D. Blyde

Cryopreservation of macropodid spermatozoa: new insights from the cryomicroscope.

This study examined the effects of cooling and cryopreservation upon macropod spermatozoa (eastern grey kangaroo, Macropus giganteus and red-necked wallaby, Macropus rufogriseus). Sperm survival during and after freezing to -30 degrees C or 70 degrees C in minimum essential medium (MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or 20% (v/v) ethylene glycol and MEM containing a mixture of 7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxide was examined by cryomicroscopy. The MEM/glycerol mixtures permitted better post-thaw sperm recovery than the other cryoprotectants. After freezing to -30 degrees C at 10 degrees C min(-1) in 20% glycerol, then rewarming at 20 degrees C min(-1), flagellar activity resumed in more than 50% of spermatozoa when the temperature increased into the range 5-10 degrees C. However, as the temperature increased, into the range 20-25 degrees C, motility declined rapidly so that less than 5% motile cells were seen at 35 degrees C. Spermatozoa in MEM without cryoprotectant were also examined by cryomicroscopy to evaluate changes in flagellar configuration, swimming behaviour and viability during cooling from 35 degrees C to approximately -7 degrees C, and rewarming to 35 degrees C. Cooling from 35 to 28 degrees C induced kangaroo spermatozoa to exhibit rigid principal-piece bending and non-linear motility, which was reversed by further cooling and the spermatozoa resumed their normal linear movement. Rewarming induced principal-piece bending in the range of 20-30 degrees C, but this effect was reversed by further warming. Although red-necked wallaby spermatozoa showed these effects, they also exhibited a tendency to form rosette-like clusters during rewarming, especially when the temperature reached approximately 14 degrees C. The clusters were induced when the flagellar end-pieces became anteriorly reflected, producing hook-like flagellar conformations, which then became interlinked.

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.

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

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

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.

9. Lack of seasonal variation in male reproductive parameters measured in a captive and wild population of common wombats in New South Wales

Despite their abundance in the wild, common wombats (Vombatus ursinus) do not breed regularly in captivity, such that there is little published about their captive reproductive management. As part of the development of an artificial insemination program in this species and in an attempt to establish important baseline reproductive information, 4 captive male common wombats at Western Plains Zoo (Dubbo, NSW) were systematically examined over a 12-month period in order to assess whether male reproductive function was seasonally dependent. The reproductive parameters investigated included, peripheral plasma testosterone secretion, testis volume and quality of semen (% motility, % live and % normal) collected by electro-ejaculation. An attempt was also made to determine the extent of seasonal change in male reproductive function of common wombats in wild populations. Plasma samples and reproductive tracts were collected from 12 wild male common wombats from the Kangaroo Valley district (NSW) in June (n = 7) and November (n = 5); plasma testosterone secretion, epididymidal sperm characteristics (% motile, % live and % normal) and measurements of testis, prostate and bulbourethral glands were compared. Our results indicated that male common wombats in captivity at Western Plains Zoo did not appear to be seasonal breeders in terms of testosterone secretion or electroejaculate quality (% motile, % live and % normal); there was, however, a significant increase in testis size between the months of June to October (F = 4.04; P = 0.05). Lack of distinct seasonal variation in male reproduction was also evident in wild common wombats in Kangaroo Valley. There was no significant difference (P > 0.05) between wild wombats collected in June or November with respect to plasma testosterone concentration, epididymidal sperm quality or testis, prostate and bulbourethral gland dimensions.