John Clulow

Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: comparisons with the homologous metanephric proximal tubule

Luminal fluids were collected in vivo by micropuncture and cannulation from the rete testis, efferent ducts and ductus epididymidis of the rat to determine the composition of efferent duct fluids and the rates of reabsorption of water and solutes by the efferent ducts. The concentration of spermatozoa increased by a factor of about 25 from 2.42 x 10(4) microliters‐1 in the fluid from the rete testis to 6.00 x 10(5) microliters‐1 in fluid at the end of the efferent ducts, indicating that 96.2% of the fluid leaving the testis is reabsorbed from the lumen of the efferent ducts. Most of this reabsorption (70.9% or 33.4 microliters h‐1) occurs in the region between the rete testis and the middle of the coni vasculosi, with only 25.1% (11.8 microliters h‐1) occurring between the coni and the beginning of the ductus epididymidis. However, reabsorption across the epithelium occurs at about the same rate in both regions, with the proximal region reabsorbing 17.2 microliters cm‐2 h‐1 (70.9% of fluid entering the region) and the distal region reabsorbing 12.2 microliters cm‐2 h‐1 (86.1% of fluid entering the region). Consequently, the fluid reabsorption rate for the whole efferent duct system (15.6 microliters cm‐2 h‐1) is similar to the values for individual regions. The principal solutes in luminal fluids from the efferent ducts are Na+ (137‐144 mM) and Cl‐ (113‐130 mM). The estimated sum contribution of Na+, Cl‐ and K+ to the osmotic pressure of luminal fluids was approximately 80% at each site sampled in the efferent ducts. The osmotic pressure of luminal fluid samples (301‐307 mosmol kg‐1) did not vary significantly along the ducts or differ significantly from that of blood plasma. The results demonstrate that there is a net reabsorption in the efferent ducts of nearly all the testicular output of water and inorganic electrolytes, and most of the protein, and that, in comparison, the ductus epididymidis is a negligible site of net fluid reabsorption. The results indicate that the ductus epididymidis, rather than the efferent ducts, is the site of accumulation of high concentrations of specific organic compounds like inositol. The efferent ducts are similar to the homologous proximal tubules of the metanephric kidney in that the luminal electrolyte composition (principal solutes Na+ and Cl‐) and osmotic pressure remain relatively stable and that fluid reabsorption is close to isotonic and occurs at the same rate as the reabsorption of Na+.

The role of Na+-H+ exchange in fluid and solute transport in the rat efferent ducts.

In vivo microperfusion techniques were used to investigate the role of Na+‐H+ exchange in the efferent ducts of the rat. Individual efferent ducts were perfused with a Krebs‐Ringer bicarbonate solution (KRB) containing 0, 1, 3, 5 or 7·5 mM amiloride. Concentrations of 1‐5 mM amiloride inhibited fluid reabsorption from the efferent ducts in a linear dose‐dependent manner with an apparent Km of 3 mM. Inhibition was maximal at 5 mM with reabsorption reduced by about 70%. The effects of amiloride were completely reversible and there was little effect of amiloride on luminal osmolality and concentrations of Na+, Cl− or K+. It is concluded that Na+‐H+ exchange is one of the principal mechanisms responsible for fluid and electrolyte reabsorption in the efferent ducts and offers a means by which the efferent ducts are able to achieve flow‐dependent, autoregulated fluid reabsorption.

In vivo microperfusion of the ductuli efferentes testis of the rat: flow dependence of fluid reabsorption

Individual ducts from the initial zone of the efferent ducts of the rat were microperfused in vivo using a double cannulation procedure, which allowed the recovery of perfused fluids for analysis and determination of the rate of fluid reabsorption from the perfused duct. The ducts were perfused at rates from 0.025 to 0.4 microliters min‐1 with either Krebs‐Ringer bicarbonate (KRB) solution or the native rete testis fluid (nRTF) that perfuses the ducts in situ. Reabsorption of KRB solution increased linearly with a perfusion rate of between 0.025 and 0.1 microliter min‐1 (from 17.4 +/− 1.5 to 34.3 +/− 3.2 nl (10 mm duct)‐1 min‐1), then increased no further. Reabsorption of nRTF increased linearly between 0.025 and 0.2 microliters min‐1 (from 17.7 +/− 1.5 to 61.4 +/− 13.5 nl (10 mm duct)‐1 min‐1) and then declined. The reabsorption rate from nRTF perfusates was significantly higher than from KRB perfusates. As a proportion of the luminal perfusate, reabsorption declined from 73.0 +/− 6.0 to 7.4 +/− 3.0% (10 mm duct)‐1 for KRB solution and from 73.1 +/− 6.0 to 4.1 +/− 1.3% (10 mm duct)‐1 for nRTF. There was no significant change in the concentration of either Na+ or Cl‐ in KRB solution or nRTF during perfusion through the efferent ducts, indicating that the reabsorption of these ions was isomolar. However, the reabsorption of K+ from nRTF occurred at a greater rate than that of water, and the initial [K+] declined from 17.2 +/− 0.4 mM in nRTF perfusates to 5.7 +/− 0.5 mM in collectates (perfusion rate, 0.1 microliter min‐1) to achieve equilibrium with blood plasma (4.7 +/− 0.4 mM). The osmotic pressure of both KRB and nRTF perfusates equilibrated with blood plasma, indicating a high permeability of the epithelium to water. The results of this study provide further evidence that fluid reabsorption in the efferent ducts is isosmotic, or close to isosmotic, and have shown that, as in the homologous proximal kidney tubule, reabsorption is dependent on luminal flow rate. In contrast to the proximal tubule, however, reabsorption in the efferent ducts is not maintained as a constant proportion of the perfusion load. It is concluded that microperfusion in vivo provides a useful technique for studying fluid reabsorption in the efferent ducts of the rat.

Sodium Chloride Inhibits the Growth and Infective Capacity of the Amphibian Chytrid Fungus and Increases Host Survival Rates

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently emerged pathogen that causes the infectious disease chytridiomycosis and has been implicated as a contributing factor in the global amphibian decline. Since its discovery, research has been focused on developing various methods of mitigating the impact of chytridiomycosis on amphibian hosts but little attention has been given to the role of antifungal agents that could be added to the hosts environment. Sodium chloride is a known antifungal agent used routinely in the aquaculture industry and this study investigates its potential for use as a disease management tool in amphibian conservation. The effect of 0–5 ppt NaCl on the growth, motility and survival of the chytrid fungus when grown in culture media and its effect on the growth, infection load and survivorship of infected Perons tree frogs (Litoria peronii) in captivity, was investigated. The results reveal that these concentrations do not negatively affect the survival of the host or the pathogen. However, concentrations greater than 3 ppt significantly reduced the growth and motility of the chytrid fungus compared to 0 ppt. Concentrations of 1–4 ppt NaCl were also associated with significantly lower host infection loads while infected hosts exposed to 3 and 4 ppt NaCl were found to have significantly higher survival rates. These results support the potential for NaCl to be used as an environmentally distributed antifungal agent for the prevention of chytridiomycosis in susceptible amphibian hosts. However, further research is required to identify any negative effects of salt exposure on both target and non-target organisms prior to implementation.

Effect of Staining and Freezing Media on Sortability of Stallion Spermatozoa and their Post-thaw Viability After Sex-sorting and Cryopreservation

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 Kenneys modified Tyrodes (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.

Composition of Luminal Fluid Secreted by the Seminiferous Tubules and After Reabsorption by the Extratesticular Ducts of the Japanese Quail, Coturnix coturnix japonica

Abstract The present report examines the composition of luminal fluid in the seminiferous tubule (STF), rete testis (RTF), and ductus epididymidis of the Japanese quail (Coturnix coturnix japonica). This subject is of particular interest, both because the reproductive ducts are intra-abdominal and because sperm production is more rapid in birds than in mammals. It was interpreted that micropuncture samples of STF contain varying amounts of contamination with intracellular solute, particularly K and protein. The concentration of solute in samples was correlated with packed cell volume (spermatocrit), and when the latter was used to assess estimates of solute concentration in STF, the magnitude of the estimates were much the same as determinations in RTF. Consequently, it is concluded that the fluid entering the rete testis of the quail is the primary secretion of the seminiferous tubules. The composition of RTF in the quail was determined to be 148 mM Na, 126 mM Cl, 9.8 mM K, 2.7 mM Mg, 1.4 mM Ca, 2.1 mM glutamate, 3.4 mM glutamine, 20.2 mM bicarbonate, 1.8 μg μl−1 of protein, pH 7.34, and 310 mmol kg−1, and it is significantly different from the composition of blood plasma. Estimates of solute output by the testis and reabsorption by the extratesticular ducts indicate, first, that most of the solutes secreted into the seminiferous tubules are subsequently reabsorbed from the extratesticular ducts and, second, that sufficient solute of testicular origin (except for protein) exists to account for the concentrations of solutes throughout the lumen of the duct system. Changes in the concentration of solute in the extratesticular ducts probably result from different reabsorption rates of solute and water. The composition of fluid from the distal end of the ductus epididymidis was 133 mM Na, 125 mM Cl, 25 mM K, 1.0 mM Mg, 0.3 mM Ca, 6.7 mM glutamate, 4.0 mM glutamine, 19.5 mM bicarbonate, 6.0 μg μl−1 of protein, pH 7.33, and 335 mmol kg−1, and it is significantly different from those of RTF and blood.

Evidence of a salt refuge: chytrid infection loads are suppressed in hosts exposed to salt

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.

Amphibian declines in the twenty-first century: why we need assisted reproductive technologies

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.

Factors influencing the “sortability” of stallion spermatozoa into X- and Y-chromosome bearing populations

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.

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1-5.0 mM theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800 g were associated with a significant reduction in motility (mean 56 +/- 3% decreasing to 27 +/- 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mM theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.