Glyphosate is the most heavily used agricultural and residential herbicide and has been detected in soil, air, surface water, and groundwater, as well as in food. Hence, it has been inadvertently getting added to our ecosystem and it certainly has reached plants, animals and food chain. In the context of India, the glyphosate is one of the mostly used as non-specific organophosphate pesticides among the agrochemicals (PAN India Pacific, 2020). It is used to control weeds in agriculture, plantations, forestry, industrial areas, houses etc. Indian Council of Agricultural Research’s report revealed that, two formulations of glyphosate, 41% SL and 71% SG are widely used in at least 22 Indian states for several food crops (cereals, pulses, vegetables, fruits, and spices) and non-food crops such as rubber plantation, and forestry activities¹. The practice is on the rise as it has become the cheap option to replace the mechanical means of weed control. Use of glyphosate has been increased manifold after introduction of genetically modified glyphosate resistant crops such as cotton, soybean, corn and canola. In India glyphosate usage has increased from 435 MT technical grades in 2010-11 to 765 M.T. in 2018-19 (Statistical Database, Pesticide Monitoring and Documentation Unit, PPQ&S, Ministry of Agriculture, Government of India). Glyphosate and its metabolite amino-methyl-phosphonate (AMPA) could be detected in green immature seed, harvested seeds and in ground water^2-4. The herbicide is being reported to have harmful effects on human, animal and environmental health. Gestational period in humans was found to be shortened by glyphosate and glyphosate-based formulations in an Indian Cohort study (USA) from women living in rural and non-rural areas⁵.

Few epidemiological studies, the dichotomous analysis of exposures revealed that glyphosate-based pesticides don’t pose developmental risks to the unborn child⁶. But, other epidemiological studies have come out with the adverse effect like infertility and subfertility in male and female along with unfavourable pregnancy. Besides that, few in vitro and in vivo studies have revealed that glyphosate and its formulations exhibit estrogen-like properties, and it indicate about the disruption of normal endocrine function, with adverse consequences to reproductive health⁷. Further deleterious effects were recorded as changes in the normal structure of ovary, delayed folliculogenesis, hormonal imbalance, increases oxidative stress, abnormal aromatase activity, implantation failure and infertility through in-vivo and in-vitro studies^8&9. Apart from that, few studies revealed that glyphosate and glyphosate-based formulations may interrupt normal endocrine function affecting the reproductive potentiality¹⁰.

The experiment was conducted for the period of 3 months in order to determine the effects of chronic toxicity of glyphosate in kuroiler chickens. The experiments were performed after obtaining the necessary permission from the Institutional Animal Ethics Committee (IAEC) of College of Veterinary Science, AAU, Khanapara, Guwahati, Assam with approval No. 770/ac/CPCSEA/FVSc/AAU/IAEC/17-18/559 dated 09.08.2017. The regulations addressing animal use were followed. Total 16 numbers of kuroiler birds of the age of 3 months were taken for this experiment. Birds were provided with ad libitum clean drinking water and standard pellet diet and were kept under constant observation during the entire period of study. Glyphosate, field grade (HIJACK, 41% glyphosate SL, (Insecticide India Ltd., Rajasthan) was used for producing experimental toxicity. One group of 4 pairs of birds were given ad libitum water and standard ration. All the experimental birds were properly vaccinated against all possible viral diseases such as, Ranikhet Disease (RD), Infectious Bursal Disease (IBD), Infectious Bronchitis (IB). Another group consisting of 8 birds were given glyphosate orally @ 27.8 mg/Kg body weight for 3 months. Doses were calculated on the basis of pilot study for LD₅₀ estimation as per OECD 425 and it was confirmed by the method as described by earlier workers¹¹. Representative tissue samples were collected for histopathological examination and formalin fixed samples were processed by standard procedure¹². The tissue sections were stained with Haematoxylin and Eosin¹³. Tissue sample especially testis in this case were considered for the ultrastructural studies (TEM). Hence, the testes were selected having most striking lesions from the glyphosate exposed birds for transmission electron microscopic examination. The specimens were fixed in 1% Karnovsky’s fixative. The samples were kept at 4°C for 4 hours. Then washed in 0.1M Sodium Cacodylate buffer (3 changes of 15 minutes in each) and stored in Sodium Cacodylate buffer¹⁴.

Then the fixed tissue samples were placed in 0.1M Karnovsky’s buffer for 15 minutes at 4°C. The step was repeated 3 times in 0.1M karnovsky’s buffer for 15 minutes each at 4°C. The samples were stored in 0.1M sodium cacodylate buffer at 4°-8 °C. The transportation of samples was done in 1.5 ml eppendorfs (microcentrifuge tube) in the 0.1M Sodium Cacodylate buffer and was submitted to the SAIF, NEHU (North Eastern Hilly University), Shilong, Meghalaya for further processing. The samples were further washed in 50% ethanol for 5 minutes and placed in saturated uranyl acetate. Then dehydrated in ascending ethanol and preparations were washed three times with propylene oxide and embedded in Epon/Araldite. Ultra-thin sections (700–800A°) were collected on uncoated copper grids, and stained with Uranyl acetate and Lead citrate. After staining, the sections were washed with distilled water. The grid was allowed to dry with the sections upward on the filter paper. The stained sections were mounted on a digital transmission electron microscope (JEM-2100) which was operated at an accelerating voltage of 120 kv. The TEM images were formed using electron transmitted through ultra thin section (50-90 nm). The achievable magnification of this instrument was from 50X up to 15,00,000 X and the highest point resolution was 0.19 nm. The images were viewed over a fluorescent screen and recorded on photographic or through a 7 megapixel CCD camera. The areas of significance were first observed under view finder, thereafter fine focused in a high-end digital monitor. The images were captured and saved electronically for further analysis.

The females of chronic study group did not lay any eggs while females from the control groups laid eggs during proper time after sexual maturity. It might be due to delayed development of the reproductive organs. No apparent gross changes were observed in testes but at the end of the experiment, the ovaries of glyphosate intoxicated group females revealed fewer follicles with presence of ovarian cysts (Fig. 1) in comparison to female birds of control group (Gr.I). Clinicopathological findings showed, significant decrease in Hb (gm%), PCV (%), TEC but leucocytosis was observed in glyphosate experimental intoxicated group of birds in different days interval as comparison to control group (Table 1 & Fig. 1).

In the second month of experiment, testis showed degeneration and necrosis in the lining epithelium of seminiferous tubule and surrounding interstitial tissue were replaced by fibrous connective tissues observed during histopathological examination (Fig. 2). The thickening of tunica albugenia and proliferation of connective tissue in the intertubular space were observed (Fig. 3). In some birds, spermatogonia and sertoli cells were found in the interstitial spaces due to rupturing of seminiferous tubules.

The ovaries of glyphosate intoxicated hens revealed numerous degenerated follicles along with mature follicles without oocytes. The ovarian oocytes without nucleus were found to have consistent histopathological features (Fig. 4). The micro vacuoles were also observed in ovaries in few hens (Fig. 5). The developing follicles and mature oocytes showed karyolysis with formation of vacuoles in the yolk-laden cytoplasm (Fig. 6).

Ultrastructural examination of testes of experimentally glyphosate induced toxicity birds revealed, disruption to the leydig cell and spermatogonium, disintegration of nuclear material, dilatation of endoplasmic reticulum (arrow) (Fig. 7), lysis of cristae of mitochondria, increase intracytoplasmic vacuolisations (Fig. 8) and presence of excess membrane bound vesicles in the cells of germinal tissues.

The female birds of experimentally induced glyphosates toxicity group (group II) failed to produce any eggs while female birds of the control groups started laying eggs around 6 months of age. Similar study in wistar rats has revealed that glyphosate altered estrogen-regulated gene expression and negatively impacted on egg production^15,16.

Haematological investigation in the present study revealed decreased Hb concentration and that might be due to haemolysis caused by glyphosate toxicity. Parallel observation was also recorded in earlier experiment in mice exposed to glyphosate¹⁷.

In experimental chronic glyphosate toxicity, testis showed degeneration and necrosis of seminiferous tubules and fibrous connective tissues proliferation. Besides that, thickening of tunica albugenia and proliferation of connective tissue in the intertubular space of testis was also recorded. In this study, the spermatogonia and sertoli cells were found in the interstitial spaces due to damage or rupture of seminiferous tubules. The changes might have been occurred due to damage of blood testis barrier and oxidative stress caused by chronic glyphosate exposure¹⁸. Similar histopathological lesions like testicular tissue damage in experimentally glyphosate exposed male rats were documented by earlier workers¹⁹.

Degenerated follicles along with mature follicle without oocytes and oocytes without nucleus as well few vacuoles with karyolysis and formation of vacuoles in the yolk-laden cytoplasm in ovaries were striking histopathological lesions in this study. In the present study the gross and microscopic alterations in ovary might have been due to oxidative stress caused by the toxin or its metabolites. The pronounced histological alterations like complete absence of newly formed corpora lutea and regressed form of luteal tissues in addition to atretic antral follicles were documented in the ovaries of female rats after 30 days post glyphosate exposure²⁰. Rounded seminiferous tubules bound by a fibrous capsule or accumulation of collagen and fibrosis surrounding the seminiferous tubules were also observed in other animals^21,22,23&24. The pathological changes like fibrosis and breach of integrity of seminiferous tubules might have been attributed negatively to spermatogenesis.

The ultrastructural examination of testes of glyphosate intoxicated birds revealed disruption of testicular cells, disintegration of nuclear material, dilatation of endoplasmic reticulum, cristeolysis of mitochondria and increase intracytoplasmic vacuolation. The similar ultrastructural alterations were documented in adult male albino rats after six weeks exposure to glyphosate²⁵.

The present study was carried out with an aim to elucidate the role or impact of glyphosate on reproductive behaviour or performances. The pathomorphological observations of gonads demonstrate that there was a definite impact of chronic glyphosate toxicity in terms of exposition of gross, microscopic and ultrastructural lesions. It is further ascertained by the compromised or detrimental reproductive performances. The pathological alterations due to glyphosate tropism to gonads may be due to special immune privileged organs and it needs to be investigated. Apart from that, this study highlights the relevance of studying the glyphosate toxicity with respect to the extent to which it impairs reproduction.

P < 0.05

P < 0.01. Means with the same letters are not significantly different.

Capital letter superscripts denote difference between groups and small letter subscripts denote difference within group.

References