bell frog (Litoria aurea) has a number of behavioural traits which suggest conspecific attraction occurs via a vocal mechanism, including a loud conspicuous call and large chorusing aggregations. To date, attempts to repopulate restored and created habitat through natural immigration and active translocation of tadpoles and juveniles have been met with limited success for this species. We used L. aurea to determine if distribution could be manipulated via conspecific attraction using artificial communication cues. We placed speaker systems in uninhabited areas of five inhabited ponds across two locations and broadcast calls of L. aurea to see if we could manipulate distribution into previously unoccupied pond areas. Surveys undertaken before and after broadcast indicate that we successfully manipulated L. aurea distribution for adults increasing both occupancy and calling around the speaker locations. This occurred in four of five replicate ponds over three months of experimental treatment, but controls remained low in abundance. We suggest that manipulation of distribution via conspecific attraction mechanisms could be a useful conservation tool for endangered amphibian habitat restoration and creation programmes, resulting in increased occupancy and programme success.

Captive bred animals often lack the ability of predator recognition and predation is one of the strongest causes of failure of breed and release projects. Several tadpole and fish species respond defensively to chemical cues from injured or dead conspecifics, often referred to as alarm pheromones. In natural conditions and in species that school, the association of chemical cues from predators to alarm pheromones released by attacked conspecifics may lead to the learning of the predator-related danger without experiencing an attack. In the laboratory, this chemical communication can also be used in associative learning techniques to teach naïve tadpoles to avoid specific predators and improve survivorship of released animals. In our experimental trials, tadpoles of the threatened green and golden bell frog (Litoria aurea) did not avoid or decrease their activity when exposed to solutions of conspecific macerate, suggesting that the chemicals released into the water by dead/injured conspecifics do not function as an alarm pheromone. This non-avoidance of dead conspecific chemicals may explain why green and golden bell frog tadpoles have seemingly not developed any avoidance behaviour to the presence of introduced mosquito fish, and may render attempts to teach naïve tadpoles to avoid this novel predator more difficult. © 2014 Springer Basel.

Sustained demographic studies are essential for early detection of species decline in time for effective management response. A paucity of such background data hindered the potential for successful conservation during the global amphibian decline and remains problematic today. The current study analysed 6 years of mark-recapture data to determine the vital demographic rates in three habitat precincts of the threatened frog, Litoria aurea (Hylidae) and to understand the underlying causes of variability in population size. Variability in population size of L.aurea was similar to many pond-breeding species; however this level of fluctuation is rare among threatened amphibians. Highly variable populations are at greater risk of local extinction and the low level of connectivity between L.aurea populations means they are at a greater risk of further decline due to stochastic extinction events and incapacity to recolonize distant habitat. We recommend that management of this species should encourage recolonization through creation of habitat corridors and reintroduction of L.aurea to areas where stochastic extinction events are suspected. © 2013 Ecological Society of Australia.

Suitability of habitat patches affects demographic processes and consequently influences the viability of populations. In order for managers to consider the potential of resources on a scale appropriate to their use, it is important to understand the processes that influence the ecology of threatened species. Differential growth rates of Litoria aurea (green and golden bell frog) at Sydney Olympic Park, Australia, may be explained by prey availability and diet. We tested: (1) whether food availability differed among precincts at Sydney Olympic Park and (2) whether the diet of L. aurea was influenced by availability of invertebrate prey. Diets were distinct among precincts and reflected the variation in biomass and richness of invertebrate assemblages. Precincts with greater biomass corresponded to areas with faster individual growth rates and greater habitat structure. The differences in diet and individual growth rates of L. aurea among precincts at Sydney Olympic Park demonstrate how caution must be applied to generalising population function, even within a small area.

Each amphibian species is evolutionarily distinct, having developed highly specialized and diverse reproductive strategies in both terrestrial and aquatic environments. These unique reproductive patterns and mechanisms, key to species propagation, have only been explored in a limited number of laboratory models. Although the development of applied reproductive technologies for amphibians has proven useful for a few threatened species, the real benefit of this technology has been new insights into the reproductive adaptations, behavior, endocrinology, and physiological mechanisms that have evolved over millions of years. As the basic fundamental database on amphibian reproductive physiology has grown, so has the applied benefit for species conservation. In particular, technologies such as non-invasive fecal and urinary hormone assays, hormone treatments for induced breeding or gamete collection, in vitro fertilization, and the ability to establish genome resource banks have all played important roles in monitoring or managing small populations of captive species. Amphibians have the ability to produce a large excess of germplasm (up to 10,000 ovulated eggs in a single reproductive event) that if not collected and preserved, would represent a wasted valuable resource. We discuss the current state of knowledge in assisted reproductive technologies for amphibians and why their extinction crisis means these available tools can no longer be implemented as small-scale, last-ditch efforts. The reproductive technologies must be established early as a key component of large-scale species recovery. © 2014 Springer Science+Business Media New York.

Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82 and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex-preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney's modified Tyrode's (KMT) and Sperm TALP (Sp-TALP) as the staining and incubation medium for stallion spermatozoa prior to sex-sorting. A significant increase in the percentage of acrosome-reacted spermatozoa occurred after staining and incubation in the clarified Sp-TALP compared with KMT. As no improvements in sorting rates were achieved using Sp-TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82 or lactose-EDTA could be employed as a cryodiluents. © 2011 Blackwell Verlag GmbH. ® ®

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozenthawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed 'attached' or 'bound' depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen-thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen-thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen-thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status. © 2010 CSIRO.

Intrinsic differences between stallions exist for semen traits such as motility, morphology fertility and the ability of spermatozoa to survive cryopreservation processes. Ejaculates from 11 stallions were used to test the differences between stallions when selecting X- and Y-chromosome bearing spermatozoa using a modified flow cytometer. Data on orientation and viability of spermatozoa were collected during sex-sorting, and motility characteristics of sex-sorted and non-sorted (control) spermatozoa were assessed before and after cryopreservation. An index was created to rank each stallion in order of their suitability for sex-sorting using the data generated by the flow cytometry software. Motility of spermatozoa was higher after sorting and cooling than in the fresh ejaculates, but was significantly lower after thawing in comparison to fresh semen for both sex-sorted and non-sorted spermatozoa. Semen samples with a high percentage of food dye positive, defined as dead, spermatozoa had a low sortability index and ranking. Thus, percentage of dead spermatozoa in the semen sample was identified as the most important factor determining sortability. We conclude that variation between stallions exists for the sortability of their spermatozoa and that the sortability index is a useful tool for the selection of suitable stallions for a sex-sorting program. Crown Copyright © 2008.

The effects of sperm freezing concentration (40 × 10 mL vs. 400 × 10 mL ), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam box vs. programmable freezing machine) were evaluated in a 2 × 2 × 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 × 10 mL was higher than for spermatozoa frozen at 400 × 10 mL . No significant differences were observed in the semen parameters assessed after cryopreservation in either 0.25 or 0.5 mL straws. However, the programmable freezer provided a more consistent and reliable freezing rate than liquid nitrogen vapour. We conclude that an effective protocol for the cryopreservation of stallion spermatozoa at low concentrations would include concentrations of 40 × 10 mL in 0.25 mL straws using a programmable freezer. This freezing protocol would be suitable for emerging sperm technologies such as sex-preselection of stallion spermatozoa as the sorting process yields only low numbers of spermatozoa in a small volume available for either immediate insemination or cryopreservation. © 2007 Elsevier B.V. All rights reserved. 6 -1 6 -1 ® 6 -1 6 -1 6 -1

In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197-201] was used to deposit low doses (6, 13 or 25 × 10 frozen-thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500 IU, Intervet). Fertility was low, with one pregnancy (13 × 10 spermatozoa, 500 µL) obtained after artificial insemination with frozen SS spermatozoa (n = 29 cycles) which resulted in the birth of a filly. Two pregnancies were obtained in mares inseminated with 6 × 10 NS spermatozoa in 250 µL (n = 31 cycles). Mares failing to conceive on two experimental cycles were allocated to the conventional insemination group. Insemination with >500 × 10 motile NS frozen-thawed spermatozoa, yielded satisfactory per cycle conception rates (35.5%, 22/62) for both stallions combined and was within the values of their normal fertility as quoted by the stud's records. This suggests that the quality of the frozen semen was acceptable and that the freezing processes yielded viable spermatozoa capable of fertilisation. The poor fertility after hysteroscopic insemination with low doses of sex-sorted or non-sorted spermatozoa from the same stallions may be directly attributable to the low dose insemination conditions with frozen-thawed rather than sex-sorted spermatozoa. © 2007 Elsevier B.V. All rights reserved. 6 6 6 6

Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. A completely randomised design was used. Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 × 10 sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 × 10 /mL. The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 × 10 /ml rather than 20 × 10 /ml. © 2007 Australian Veterinary Association. 6 6 6 6