Background. Cyclic mastalgia (CM) is premenstrual bilateral and diffuse breast pain that presents cyclically and affects women in their reproductive years. It may associate with latent hyperprolactinemia due to the insufficient inhibitory effect of dopamine on the pituitary gland. Vitex agnus-castus (VAC) is known for its dopaminergic activity and its possible actions on CM and latent hyperprolactinemia. However, the treatment effect of VAC on CM remains unclear.Materials and methods. To perform a systematic review and meta-analysis of clinical trials that report on the efficacy ofVAC treatment in CM patients, literature search was performed in major research databases.Results. This review includes 25 studies (17 randomized control trials plus eight nonrandomized trials). VAC was effective in relieving breast pain intensity and lowering the increased serum prolactin level in reproductive age CM patients (18-45 years) with or without premenstrual syndromes. Typical dosage was 20-40 mg/day with a treatment duration of 3 months. A conservative meta-analysis included only six studies (n = 718: VAC - 356, placebo - 362) and revealed a moderate effect size (SMD 0.67, 95% CI 0.5­0.85) favoring VAC over a placebo. Seven trials demonstrated VAC to be a noninferior alternative to pharmaceutical therapies for CM, including dopamine agonists, nonsteroidal anti-inflammatory drugs, serotonin reuptake inhibitors, and hormonal contraceptives. VAC was safe and associated with only mild and reversible adverse events. However, the risk of bias in most studies was unclear due to insufficient information.Conclusion. VAC is a safe and effective treatment option for CM. More high-qualityclinicaltrialsareneededto strengthentheevidencebase.

Загальна інформація. Циклічна масталгія (ЦМ) – це передменструальний двосторонній дифузний біль у молочних залозах, що виникає циклічно в жінок репродуктивного віку. Він може бути пов’язаний із латентною гіперпролактинемією внаслідок недостатнього гальмівного впливу дофаміну на гіпофіз. Відомо, що вітекс священний (Vitex agnus-castus, VAC) має дофамінергічну активність і може впливати на перебіг ЦМ і латентної гіперпролактинемії. Проте терапевтичний ефект VАС при ЦМ залишається не до кінця зрозумілим. Матеріали та методи. З метою систематичного огляду та мета-аналізу клінічних випробувань, у яких повідомляється про ефективність лікування VAC пацієнток із ЦМ, було проведено пошук наукової літератури в основних базах даних клінічних досліджень. Результати. Цей огляд включає 25 досліджень (17 рандомізованих контрольованих випробувань і 8 нерандомізованих). VAC ефективно зменшував інтенсивність болю в молочних залозах і знижував підвищений рівень пролактину в плазмі крові пацієнток репродуктивного віку (18–-45 років) з передменструальним синдромом або без нього. Звичайна доза становила 20–40 мг/добу, а тривалість лікування – 3 місяці. Попередній мета-аналіз включав тільки шість досліджень (n = 718: VAC – 356, плацебо – 362) і виявив помірний розмір ефекту VAC (стандартизована різниця середніх значень 0,67; 95% довірчий інтервал: 0,5–0,85) у порівнянні з плацебо. У семи випробуваннях було продемонстровано, що VAC є не менш ефективною альтернативою медикаментозній терапії ЦМ, що включає агоністи дофаміну, нестероїдні протизапальні засоби, інгібітори зворотного захоплення серотоніну та гормональні контрацептиви. Лікування VAC було безпечним, при застосуванні препарату виникали тільки легкі та оборотні небажані явища. Проте в більшості досліджень через недостатню кількість інформації неможливо точно оцінити ризик систематичної помилки. Висновок. VAC – це безпечний і ефективний спосіб лікування ЦМ. Для покращення доказової бази потрібно провести якісніші клінічні випробування. Ключові слова: вітекс, авраамове дерево, масталгія, гіперпролактинемія, систематичний огляд, мета-аналіз.

Evidence based practice (EBP) is a system of applying the most current and valid high quality evidence to support clinical decision making in a healthcare setting. In the twenty five years since its inception, EBP has become the accepted benchmark for excellence in healthcare. Although the system emerged within the biomedical sciences, in the years since EBP has become normative across all healthcare modalities from dentistry, allied health to complementary and alternative medicine (CAM). Practicing evidence based medicine within any modality potentially offers the patient the best available care based on high quality evidence. Yet it is the nature of the evidence that provokes some questions about the suitability of EBP across all modalities of healthcare. The meta analysis of randomized controlled trial (RCT) stands at the pinnacle of the hierarchy of evidence in EBP. This forms a challenge to CAM due to the difficulty in reducing the elementals of a holistic naturopathic assessment of a patient into an answerable question to be tested within a RCT. On one level this makes EBP paradigmatically incompatible with CAM, yet on another level it presents the opportunity to redefine the parameters of what is considered high level evidence. EBP has become a tool, and at times a weapon wielded by governments and health insurance companies to direct healthcare funding and policy. The implications of the nature of accepted evidence are becoming far reaching. The pursuit of the best available healthcare for each individual is the focus of EBP. However, the injudicious use of this system to direct health policy is fraught with biomedical bias and dominance. This issue raises the challenge to CAM to present high level evidence according to the rules of evidence, or face the annihilation of centuries of empirical knowledge.

Evidence based medicine involves using both the individual clinician's expertise and the current best available external clinical evidence from systematic research in deciding on the appropriate care for individual patients. The current approach to evidence based practice in healthcare adds a third component which is patient values. Evidence based practice is thus a triad, in which the practitioner's expertise, research evidence and the patient's values are all given consideration. The balance to be struck between them depends on the individual case. The literature indicates that complementary medicine practitioners are moving away from traditional knowledge and towards the use of evidence based practice in their clinical discussions. In the context of the daily practice of complementary medicine practitioners and their continuing development of their knowledge base of evidence based practice, this short review discusses the good and bad of a review journal article.

Science classes for health science degrees are some of the most challenging any lecturer will undertake. In many institutions they act as the ‘gate-keeper’ subjects for the degrees they serve and are often deemed the reasons for high attrition and fail rates. This paper focuses on a suite of four biomedical science courses that run over the first two years of various healthcare degrees at Charles Sturt University. The majority of our regional students are enrolled in nursing or paramedic undergraduate degrees and have entered through non-traditional pathways. Students study these courses either internally on campus or via distance education with many moving between modes of delivery. In an attempt to improve student performance we set out to realign the course content and assessments of these key subjects using Bloom’s taxonomy. A review is presented of the teaching teams’ experiences and responses to the challenges in teaching human bioscience, pathophysiology and pharmacology. The review considers the data generated over 12 semesters of teaching between 2007 and 2012 inclusive and assesses the impact of the content realignment. It includes the trends in student subject evaluations and historical data relating to student success, attrition and failure. Although student opinion towards these subjects has in general improved, the review highlighted problems associated with analysis of trends over time when centralised raw data is unavailable. Despite this limitation, it has enabled the team to identify where future efforts need to be directed; the student transition from level 1 to level 2.

This study examined the effect of cryoprotectants (20% DMSO, a 10% DMSO/10% glycerol mixture, 20% glycerol and 1 M 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 °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 °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 °C and MMP would halve after 19.4 min at 4 °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 °C and PMI would halve after 21.1 min at 4 °C. These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermatozoa then it is best administered at 4 °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.

Kangaroo spermatozoa have proven extremely difficult to cryopreserve such that despitenumerous empirical studies using high concentrations of glycerol and/or DSMO, the bestpost-thaw motility has only been in the order of 10%. The efficacy of glycerol as acryoprotectant for marsupial spermatozoa is somewhat paradoxical, being necessary athigh concentrations for adequate cryopreservation, but at the same time “cytotoxic.” Theunderlying objectives of this project were to understand the causes of cryoinjury tokangaroo spermatozoa and to apply this information in an attempt to develop a reliablemethod for cryopreservation. These objectives were addressed through strategically linkedstudies incorporating the documentation of cryoinjury, hypothesis driven investigations intothe causes of cryoinjury and the application of novel cryopreservation protocols.The ultrastructure and freeze-fracture of caput and cauda epididymal Eastern greyKangaroo (EGK) spermatozoa at 35°C, 4°C and following cryopreservation with andwithout 20% glycerol was investigated. The addition of 20% glycerol resulted in significantdamage to the sperm plasma membrane and mitochondria compared to no glycerol at thesame temperatures (P < 0.05). Following cryopreservation, 20% glycerol significantlyimproved the preservation of the cauda epididymal sperm plasma membrane andmitochondria and reduced the incidence of axonemal damage and periaxonemal spaces.For caput epididymal spermatozoa, glycerol only improved the preservation of the plasmamembrane following cryopreservation (P < 0.05).Freeze fracture microscopy revealed a pattern of helically wound intramembranousparticles in the plasma membrane over the fibre network of the mid piece of the sperm tail.After thawing, the plasma membrane was damaged such that this structure was missing iniipatches, and the helical rows of particles were mal-aligned. On the principal piece,particles were arranged randomly at physiological temperatures; however, upon cooling to4°C with 20% glycerol, the particles become aggregated. Once re-warmed (35°C),particles over the principal piece resumed their random organisation. This finding is furtherevidence of a reversible phase transition of the macropod sperm plasma membrane duringcooling that is not associated with a loss of motility or membrane integrity.In attempt to investigate cryoinjury caused by alternative cryoprotectants the next studiesexamined the effect of cryoprotectants (20% DMSO, a 10% DMSO / 10% glycerol mixture,20% glycerol and 1 M sucrose solution) on kangaroo sperm structure and function, alongwith the effect of varying concentrations of glycerol on sperm mitochondrial function.Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 mins at35°C in each cryoprotectant and the plasma membrane integrity (PMI) and motilityassessed using light microscopy. The same samples were fixed for TEM and theultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerolon the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubatedwith JC-1 in Tris-citrate media at 35°C for 20 mins in a range of glycerol concentrations (0,5, 10, 15 and 20%) and the mitochondrial membrane potential (MMP) and plasmamembrane integrity determined. As expected, incubation of spermatozoa in 20% glycerolfor 10 mins resulted in a significant reduction in motility, PMI and ultrastructural integrity.Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility orPMI but a significant loss of structural integrity when compared to the control spermatozoa(0% Glycerol). However, 20% DMSO was overall less damaging to sperm ultrastructurethan glycerol, 10% glycerol and 10% DMSO, and sucrose. While all glycerolconcentrations had an adverse effect on mitochondrial function, the statistical modelspresented for the relationship between MMP and glycerol predicted that sperm, wheniiiadded to 20% glycerol, would loose half of their initial MMP immediately at 35°C and after19.4 mins at 4°C. Models for the relationship between PMI and glycerol predicted thatsperm would loose half of their initial PMI after 1.8 mins at 35°C and 21.1 mins at 4°C.These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermthen it is best administered at 4°C and that mitochondrial function is more sensitive toglycerol than PMI. Further studies were directed at investigating strategies that reduceexposure of spermatozoa to glycerol during processing and evaluating the cryoprotectiveproperties of 20% DMSO for kangaroo sperm.The first hypothesis to be tested with respect the causes of cryopathology examined thepossibility that the ultrastructural changes that occur to the kangaroo sperm duringepididymidal maturation reduced the tolerance of the sperm cell organelles to respond toosmotic flux, glycerol cytotoxicity and ice-crystal damage. Caput and cauda epididymidalspermatozoa were recovered from red-necked wallabies (Macropus rufogriseus) and EGK.In Experiment 1, caput and cauda epididymal spermatozoa were frozen and thawed usinga standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol.Although cryopreservation of caput epididymidal spermatozoa resulted in a significantincrease in sperm plasma membrane damage, they were more tolerant of the procedurethan spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2,caput and cauda epididymidal spermatozoa were diluted into phosphate-buffered salinemedia of varying osmolarity and their osmotic tolerance was determined. Plasmamembranes of caput epididymidal spermatozoa were clearly more tolerant of hypo-osmoticmedia than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput andcauda epididymidal spermatozoa were incubated in Tris-citrate buffer with and without20% glycerol at 35 and 4°C to examine the cytotoxic effects of glycerol. At bothtemperatures, caput epididymidal spermatozoa showed less plasma membrane damageivcompared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05).The results from these experiments clearly indicate that epididymal maturation of kangaroospermatozoa resulted in a decreased ability to withstand the physiological stressesassociated with cryopreservation.The second hypothesis to be tested with respect the causes of cryopathology examinedwhether filamentous (F) actin associated with the complex cytoskeleton of the kangaroosperm head and tail may be contributing to lack of plasma membrane plasticity and aconsequent loss of membrane integrity during cryopreservation. In the first study, thedistribution of G and F actin within EGK cauda epididymidal spermatozoa was successfullydetected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam),respectively. G-actin staining was most intense in the acrosome but was also observedwith less intensity over the nucleus and mid-piece. F-actin was located in the spermnucleus but was not discernable in the acrosome or sperm tail. To investigate whethercytochalsin D (a known F-actin depolymerising agent) was capable of improving theosmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypoosmoticmedia (61 and 104 mOsm) containing a range of cytochalasin D concentrations(0-200μM). Cytochalsin D had no beneficial effect on the plasma membrane integrity ofsperm incubated in hypo-osmotic media. The results of this study indicated that the F-actindistribution in cauda epididymidal spermatozoa of the EGK was surprising different to thatof the Tammar Wallaby (M. eugenii) and that cytochalsin-D does not appear to improvethe tolerance of EGK cauda epididymidal sperm to osmotic induced injury.The aims of the final study in this thesis were to investigate alternative techniques for thecryopreservation of kangaroo spermatozoa that reduce or eliminate the need for glycerol.This was investigated by (1) freezing sperm with 20% glycerol in pre-packaged 0.25 mLCassou straws in such a way as to enable rapid dilution of the glycerol post-thaw, (2)vinvestigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide(DMA - 10, 15 and 20% v/v) as cryoprotectants and (3) to evaluate the potential use ofultra-rapid small volume vitrification cooling of kangaroo spermatozoa with or withoutcryoprotectant (20% v/v Glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-strawpost-thaw dilution (1: 20) of 20% glycerol produced no significant improvement in postthawviability of kangaroo spermatozoa. Cryopreservation of kangaroo spermatozoa in20% DMSO resulted in only 2% motile sperm and 5% of sperm with an intact plasmamembrane. While kangaroo spermatozoa frozen by ultra-rapid freezing techniquesshowed no evidence of post-thaw viability irrespective of whether cryoprotectant was usedor not, sperm frozen in 10 - 20% DMA showed post-thaw motility and plasma membraneintegrity of 11 - 13% and 18 - 23% respectively. Although still modest compared to othermarsupial and eutherian spermatozoa, these results represent a significant improvementin the development of a cryopreservation procedure for kangaroos and provide the basisfor further investigation using other amide-based cryoprotectants.

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.

Macropod spermatozoa have proven difficult to cryopreserve such that empirical studies using high concentrations of glycerol and/or DSMO have resulted in only 10% post-thaw motility. We examined the ultrastructure and freeze-fracture of caput and cauda epididymal macropod spermatozoa at 35, 4°C and following cryopreservation with and without 20% glycerol. The addition of 20% glycerol resulted in significant damage to the sperm plasma membrane and mitochondria compared to no glycerol at the same temperatures (P<0.05). Following cryopreservation, 20% glycerol significantly improved the preservation of the cauda epididymal sperm plasma membrane and mitochondria and reduced the incidence of axonemal damage and axonemal spaces. For caput epididymal spermatozoa, glycerol only improved the preservation of the plasma membrane following cryopreservation (P<0.05). Freeze fracture microscopy revealed a pattern of helically wound intramembranous particles in the plasma membrane over the fibre network of the mid piece of the sperm tail. The fibre network is an interconnecting cytoskeletal structure found underneath the plasma membrane of the kangaroo sperm midpiece and is thought to add rigidity to the proximal portion of the sperm tail. After thawing, the plasma membrane was damaged such that this structure was missing in patches, and the helical rows of particles were mal-aligned. On the principal piece, particles were arranged randomly at physiological temperatures; however, upon cooling to 4°C with 20% glycerol, the particles become aggregated. Once rewarmed (35°C), particles over the principal piece resumed their random organisation. This finding is further evidence of a reversible phase transition of the macropod sperm plasma membrane during cooling that is not associated with a loss of motility or membrane integrity.

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm’s response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5μM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n=4) and wombat (n=4) spermatozoa packaged in 0.25mL straws were cryopreserved using two freezing rates (fast—3cm above liquid N2 interface; slow—6°C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris–citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P<0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.

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.

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t=3.81; df=4; p⩽0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Variation in localization and distribution of saccharides on the sperm surface of a marsupial, the brushtail possum, Trichosurus vulpecula, was compared between spermatozoa from the caput and cauda epididymides. Spermatozoa were subjected to the following treatments: (i) unfixed and fixed spermatozoa were stained with fluorescein-labelled lectins; (ii) unfixed spermatozoa were incubated with lectins for determination of agglutination; and (iii) spermatozoa were incubated with detergent to remove the plasmalemma, the glycoproteins were separated on SDS-PAGE and western blots were stained with biotinylated lectins. Many of the fluorescein isothiocyanate (FITC)-labelled lectins bound selectively to the sperm surface, and marked differences were found in lectin staining affinity between caput and cauda epididymal spermatozoa. Incubation of spermatozoa from the cauda epididymidis with neuraminidase reversed many of the differences in staining of the cauda epididymal spermatozoa, indicating masking of some terminal saccharides by sialic acid. Agglutination of spermatozoa from the caput epididymidis occurred after incubation with Concanavalin A (ConA) and soybean agglutinin (SBA), but agglutination was less extensive for spermatozoa from the cauda epididymidis. Western blot analysis indicated several ConA-positive bands in caput sperm extracts, but fewer positive bands in the cauda sperm extracts, whereas SBA stained four bands from caput but none from the cauda epididymal spermatozoa. These results demonstrate extensive glycosylation of the surface proteins of spermatozoa from the caput epididymidis and significant differences in spermatozoa from the cauda epididymidis. In general, the findings indicate similar glycosylation of the surface of marsupial spermatozoa to those from eutherian mammals despite marked differences in their morphology and early divergence of marsupials from eutherian mammals. It would appear that this situation differs markedly from that in sub-mammalian vertebrates.