Histopathological changes in Oreochromis mossambicus (Peters, 1852) ovaries after a chronic exposure to a mixture of the HIV drug nevirapine and the antibiotics sulfamethoxazole and trimethoprim.

The burden of the human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS) infection has transformed the African continent into a major consumer of antiretrovirals (ARVs) drugs. In addition to HIV burden, the African continent has also a high incidence of tuberculosis (TB) and has been experiencing recurring outbreaks of several other viral, bacterial, and parasitic epidemic diseases. The novel severe acute respiratory syndrome coronavirus 2 (SARS-COV-2 or Covid-19) pandemic outbreak is adding to the continent's infectious diseases burden as experts are predicting that it will be here for a long time. One of the consequences of these infectious diseases is that antiviral and antibiotic compounds have become some of the most consumed pharmaceuticals on the continent. Many of these drugs have been frequently detected in surface waters across Africa. There is limited information available on the adverse effects of the mixtures of different types of pharmaceuticals in African aquatic environments on fish reproduction. The present study investigated the effects of the ARV drug nevirapine (NVP - 1.48 and 3.74 µg/L) and its mixture with the antibiotic sulfamethoxazole (3.68 µg/L) and trimethoprim (0.87 µg/L) on O. mossambicus gonads using histopathological endpoints as biomarkers. The fish (n = 52) were exposed for 30 days in a static renewal system. Female O. mossambicus exposed to nevirapine (3.74 µg/L) and to NVP - antibiotic mixture recorded higher ovary indices. Statistically significant differences were found in female ovary indices between the fish exposed to NVP (3.74 µg/L) and the control fish (p = 0.002) as well as between the fish exposed to the NVP - antibiotic mixture and the control fish (p = 0.009). The main observed histopathological changes in the ovaries were increased vitellogenic oocyte atresia and vacuolation of the interstitial tissue in the fish exposed to NVP - antibiotic mixture. It is evident that the presence of NVP - antibiotics mixture in water triggered the observed histopathology in female fish ovaries. The detected abnormal high rate of atretic oocytes could result in impaired fish reproduction.

Hatching success and survival of fish early life stages in a chronic exposure to nevirapine: a case study of the Mozambique tilapia.

The anti-retroviral nevirapine has been detected in surface waters throughout South Africa and its effects on non-target aquatic animals are still unknown. The aim was to investigate the potential effects of nevirapine on the hatching success and survival of Oreochromis mossambicus early life stages through a chronic exposure. The exposer started with newly fertilized O. mossambicus eggs and concluded 30 days after hatching. Environmental relevant concentration of nevirapine (1.48 µg/l) was used in a static renewal system and a controlled environment (27 ± 1°C; 14:10 day/night cycle). The main endpoints assessed included hatching success and survival; a morphological assessment was also done on whole individual on day 1 and 30 post-hatching to identify any physical abnormality. Nevirapine had no noticeable effects on the hatching success and survival of O. mossambicus larvae; no statistically significant differences were observed between the control and the nevirapine exposed fish (p > 0.05).

The effects of environmentally relevant concentrations of aldrin and methoxychlor on the testes and sperm of male Clarias gariepinus (Burchell, 1822) after short-term exposure.

The organochlorine pesticides aldrin (0.14 µg/L) and methoxychlor (0.23 µg/L) were both present in the Albasini Dam, Limpopo Province, South Africa, during a field survey in 2014. The use of aldrin has been banned in the USA since 1987 and restricted in South Africa since 1992. The use of methoxychlor, however, remains undefined with little information available about its registration in South Africa despite being banned in Europe (2002) and USA (2003). The aim of this study was to determine the potential effects of environmentally relevant concentrations of aldrin and methoxychlor on the reproductive system of male catfish, Clarias gariepinus. Males were exposed for 96 h to the two pesticides under controlled laboratory conditions. Following exposure, each fish was weighed and measured, and a necropsy performed to determine any macroscopic abnormalities and the general health of the fish. The fish were killed and dissected and the testes removed, weighed and measured to determine the gonadosomatic index (GSI). The right testis of each fish was sectioned for histopathological assessment and to calculate the testes index (IT). The left testis was used for computer-assisted sperm analysis (CASA). The histopathological assessment of the testes showed histopathological changes such as of melano-macrophage centres (MMCs) and vacuolation of spermatogonia and spermatocytes. However, the classification of these changes indicated that the testes tissue structure was normal with slight histological changes. No statistically significant differences (p > 0.05) were found in the CASA parameters between exposure groups. The results of this study showed that the environmentally relevant concentrations of aldrin and methoxychlor did not have a negative effect on the motility of the mature sperm, but adverse effects were noted in the early stages of spermatogenesis, indicating possible effects over longer exposure periods.

The potential effects of efavirenz on Oreochromis mossambicus after acute exposure.

Antiretroviral drugs (ARVs) are hazardous therapeutic pharmaceuticals present in South African surface water. Efavirenz is an ARV commonly used in human immunodeficiency virus (HIV) treatment in South Africa. Although little is known about the toxic effects of efavirenz on fish health, threats of toxicity to the aquatic environment have been reported. Oreochromis mossambicus were exposed under controlled conditions to environmentally-relevant efavirenz concentrations (10.3ng/l) as measured in rivers that flow into the Nandoni Dam in the Vhembe District, South Africa. Acute (96h) exposures were conducted using efavirenz concentrations of 10.3ng/l and 20.6ng/l. The overall health of exposed fish was determined using a histology-based fish health assessment index. Necropsies and haematology were conducted and somatic indices calculated after which the liver, kidney, heart, gills and gonads were microscopically quantitatively assessed. Results indicated that fish exposed to 20.6ng/l efavirenz had significantly (p<0.02) higher liver indices than the control fish, indicating increased liver damage including steatosis and frank necrosis. Fish exposed to 20.6ng/l efavirenz presented with significantly (p<0.02) higher total fish indices, representative of declined overall health compared to control fish. It was concluded that the exposure of O. mossambicus to efavirenz resulted in liver damage and overall decline in fish health. These novel findings may indicate a health risk for O. mossambicus and other biota exposed to efavirenz in aquatic ecosystems. Thus, ARV's in water sources of South Africa pose a definite threat to wildlife and ultimately human health.

Organochlorine pesticide levels in Clarias gariepinus from polluted freshwater impoundments in South Africa and associated human health risks.

There are increasing concerns regarding the safe human consumption of fish from polluted, freshwater impoundments. The aim of this study was to analyse the muscle tissue of the sharptooth catfish Clarias gariepinus for selected organo-chlorine pesticides (OCPs) and to perform a human health risk assessment using a standard protocol described by the United States Environmental Protection Agency (US EPA). Fish were collected from the polluted Roodeplaat-(RDPD), Rietvlei-(RVD) and Hartbeespoort (HBPD) Dam impoundments located in the north-eastern regions of South Africa. GC-MS analyses showed levels of various OCPs in fish muscle samples from all three impoundments. For fish collected from the RDPD, p,p'-DDE, endosulfan, lindane and ß- and δ-HCH were the most prevalent OCPs detected, while p,p'-DDE and endosulfan were the most predominant in fish from the RVD. Lindane and ß- and δ-HCH were the main OCPs detected in fish from the HBPD. Dieldrin was the only OCP detected at concentrations for which a cancer risk and a hazard index above the acceptable risk levels were estimated. This was the case for fish from both the RDPD and RVD impoundments. No toxic risk was estimated should fish from the HBPD be consumed.

Sperm-egg interaction and functional assessment of springbok, impala and blesbok cauda epididymal spermatozoa using a domestic cattle in vitro fertilization system.

The study assesses the possibility to estimate the potential fertility of post-thawed antelope (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi) epididymal sperm using homologous and heterologous IVF and the functioning of cattle IVF system to produce antelope embryos. Cauda epididymal sperm were collected from the antelope and cryopreserved under field conditions. In vitro matured domestic cow, blesbok and springbok oocytes were co-incubated in modified-Tyrode Lactate (m-TL) IVF media with springbok, impala and blesbok sperm for heterologous IVF and springbok and blesbok sperm for homologous IVF. A group of presumptive zygotes from each treatment were examined for sperm penetration and male pronuclear formation after 18h and the remainder were cultured and evaluated for embryo cleavage 22h later. The study shows that Modified Tyrode Lactate in vitro fertilization media supports survivability, capacitation and hyperactivation of springbok, impala and blesbok sperm. Springbok, impala and blesbok post-thawed epididymal spermatozoa are capable of fertilizing domestic cow oocytes under conditions that support domestic cattle IVF. Penetration, male pronuclear formation and embryo cleavage did not differ (p>0.05) between cow oocytes inseminated with sperm from springbok, impala or blesbok however these parameters were higher (p<0.05) for oocytes inseminated with bull sperm. Modified Tyrode Lactate IVF media supported homologous fertilization and embryo development in springbok and blesbok however did not support blastocyst development. These findings suggest that cattle provide a useful model for evaluating springbok, impala and blesbok post-thawed cauda epididymal sperm functionality. Domestic cattle embryo culture conditions need to be modified to promote blastosyst development in these antelope species. Such research provides an important tool in assisted reproductive technology development when high biological value material is utilized for wild species recovery plans.

Comparative histomorphological assessment of the testes of two Clarias species from the Okavango Delta Panhandle, Botswana.

The testes of two Clarias species from the Okavango Delta Panhandle were histomorphologically assessed for differences in structure mainly because the testes varied externally in colour, with Clarias ngamensis having black testes while those of Clarias gariepinus were off-white. Although a detailed histological description of normal testes in C. gariepinus has been compiled based on laboratory experimentation, there is limited histomorphological field data available on both species which could be used as reference material in the histology and histomorphology assessment of gonads both in the laboratory and field experiments. In August 2006 and 2007 fifteen fish per species of C. ngamensis and C. gariepinus were collected from the Shakawe Panhandle in the Okavango Delta. Testicular samples excised from the fish were fixed in 10% neutral buffered formalin and later transported to the University of Johannesburg where laboratory analyses using standard histological procedures were conducted. Microscopic assessments were used to describe the testicular morphology and the reproductive developmental stages. Haematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Perl's Prussian Blue and Gordon and Sweets Silver Stain Solution were used to enhance finer cellular detail and the morphology of various structures present in the catfish testes. Results emanating from this study (a) form a histomorphological baseline set of data from an undisturbed wetland system for comparison between normal morphology and anomalies within the catfish species, (b) contribute to the limited database of the normal histomorphology of gonads in Southern African freshwater fish species and (c) expand the academic knowledge and skills required for the conservation of wetland ecosystems in Southern Africa.

Computer assisted sperm analysis of motility patterns of postthawed epididymal spermatozoa of springbok (Antidorcas marsupialis), impala (Aepyceros melampus), and blesbok (Damaliscus dorcus phillipsi) incubated under conditions supporting domestic cattle in vitro fertilization.

The need for information on the reproductive physiology of different wildlife species is important for ex situ conservation using such methods as in vitro fertilization (IVF). Information on species reproductive physiology and evaluation of sperm quality using accurate, objective, repeatable methods, such as computer-assisted sperm analysis (CASA) for ex situ conservation has become a priority. The aim of this study was to evaluate motility patterns of antelope epididymal spermatozoa incubated for 4 h under conditions that support bovine IVF using CASA. Cauda epididymal spermatozoa were collected postmortem from testicles of springbok (N=38), impala (N=26), and blesbok (N=42), and cryopreserved in biladyl containing 7% glycerol. Spermatozoa were thawed and incubated in Capacitation media and modified Tyrode lactate (m-TL) IVF media using a protocol developed for domestic cattle IVF. The study evaluates 14 motility characteristics of the antelope epididymal sperm at six time points using CASA. Species differences in CASA parameters evaluated under similar conditions were observed. Several differences in individual motility parameters at the time points were reported for each species. Epididymal sperm of the different antelope species responded differently to capacitation agents exhibiting variations in hyperactivity. Motility parameters that describe the vigor of sperm decreased over time. Spermatozoa from the different antelope species have different physiological and optimal capacitation and in vitro culture requirements. The interspecies comparison of kinematic parameters of spermatozoa between the antelopes over several end points contributes to comparative sperm physiology which forms an important step in the development of species specific assisted reproductive techniques (ARTs) for ex situ conservation of these species.

Liver histopathology of the sharptooth catfish Clarias gariepinus as a biomarker of aquatic pollution.

This paper reports on a comparative perspective of liver histopathological data of the sharptooth catfish Clarias gariepinus. The data was collected from a spectrum of relatively un-impacted and isolated, to polluted, eutrophic freshwater ecosystems. Results were compared between regional areas, by combining data from freshwater systems which has a similar pollution status and/or is located within the same geographical region. Measurements included necropsy observations, semi-quantitative liver histopathology (Liver Index), and selected biometrical indices. The aim was to establish whether the results of these measurements would differ between, and/or reflect the pollution status of, the different freshwater aquatic ecosystems. The histopathological analysis showed a higher prevalence of toxicopathic non-neoplastic, and pre-neoplastic alterations in C. gariepinus from the polluted sites. We also found a significant difference between the Liver Index, hepatosomatic index, and condition factor values of fish inhabiting impoundments known to be polluted, compared to the same species from the selected reference sites. Fish from polluted sites also had more macroscopic liver abnormalities. The results suggest that the liver histopathology of this bio-indicator fish species could be a useful biomarker of freshwater aquatic pollution.