Browsing by Author "Maree, Liana"
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Item Effect of copper sulphate and cadmium chloride on non-human primate sperm function in vitro(MDPI, 2021) Hardneck, Farren; de Villiers, Charon; Maree, LianaAbstract: In order to address the large percentage of unexplained male infertility in humans, more detailed investigations using sperm functional tests are needed to identify possible causes for compromised fertility. Since many environmental and lifestyle factors might be contributing to infertility, future studies aiming to elucidate the effect of such factors on male fertility will need the use of appropriate research models. The current study aimed to assess the effects of two heavy metals, namely copper sulphate, and cadmium chloride, on non-human primate (NHP) sperm function in order to establish the possibility of using these primate species as models for reproductive studies. Our combined results indicated that the functionality of NHP spermatozoa is inhibited by the two Citation: Hardneck, F.; de Villiers, C.; Maree, L. Effect of Copper Sulphate and CadmiumChloride on Non-HumanPrimate Sperm Function In Vitro. Int. J. Environ. Res. Public Health 2021, 18, 6200. https:// doi.org/10.3390/ijerph18126200 Academic Editor: Mei-Fang Chien Received: 9 April 2021 Accepted: 21 May 2021 Published: 8 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). heavy metals investigated. After in vitro exposure, detrimental effects, and significant lowered values (p < 0.05) were obtained for sperm motility, viability and vitality, acrosome intactness, and hyperactivation. These metals, at the tested higher concentrations, therefore, have the ability to impair sperm quality thereby affecting sperm fertilizing capability in both humans and NHPs.Item Effects of GnRH vaccination in wild and captive African Elephant bulls (Loxodonta africana) on reproductive organs and semen quality(Public Library of Science, 2017) Lueders, Imke; Young, Debbie; Maree, Liana; van der Horst, Gerhard; Luther, IlseOBJECTIVES: Although the African elephant (Loxodonta africana) is classified as endangered by the International Union for Conservation of Nature (IUCN), in some isolated habitats in southern Africa, contraception is of major interest due to local overpopulation. GnRH vaccination has been promoted as a non-invasive contraceptive measure for population management of overabundant wildlife. We tested the efficacy of this treatment for fertility control in elephant bulls. METHODS: In total, 17 male African elephants that were treated with a GnRH vaccine were examined in two groups. In the prospective study group 1 (n = 11 bulls, ages: 8±36 years), semen quality, the testes, seminal vesicles, ampullae and prostate, which were all measured by means of transrectal ultrasound, and faecal androgen metabolite concentrations were monitored over a three-year period. Each bull in the prospective study received 5 ml of Improvac® (1000 μg GnRH conjugate) intramuscularly after the first examination, followed by a booster six weeks later and thereafter every 5±7 months. In a retrospective study group (group 2, n = 6, ages: 19±33 years), one examination was performed on bulls which had been treated with GnRH vaccine for 5±11 years. RESULTS: In all bulls of group 1, testicular and accessory sex gland sizes decreased significantly after the third vaccination. In six males examined prior to vaccination and again after more than five vaccinations, the testis size was reduced by 57.5%. Mean testicular height and length decreased from 13.3 ± 2.6 cm x 15.2 ± 2.8 cm at the beginning to 7.6 ± 2.1 cm x 10.2 ± 1.8 cm at the end of the study. Post pubertal bulls (>9 years, n = 6) examined prior to vaccination produced ejaculates with viable spermatozoa (volume: 8±175 ml, sperm concentration: 410-4000x106/ml, total motility: 0±90%), while after 5±8 injections, only 50% of these bulls produced ejaculates with a small number of immotile spermatozoa. The ejaculates of group 2 bulls (vaccinated >8 times) were devoid of spermatozoa. Faecal androgen metabolite concentrations measured in captive males decreased significantly after the fourth vaccination. None of the males entered musth during the treatment period. CONCLUSIONS: Our results showed a marked decrease in semen quality, testicle and secondary sex gland sizes following repeated GnRH vaccinations. After 2±4 years of continuous treatment every 5±7 months, the effects were similar to surgical castration.Item Evaluation of standard and development of new sperm function tests in selected primate species(University of the Western Cape, 2017) Prag, Farren Chelsea; Maree, LianaMale infertility in humans has increased in the last few decades and could be as high as 40%, while up to 50% of these men have ''unexplained'' (idiopathic) infertility. Although newly developed molecular techniques have great value in detecting subtle causes of male infertility, more detailed sperm functional tests are required to identify compromised fertility, especially in a clinical set-up. Since ethical constraints often preclude the pursuit of many basic research questions in humans, non-human primates (NHPs) have been identified as key models in human-related studies. NHPs are often used in studies on male fertility/infertility, IVF or assisted reproductive technology (ART) procedures, male contraception and reproductive toxicology. However, comparing results of NHP and human studies require that techniques used for assessment must be objective, standardized and sensitive to recognize compromised sperm function. The aim of this study was to evaluate standard sperm functional tests and develop new functional tests using NHP sperm, specifically from vervet monkeys (Chlorocebus aethiops), chacma baboons (Papio ursinus) and rhesus monkeys (Macaca mulatta), for application in human and NHP studies and to ultimately develop a basic primate model. The sperm functions investigated included sperm motility, longevity, vitality, DNA integrity, acrosome reaction, and hyperactivation. The sperm functional tests evaluated were: CASA motility analysis; Sperm Longevity test; Eosin-Nigrosin and Hoechst and Propidium Iodide staining, as well as the use of WST-1 cytotoxicity assay for vitality; the TUNEL assay for DNA integrity; Acrosome Intactness Test; and induction of hyperactivation via stimulants. The validity of each test was investigated by inhibiting sperm function through the use of copper sulphate and cadmium chloride. All functional tests were successfully performed across all three species, except the TUNEL assay for DNA integrity, and was further used for validation testing. Validation testing proved that all sperm functional parameters were significantly affected by the highest concentrations of the chemicals (250 µg/ml CuSO4 and 500 µg/ml CdCl2) and if not significant, trends of reduction were seen. The tests employed were therefore sensitive to the inhibitory effect of the metals. By evaluating these established sperm functional tests we found that primates would serve as good models for research study. Furthermore, we optimized and modified techniques for sperm and functional analysis in these three primate species and this study will standardize protocols for use in future studies on male infertility. Additionally, comparing human and NHP sperm function can possibly reveal or explain the high infertility rates in humans.Item Evaluation of sperm functionality in non-human primates, focussing on sperm capacitation(University of the Western Cape, 2019) Mabotha, Luke Allen; Kotze, Retha; Maree, Liana; de Villiers, CharonThe incidence of male infertility is increasing, with up to 50% of infertile males having “unexplained” (idiopathic) infertility. Newly developed molecular techniques have great value in detecting subtle causes of male infertility, as compared to idiopathic infertility which may be explained by standardizing and optimizing sperm functional and structural tests in non-human primate (NHP) sperm. The aim of the study was to evaluate sperm functionality utilizing the sperm of two NHP species, i.e.1) the rhesus monkey (Macaca mulatta) and 2) the vervet monkey (Chlorocebus aethiops), and further evaluate the effect of physiological media (including commonly used, and newly formulated sperm wash and sperm capacitating media) on NHP sperm functionality. Sperm functionality was evaluated by investigating the following sperm functions i.e.: sperm motility, vitality, acrosome reaction (AR), hyperactivation, and mitochondrial membrane potential (MMP). Sperm functional tests included computer-aided semen analysis (CASA), motility analysis, BrightVit staining for sperm vitality, flourescenin isothiocyanate (FITC)- conjugated peanut agglutinin (PNA) staining for sperm acrosome integrity, induction of hyperactivation by stimulants (sperm preparation media containing capacitating ingredients), and mitochondrial inhibitor (Oligomycin-A) for testing MMP. All functional and structural tests were investigated in both species, except for acrosome integrity, mitochondrial inhibition and functional tests compared over time that could not be successfully completed and investigated in the rhesus species. Motility analysis tests proved that within the vervet species, the use of different physiological media results in statistically significant differences in motility and kinematic parameters over a 1 hour time period. Hyperactivation tests proved that capacitating physiological media produced significantly higher percentages hyperactivation when compared to sperm wash media within the vervet species over a 1 hour time period. Furthermore, within both NHP species, sperm structural analysis (vitality and acrosome integrity) results showed that no significant differences are present when making use of different physiological media over a period of 1 hour incubation. The incubation of vervet sperm with different concentrations of mitochondrial inhibitor, Oligomycin-A (0 μM, 5 μM, and 25 μM), resulted in motility inhibition over a 1 hour incubation period. By the evaluation of these tests it was found that the use of different sperm wash [Human tubal fluid (HTF), Ham‟s F-10® and HD Sperm Wash Plus (HDSWP)] and sperm capacitation media [Human tubal fluid with added caffeine (HTFC) and HD Sperm Capacitating Plus (HDSCP)] resulted in significantly different results within sperm functional tests as compared to sperm structural tests. The study indicates that the composition of media, varying from simple to more complex, used for semen preparation plays an important role in determining NHP sperm functionality. Based on these findings further investigation in larger NHP sample groups and human sperm are required to evaluate the role of certain ingredients in the development of more cost-effective media producing satisfactory results in terms of sperm functionality for artificial reproductive technologies (ART).Item Interventions to reduce pesticide exposure from the agricultural sector in Africa: A workshop report(MDPI, 2022) Röösli, Martin; Fuhrimann, Samuel; Maree, LianaDespite the fact that several cases of unsafe pesticide use among farmers in different parts of Africa have been documented, there is limited evidence regarding which specific interventions are effective in reducing pesticide exposure and associated risks to human health and ecology. The overall goal of the African Pesticide Intervention Project (APsent) study is to better understand ongoing research and public health activities related to interventions in Africa through the implementation of suitable target-specific situations or use contexts. A systematic review of the scientific literature on pesticide intervention studies with a focus on Africa was conducted. This was followed by a qualitative survey among stakeholders involved in pesticide research or management in the African region to learn about barriers to and promoters of successful interventions.Item Origin, Migration, and Reproduction of Indigenous Domestic Animals with Special Reference to Their Sperm Quality(MDPI, 2022) Van der Horst, Gerhard; Maree, Liana: Indigenous domestic animals such as cattle, sheep, goats, pigs, and chickens have a natural resistance to endo- and ecto-parasites and are tolerant in terms of harsh environmental conditions. These species orginated from the Fertile Cresent between 12,000 and 10,000 BP before migrating into surrounding continents. In view of limited information on the reproductive status of indigenous breeds, it is important to examine their semen characteristics in order to select males to improve livestock production. We have largely relied on existing literature but also our published and ongoing research on sperm quality assessment of several indigenous breeds. The sperm quality of these breeds is similar to current commercial breeds and has been quantified using cutting-edge methods. In this context, we have presented sperm functional tests which provide a better estimate of semen quality than just a standard semen analysis. Initial results suggest that the indigenous breeds have a high sperm quality and sperm functionality similar to currently farmed exotic or crossbreeds. In the long-term, the importance of preserving the favorable traits of these breeds is a priority in view of crossbreeding with existing good meat and milk producers.Item Progesterone, myo-inositol, dopamine and prolactin present in follicular fluid have differential effects on sperm motility subpopulations(MDPI, 2021) Keyser, Shannen; van der Horst, Gerhard; Maree, LianaConsidering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.Item Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed(Taylor & Francis, 2013) Maree, Liana; van der Horst, GerhardMotility is an essential characteristic of all fl agellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer ® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identifi ed. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.Item Quantitative assessment of heavy metal effects on sperm function using computer‐aided sperm analysis and cytotoxicity assays(Wiley, 2018) Hardneck, Farren; Israel, Gadieja; Pool, Edmund; Maree, LianaOne known environmental risk factor impacting on human reproduction is heavy metal pollution. Although some metals (e.g., Cu, Se and Zn) have protective effects on the male reproductive system in low doses, heavy metals can accumulate to toxic levels and result in poor semen quality and decreased sperm function. We investigated the effect of CuSO4 and CdCl2 (10, 50, 100 and 250 μg/ml or 500 μg/ml) on human sperm motility and vitality by using computer‐aided sperm analysis (CASA) and two cytotoxicity assays (WST‐1 and XTT). Several sperm motility parameters were significantly reduced after 5 hr of exposure to the highest concentrations of CuSO4 (250 μg/ml) and CdCl2 (500 μg/ml). The WST‐1 assay also revealed significantly lower absorbance values for 50, 100 and 250 μg/ml CuSO4 and for 500 μg/ml CdCl2; however, no significant effect was seen with XTT. The calculated average IC50 value was 50.31± 4.34 μg/ml for CuSO4 and 392.32 ±76.79 μg/ml for CdCl2. The effects of these metals were confirmed with MgCl2, a positive control. This study provides threshold concentrations for the harmful effect of CuSO4 and CdCl2 on human spermatozoa and recommends the use of WST‐1 as vitality assay in future in vitro studies.Item Seasonal differences in semen characteristics and sperm functionality in Tankwa goats(University of the Western Cape, 2018) Ngcauzele, Asanele; Maree, Liana; van der Horst, Gerhard; Kotze, AntoinetteTankwa goats have been free-ranging in the Tankwa Karoo National Park in the Northern Cape for more than 80 years. A genetic study concluded that these feral goats are a unique genetic resource compared to other goat breeds in South Africa and should be conserved as a distinctive population. A decision taken by the South African National Parks who is the managing authority in the park, was to remove all alien species, which included the Tankwa goats. Several animals were translocated to the Carnarvon Research Station by the Northern Cape Department of Agriculture, Land Reform & Rural Development, where the Tankwa goat population has grown to a few hundred individuals. Currently, sound scientific decisions including the application of a wide range of technologies and approaches are applied to conserve the population, such as an informed understanding of the reproductive biology of these goats. The aim of this study was to define sperm quality in Tankwa goats using various macroscopic and microscopic evaluation techniques.