Stereological and Histopathological Effects of Trigonella foenum-graecum Seeds on Rabbits Ovary

This study aimed to investigate the impacts of the Trigonella foenum-graecum (T. foenumgraecum) seeds on the female gonad. A total of twenty local rabbits were used in this study; were divided into four groups (5 each): first group (G1) was considered as the control group. The second group (G2), third group (G3) and fourth group (G4) were fed daily1.5%, 3%, and 4.5% of T. foenum-graecum seeds respectively for 60 days (twice daily). At the end of the experiment, the animals were euthanized by diethyl ether (C2H52O). Then the abdomen was incised, and the samples of ovaries were collected and fixed by 10% neutral buffered formalin. The histological assessment was done with a paraffin embedding technique and the histological sections were stained with Hematoxylin and Eosin stain. The result showed that the numbers of primary and secondary follicles were significantly P< 0.05 decreased in G3and G4 compared with the control (G1) and G2. The numbers of Graafian follicles were significantly P<0.05 decreased G4 compared with other groups. The diameters of the primary, secondary, and Graafian follicles were significantly lower than the other groups. The thickness of the granulosa cell layer in G3and G4 were significantly lower than the other groups. The histological figures declared that the ovary of G2 was similar to that in G1. The histological sections of G3 and G4 were revealed marked cortical and medullary vascular congestion and focal hemorrhage; there were also marked follicular degeneration and cystic necrosis. The study concluded that the low concentration of T. foenum-graecum (fenugreek) seeds do not have any positive effect in terms of ovarian stimulation. T *Correspondence: tamara.natiq@covm.uobaghdad.edu.iq Received: 23 March 2020 Accepted: 1 September 2020 Published: 28 December 2020 DOI: https://doi.org/10.30539/ijvm.v44i(E0).1016 This article is an open access distributed under the terms and conditions of the Creative Common Attribution License (CC BY 4.0)


INTRODUCTION
he chief objective of the plants' is the food source, several types of uses as the provenance of medicine (1). Trigonella foenum-graecum(Fenugreek) is a leguminous grass cultivated in different countries around the world such as Asia and Africa; seeds of Fenugreek are known in ancient before 2500 years ago; it is utilized as a classical feed and feed additive in addition to its medicinal uses flatulence, dyspepsia, bronchial asthma and antitussive (2), other uses in cook as flavoring agents (3).The most active medical ingredient of fenugreek seeds, progesterone and estrogen-like effect (4,5).The rabbit ovaries are relatively small in size and have flattened oval shape. Each ovary cell surface is separated from underlying tissue by tunica albuginea (fundamental basement

A B S T R A C T
This study aimed to investigate the impacts of the Trigonella foenum-graecum (T. foenumgraecum) seeds on the female gonad. A total of twenty local rabbits were used in this study; were divided into four groups (5 each): first group (G1) was considered as the control group. The second group (G2), third group (G3) and fourth group (G4) were fed daily1.5%, 3%, and 4.5% of T. foenum-graecum seeds respectively for 60 days (twice daily). At the end of the experiment, the animals were euthanized by diethyl ether (C2H52O). Then the abdomen was incised, and the samples of ovaries were collected and fixed by 10% neutral buffered formalin. The histological assessment was done with a paraffin embedding technique and the histological sections were stained with Hematoxylin and Eosin stain. The result showed that the numbers of primary and secondary follicles were significantly P< 0.05 decreased in G3and G4 compared with the control (G1) and G2. The numbers of Graafian follicles were significantly P<0.05 decreased G4 compared with other groups. The diameters of the primary, secondary, and Graafian follicles were significantly lower than the other groups. The thickness of the granulosa cell layer in G3and G4 were significantly lower than the other groups. The histological figures declared that the ovary of G2 was similar to that in G1. The histological sections of G3 and G4 were revealed marked cortical and medullary vascular congestion and focal hemorrhage; there were also marked follicular degeneration and cystic necrosis. The study concluded that the low concentration of T. foenum-graecum (fenugreek) seeds do not have any positive effect in terms of ovarian stimulation. T *Correspondence: tamara.natiq@covm.uobaghdad.edu.iq membrane) which is divided into medulla (inner part) and cortex (outer part). Cortex is divided into follicles and stroma components (6,7). Seeds of T. foenum-graceum possess estrogen-like activity, so it can increase the size of ovary through folliculogenesis enhancement. The increment of ovarian size is promoted by a growing and increase the number of resurgent follicles types as a result to estrogenic effect dominants (5). In the female, the main site of estrogen secretion is the ovarian granulosa cells and theca interna 8). Therefore, this study was aimed to determine the effect of T. foenum-graecum seeds on the female gonads.

Experimental Animals
The procedures used in this study were reviewed and approved by the scientific committee at the University of Baghdad's College of Veterinary Medicine in compliance with animal welfare ethical standards.
This study used a total of twenty local female rabbits that were 8-10 months old and weighed 1.6-1.8 kg. The animals were housed in the animal house of the College of Veterinary Medicine, University of Baghdad during September to November 2019, at a temperature of 25±2 °C, with a light-dark cycle of 12:12 hours, and standard ventilation system and humidity (2). Rabbits were divided into four groups; (of 5 each). The first group (G1) was served as a control group which fed 100 g/daily concentrate diet (pellets) and green forage. The second group (G2) was fed 100g/ daily of ration supplemented with 1.5% T. foenum-graecum seeds. The third group (G3) was fed daily of 100 g of ration supplemented with 3% T. foenum-graecum seeds and the fourth group (G4) was fed daily 100 g of ration supplemented with 4.5% T. foenumgraecum seeds. The study lasted for 60 days. Rabbits fed on a concentrate diet (pellets) and green forage as a preliminary period for 2 weeks for acclimatization.

Plant
The T. foenum-graecum seeds were obtained from a local market at Baghdad province, Iraq, cleaned and authenticated in the Iraqi National Herbarium.

Histopathological and Stereological Evaluation
Histopathological processes were performed at the end of the experiment after euthanizing the rabbits by diethyl ether and incising the abdomen of animals. The ovaries were obtained and fixed with 10% neutral buffered formalin, then prepared for paraffin embedding technique and sectioned at 5-6µm and stained with hematoxylin and eosin stain (9). Tissue sections were examined and photographed by Olympus microscope SC 35 camera.
The stereological assessment was included the numbers, diameters of different types of ovarian follicles and thickness of their granulosa cells layer. The images was analyzed and scored using Fiji image analyzer system (10).

Statistical Analysis
Data were analyzed by SPSS (version 24.0). All numerical results were expressed as the mean± standard error (SE). For comparisons, the statistical significance was assessed by ANOVA. The significance level was set at P<0.05 (11).
This study agreed with the result of (12, 13) who refereed to a decrease in the number of primary and secondary follicles and the diameter of secondary and antrum follicles, on the other hand this result disagreed with the result of (13). The current results agree with opinion of (14) who referred to the adverse effect of methanolic extract of Rumexsteudelii which induced decreasing the number of follicles, corpora lutea and atrophic changes in the uterus. The decrease in numbers of ovarian follicles is attributed to the effect of Toenum-graecum seeds on the levels of FSH and LH that cause disturbance of folliculogenesis (14) and this disturbance could be attributed to the affecting of the normal negative feedback mechanism. (15). Additionally, it could be attributed to the plant extract for containing phenolic compound effects on the secretion of estrogen and progesterone. Jhon et al. (16) showed that plant extract may have an effect on the production and secretion of FSH which secret from the pituitary gland and these changes in the secretion of the FSH and LH may occurred as a result from the effect of caffaic acids and cause a reduction in reproduction. On the other hand (17) revealed that the exogenous sources of estrogen may lead to decrease the number of different types of follicles, cause of cystic follicle and decrease in the number of the corpus luteum in female rat. Other study revealed that 3% of T. foenum-graecum seeds increased the concentration of estrogen and the concentration of progesterone has significantly decreased at 1.5% of T. foenum-graecum (7). The research also revealed that the offspring percentage increased significantly at 1.5% and 3% (19).

Histopathological Examination
Histopathological examination of ovaries in local female rabbits fed 1.5%, of T. foenum-graecum seeds for 60 days revealed that there were no significant effects when compared to the control group (Figures 1 and 2). However, the histological sections of female rabbits fed 3% and 4.5% seeds of T. foenum-graecum showed marked cortical and medullary pathological changes including marked vascular congestion, focal hemorrhage, follicular degeneration, and necrosis with cystic like-depletion. The degenerated follicle revealed marked vacuolar degeneration and necrosis of granulosa cells in addition to sloughing of granulosa cell layer from zona pellucida, edema of theca externa, and the stromal cells showed severe cellular swelling and necrosis (Figures 3, 4, 5, and 6). Such result agreed with the result of (1) who referred to many histological changes involved atretic changes. The histopathological changes were related to the presence of the saponins, steroids, and amino acids in fenugreek seeds extract in a dose of 200 mg/kg BW (6). On the other hand, in male reproductive and adenohypophysis, there were significant effects of aqueous extract of fenugreek seeds which may lead to decrease the acidophilic cells and increase the basophilic cells with great numbers of round cell debris may be seen in the lumen of epididymis; an increase in desquamation of epithelial cells within fluid secretion of the seminal vesicle (15). In present result the follicular number decreased in the groups G3 and G4 that treated with 3% and 4.5% of T. foenum-graecum seeds, respectively. The above results agreed with (15) who mentioned that administration of T foenum-graecum extract may affect the fertility and prolificacy percentages. Kaisser et al. (19) showed that in treated rabbits, the number of Graafian follicles increased in addition to granulosa cell became associated with clear decrease in the fat percentage and their plasma membrane, while the primary and secondary follicle number was also decreased. On the other hand the current result disagreed with the result of (2) who revealed that the extract of Toenumgraecum seeds act as antioxidative and anti-inflammatory and potential role in preventing hepatic and renal damage which induced by chronic NaNO2 administration.
From the results of current study it could be concluded that low dietary concentration of fenugreek seeds do not have positive effect in terms of ovarian stimulation. On the other hand, higher dietary concentration of T. foenumgraecum seeds could produce adverse effects on the reproductive efficiency and follucologenesis.