Studies on reproductive pathology and teratogenic effects in experimentally induced hypothyroidism in Sprague Dawley rats and amelioration with Withania Somnifera and Shilajit Ramya B.1,*, Kumar Anand A.2,**, Reddy Gopala A.3, Purushotham G.4, Shivakumar P.5 1Veterinary Pathology, VCC, College of Veterinary Science, Mamnoor-506 001 2Department of Veterinary Pathology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati-517 502 3PV Narasimha Rao Telangana Veterinary University, Hyderbad-500 030 4College of Veterinary Science, Mamnoor, PV Narasimha Rao Telangana Veterinary University, Telangana-506 001 5Department of Veterinary Pharmacology and Toxicology, AHP, Mamnoor-506 166 *Address for Correspondence Ramya B., Assistant Professor, Veterinary Pathology, VCC, College of Veterinary Science, Mamnoor-506 001, India, E-mail: drramyavet@gmail.com
**Anand A. Kumar, Professor and Head, Department of Veterinary Pathology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati-517 502, India, E-mail: 7aakumar@gmail.com
Online Published on 27 March, 2024. Abstract The experiment was conducted to study the reproductive pathology and teratogenic effects in methimazole induced hypothyroidism in rats and its amelioration. Twenty one days old weaned, female Sprague Dawley rats (96) were divided into 8 groups with 12 animals in each group. They were treated as Group 1: Euthyroid, Group 2: Hypothyroidism induced with methimazole @ 0.02%, Group 3: Hypothyroid + LT4, Group 4: Withania somnifera root extract control, Group 5: Hypothyroid + Withania somnifera root extract, Group 6: Shilajit control, Group 7: Hypothyroid + Shilajit and Group 8: Hypothyroid + Withania somnifera root extract + Shilajit, both @ 100 mg/Kg b. wt. At the end of 3 months, 6 rats from each group were sacrificed. Blood was collected to estimate thyroid profile and estrogen levels. Organs (thyroid, uterus and ovaries) were collected for histopathology studies. The remaining rats were mated with euthyroid adult males and all 48 pregnant rats were administered with treatments as above till the 17th day of gestation. Progesterone levels in pregnant dams and pup survival and mortality rates were estimated. Half of the pups from each group on zero day of their birth were preserved in 10% neutral buffered formalin (NBF) for histopathology (whole pup as a unit) and skeletal staining was done to view the teratogenicity. The remaining half were reared for 21 days and were then sacrificed and various organs (uterus, testis and thyroid) were collected for histopathology studies. The sections of the thyroid gland, reproductive organs (parent stock and F1 generation) and visceral organs of the whole pup of group II rats revealed marked degenerative changes. Pups stained with Alizarin red S from group II showed skeletal deformities. The present study showed teratogenicity and deviations in reproductive organs in the hypothyroidism model and the usage of herbal drugs have shown improvement in alleviating the toxic effects of hypothyroidism. Top Keywords Histopathology, Hypothyroidism, Methimazole, Shilajit, Teratogenicity, Withania somnifera. Top |
Introduction Thyroid hormones are metabolic hormones produced by the thyroid gland and are present in two main forms: the prohormone thyroxine (T4) and the biologically active form triiodothyronine (T3). THs play a crucial role in various aspects of an individual's life, for example, development, metabolism and reproduction, across vertebrates, including humans1. Maternal hypothyroidism (MH) is the most common cause of transient congenital hypothyroidism. Regarding development of fetal body systems during pregnancy, interference at different times provides different results and the appropriate time for induction of hypothyroidism should be selected based on accurate time of development of the system under assessment2. In females hypothyroidism can fabricate reproductive problems and results in improper progress of offspring with stunted growth and mental retardation if untreated3. |
Scientific investigations towards the mitigation of thyroid disorders by the plant extracts are running fast but, the solitary hitch was that, only one thyroid hormone among T3, T4 or TSH was altered by the plant extract4. Though many herbs are available for the effective function of thyroid hormones, the present study was conducted to find out the efficacy of the herbs that can regulate the levels of thyroid hormones independently or in combination by using Shilajith and Withania somnifera. |
Top Materials and Methods Ninty six female rats (21 days old weaned) Sprague-drawly rats were picked up from Mahaveera Enterprises, Hyderabad. Rats were nursed in the modern animal house of the Department of Veterinary Pharmacology and Toxicology with a 12 h - 12 h dark and light cycle at a temperature of 22-24OC. Rats were enclosed in hard base polypropylene cages, placed on standard pellet feed and provided ad libitum water. Rats were adapted for five days before the experiment. The study was conducted with prior approval of the Institutional Animal Ethics Committee, CVSc, Rajendranagar (I/6/16/05-01-16). |
Rats were distributed into 8 groups with 12 in each group and the experiment was conducted for 3 months as per the technical program given below: |
Group 2: Hypothyroidism induced with Methimazole @ 0.02% |
Group 3: Hypothyroid + Levothyroxine |
Group 4: Withania somnifera root extract control @ 100 mg/Kg b. wt. |
Group 5: Hypothyroid + Withania somnifera root extract @ 100 mg/Kg b. wt. |
Group 6: Shilajit control @ 100 mg/Kg b. wt. |
Group 7: Hypothyroid + Shilajit @ 100 mg/Kg b. wt. |
Group 8: Hypothyroid + Withania somnifera root extract @ 100 mg/Kg b. wt. + Shilajit @ 100 mg/Kg b. wt. |
Thyroid profile and estrogen levels were estimated from sera of blood collected at the end of the third month by Radio Immuno assay employing DiaSorin S.p.A. kits, USA. At the end of 3 months, 6 rats from each group were sacrificed and various organs (uterus, ovary and thyroid) were collected in 10% neutral buffered formal saline for histopathological studies. |
Top Experiment Left over 48 female rats (6 from each group) from experiment 1 were mated with 24 euthyroid adult (above 3 months) males (from Mahaveera Enterprises, Hyderabad) and all the 48 pregnant rats were administered with treatments as in experiment 1, up to 17th day of gestation. |
Progesterone levels in pregnant dams were estimated in the collected serum samples by Radio Immuno assay employing DiaSorin S.p.A. kits, USA. Pups from all the groups were screened for survival and mortality rates. Half the number of the pups from each group on the day of their birth was preserved in 10% neutral buffered formalin (NBF). A few of the preserved pups were processed for histopathology5, to study the section of whole pup as a unit and a few pups were subjected to skeletal staining with alizarin red-S6 to view the teratogenicity. The remaining half balance pups were reared till 21 days. The rats were then sacrificed and various organs (uterus, testis and thyroid) were collected in suitable preservatives for histopathology studies. |
Top Results Thyroid Profile The serum T3 and T4 hormone levels (ng/dl) of group 2 were significantly (p<0.05) lesser than group 1 while the values in groups 3, 4, 5, 6 and 8 were analogous to group 1. T3 and T4 concentration in group 7 was significantly (p<0.05) inferior to that of group 1. The serum TSH hormone concentration (p IU/ml) of group 2 was significantly (p<0.05) elevated than group 1 and was significantly lesser in groups 3 to 8 (Table 1). Serum Estradiol (pg/ml) The serum estradiol concentration (pg/ml) of rats in group 2, was significantly (p<0.05) elevated than control groups and treatment groups (Table 2). Serum Progesterone (ng/ml) The serum progesterone concentration (ng/ml) in groups 1, 3, 4 and 5 was lower than in groups 2, 6 and 7, while it was significantly (p<0.05) higher in group 8 (Table 2). Pup survival/mortality Mortality was not observed in pups and the survival rate was maintained till weaning in all the groups. In parent stock very mild reduction in the size of thyroid in group II rats and no appreciable or significant gross or histological lesions were observed in any of the experimental groups. Thyroid gland The section of thyroid in group II, showed vacuolation, a few follicles contained varied amounts of colloid and the others were devoid of colloid (Fig. 1). In groups V and VII the alterations were mild, nearly comparable with that of control groups (Fig. 2) and in group VIII, the changes are comparable with that of control groups. Ovary In group II, moderately degenerated tertiary follicles mainly along with primordial, primary and secondary follicles, increase in thickness of tunica albuginea, atretic/cystic spaces, oedema, vacuolar degeneration, degenerated oocyst in tertiary follicle were seen (Figs. 3 and 4). In group V and VII, the changes were mild (Figs. 5 and 6) and in group VIII, the changes were almost comparable to normal cosntrol groups. Uterus In group II, glandular hyperplasia and fibroblasts proliferation, mono nuclear cell infiltration, thickened endometrium, disruption, oedema, congestion and hemorrhages were noted (Figs. 7-12). In groups V and VII, discontinuity in surface epithelium with mild to moderate congestion and hyalinization in the circular muscle layer of myometrium was noted (Fig. 13). In group VIII, the surface epithelium was thrown into folds and other changes were comparable to that of normal control groups. Histopathology of whole pup as a unit In group II, the section of the liver showed fatty change, an increase in sinusoidal spaces with mild degenerative changes, central vein congestion and mononuclear cell infiltration (Figs. 14 and 15) and the kidney showed degenerative changes in developing tubules and glomeruli (Fig. 16). In groups V and VII, mild degenerative changes were noted in the brain (Fig. 17). In F1 Generation:No appreciable or significant gross or histological lesions were observed in any of the experimental groups. Thyroid gland The section of thyroid in group II showed altered follicles with variation in shape and size, an increased number of C cells focally and an increase in interfollicular space (Fig. 18) and in group VIII the changes were almost comparable with that of normal control groups. Testis In group II, semniferous tubules degeneration, Leydig cells accumulation in the center, disruption of basement membrane along with tubular epithelium and cuboidal cells at the center were prominent. Different stages of spermatozoa with few defects and moderate hyperplasia were noted (Figs. 19 and 20). In groups V and VII, mild to moderate hyperplasia and hypertrophied semniferous tubules with mild changes in the basement membrane were noted. In group VIII the changes were almost comparable with that of normal control group (Fig. 21). Uterus In group II, vacuolation and hyperplasia of glands were recorded (Fig. 22). In groups V and VII, discontinuity in surface epithelium, mild to moderate hyalinisation in the circular muscle layer of myometrium and mild to moderate hyperplasia was noted. In group VIII, the changes were comparable to that of normal control groups (Fig. 23). Teratogenecity Skeletal staining with Alizarin red S was performed to pups belonging to all groups. The pups from group II, showed skeletal deformities like reduction/absence of growth plate thickness, bending of bones like femur, hidden humerus, lateral deviation of distal half of spine, variation in inter vertebral space with undeveloped/ underdeveloped vertebrae, deviation in collateral alignment of ribs, increased intercostals spaces and thinning of ribs (Figs. 24 and 25). Pups from group V, showed normal growth plates with mild lateral deviation at the distal part of spine (Fig. 26). Pups from group VII showed improved intercostal space with developed growth plates and mild widening of proximal half of the vertebral column (Fig. 27). Pups from group VIII showed well developed growth plates improved intercostal space (Fig. 28). Top Discussion Thyroid profile The significant (p<0.05) decrease in serum T3 and T4 hormone concentration (ng/dl) and significant (p<0.05) increase in TSH hormone concentration (p IU/ml) of the group 2 might be due to methimazole's inhibition of the enzyme thyroperoxidase, that acted in thyroid hormone synthesis by oxidizing the anion iodide (I-) to iodine (I2), hypoiodous acid (HOI), enzyme linked hypoiodate (EOI) facilitating iodine's addition to tyrosine residues on the hormone precursor thyroglobulin, a necessary step in the synthesis of triiodothyronine (T3) and thyroxine (T4). The decrease in T3 and T4 through negative feedback mechanism might have stimulated pituitary to release more thyroid stimulating hormone7. Increase in thyroid hormone synthesis in group 5 rats might be due to immunomodulation effect of herb that has a stimulatory effect on a sluggish thyroid and increased serum T4 concentration8 . Shilajit improved thyroid profile by controlling thyroid gland function through it's adaptogenic activity9. Estrogen and Progesterone The significant (p<0.05) increase in serum estradiol concentration (pg/ml) in hypothyroid group might be due to the result of a permissive action of prolactin that augments the number of luteal LH receptors which favours gonadotrophic action that in turn promotes the conversion of progesterone into androstenodione and then to estradiol10. Further, increased circulating estradiol stimulates expression of cyp19A1 aromatase that binds elevated estrogen receptor beta (ER(3) in granulosa and luteal cells that in turn stimulates estradiol synthesis and release. Thus, a positive feedback loop might have established and lead to a sustained increase in circulating estradiol during hypothyroidism11. The serum progesterone concentration (ng/ml) in group 2 on days 15-21 of pregnancy was higher than other groups might be due to increased prolactin concentrations in altered thyroid profile3. The effect of the ashwagandha and shilajit on gonadal hormone levels might be due to antioxidant effect of herbs which as a reproductive function probably was performed via inhibition of oxidative stress. These can also be correlated with the histopathological studies of the reproductive organs of the present study8. Pup survival/mortality Mortality was not observed in pups and survival rate was maintained till weaning in all the groups might be due to absence of iodine deficiency and presence of only thyroid hormone deficiency which alone cannot result mortality in pups12. Thyroid Gland The lesions observed in the section of thyroid gland in group II of both parent and F1 generation were similar with degree of variation. Follicles shape and size, amount of colloid varied. Some follicles were devoid of colloid Conversion of cell type in the follicular epithelium with presence of C cells and para follicular cells. The changes observed might be due to functional induction of methimazole on thyroid gland13. In groups V and VII, improvement in the structure of follicles and their function was observed and in group VIII, no lesions were noted. These findings might be due to improved functioning of thyroid gland by stimulating the thyroid stimulating hormone by action of Withania somnifera14. Uterus, Ovary and Testis In group II the sections of the reproductive organs in parent stock as well as F1 generation revealed almost in similar nature with degree of variation. In uterus congestion, hemorrhages in endometrium, hyperplasia of endometrial and myometrial glands, mononuclear cell infiltration, fibroblasts proliferation and hyalinization were noted15. In ovary (parent stock) interfollicular thickening, edema, thickened endothelium, degenerated primordial, primary, secondary and tertiary follicles16, degenerated oocysts were recorded17. In testis (F1 generation), semniferous tubule degeneration, leydig cells accumulation towards centre and degenerated cells16, disruption of basement membrane and various stages of defective spermatozoa were observed18. These findings might be due to inhibitory action of thyroid hormones in turn on estrogen and progesterone secretions that can be correlated with the results of present study. The improved lesions in group V and VII and almost no lesions in group VIII might be due to improved functioning of endocrine glands in turn maintenance of balance of hormonal secretion by the action of Withania somnifera, shilajit and their combination19, 20. Whole pup The histopathological lesions in various organs processed as whole pup in group II revealed neurofibrillary degeneration in brain, mild degeneration and increase in interfibrillary spaces with developing myocytes in heart, altered follicles and presence of colloid in few follicles in thyroid gland, severe fatty change, increased sinusoidal space with mild degenerative changes, mononuclear cell infiltration in liver and fatty change, degenerative changes with developing tubules and glomeruli in kidney. The changes observed might be due to structural and functional inhibition of immune organs as well as others that might have acquired from parent stock that were treated with methimazole for inducing hypothyroidism and further the thyroid gland functioning initiates from 15th day onwards in fetus21. So this can be attributed as up to first two weeks of fetal stages the changes in the organs must be due to inheritance and further the improvement might be due to function of its own thyroid gland. No literature on this aspect was available and may be the first report. The improved status in groups V and VII and in group VIII can also be explained as above with relevancy. Teratogenisity Pups from group 2 showed skeletal deformities like reduction/absence of growth plate thickness, bending of bones like femur, hidden humerus, lateral deviation of distal half of spine, variation in inter vertebral space with undeveloped/underdeveloped vertebrae, deviation in collateral alignment of rib 6, increased intercostal spaces and thinning of ribs. These abnormalities might be due to administration of methimazole that might have reulted developmental hypoplasia and functional hypothyroidism, leading to severe embryopathies in fetuses22. Skeletal changes in pups might be due to inhibition of GSH synthesis or reduced GSH, that might act as detoxificant of a xenobiotic free radical intermediate and protects embryo from tereatogenic effects. GSH might also act as cytoprotective against oxidative stress23. Pups from rats of groups 5 and 7 revealed less teratogenic effects compared to group 2 due to antioxidant actions of Withania somnifera and shilajit along with free radical scavenging nature, thus reduced free radicals decrease GSH which was a major terarogenic agent8, 24. In the present study, amelioration effect of Withania somnifera was more than Shilajit might be due to its slow action on the injured cell and the combination of both drugs had synergystic effect resulted in high amelioration than Withania alone as ayurvedic medicine generally prefers combinations of herbs rather than single herbs25. Top Conclusion Hormonal and histopathological alterations in the present study indicated reproductive organ damage at the molecular level. Whole pup histopathology indicated organ damage might be due to induced hypothyroidism. The skeletal abnormalities can be well attributed for induced hypothyroidism. Ameliorating actions of Shilajit alone were less significant than Withania somnifera alone and combination of the herbs. Administration of Withania somnifera and Shilajit individually and combined alleviated the deleterious effects caused by hypothyroidism due to their antioxidant and endocrine stimulant properties. Synergistic action of herbs in ameliorating the effects of hypothyroidism is far better than administration of individual herbs. |
Top Figures | Fig. 1:: Fig. 1 Section of thyroid gland showing vacuolation, some follicles contained varied amounts of colloid, the others were devoid of col-lod in group II (H&E X400)
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| | Fig. 2.: Section of thyroid gland showing mild changes in follicles with varied amounts of colloid in group VII (H&E X400)
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| | Fig. 3.: Section of ovary showing moderately degenerated tertiary follicle mainly along with primordial, primary and secondary follicles. Increase in thickness of tunica albuginea, atretic/cystic spaces, oedema, vacuolar degeneration, degenerated oocyst in tertiary follicle in group II (H&E X400)
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| | Fig. 4.: Section of ovary showing fibroblasts proliferation with thickened endothelium in group II (H&E X400)
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| | Fig. 5.: Section of ovary showing mild oedema and degeneration of follicles in group V (H&E X400)
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| | Fig. 6.: Section of ovary showing mild degenerations in follicular granulosa and follicles in group VII (H&E X400).
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| | Fig. 7:: Section of uterus showing glandular hyperplasia and fibroblasts proliferation in group II (H&E X400)
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| | Fig. 8.: Section of uterus showing mono nuclear cell infiltration in group II (H&E X400)
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| | Fig. 9.: Section of uterus showing thickened endometrium, disruption, oedema, congestion and hemorrhages in group II (H&E X100)
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| | Fig. 10.: Section of uterus showing severe mononuclear cell infiltration and lymphoid aggregates in group II (H&E X400)
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| | Fig. 11.: Section of uterus showing glandular hyperplasia in endometrium in group II (H&E X400)
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| | Fig. 12.: Section of uterus showing thick luminal epithelium and fibroblasts proliferation in group II (H&E X400).
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| | Fig. 13:: Section of uterus showing discontinuity in surface epithelium with mild to moderate congestion and hyalinisation in circular muscles in group VII (H&E X400)
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| | Fig. 14.: Section of liver showing fatty change, increase in sinusoidal spaces with mild degenerative changes in group II (Whole pup) (H&E X400)
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| | Fig. 15.: Section of liver showing central vein congestion and mononuclear cell infiltration in group II (Whole pup) (H&E X400)
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| | Fig. 16.: Section of kidney showing fatty change, degenerative changes with developing tubules and glomeruli in group II (Whole pup) (H&E X400)
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| | Fig. 17.: Section of brain showing mild degenerative changes in group VII (Whole pup) (H&E X400)
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| | Fig. 18.: Section of thyroid gland showing altered follicles with variation in shape and size, increase number of C cells focally and increase in interfollicular space in group II (H&E X400).
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| | Fig. 19:: Section of testis showing different stages of spermatozoa with few defects in group II (H&E X400)
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| | Fig. 20.: Section of testis showing semniferous tubular degenerations, accumulated ley dig cells in the centre and disruption of basement membrane along with tubular epithelium in group II (H&E X400)
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| | Fig. 21.: Section of testis almost comparable with that of normal control groups in group VIII (H&E X400)
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| | Fig. 22.: Section of uterus showing vacuolation and hyperplasia of glands in group II (H&E X400)
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| | Fig. 23.: Section of uterus showing no changes and can be comparable to that of normal control groups in group VIII (H&E X400)
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| | Fig. 24.: Pup showing moderate lateral deviation at distal half of spine with under developed vertebrae, thin ribs, loss of growth plate, improper bone alignment in hind limbs in group II.
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| | Fig. 25:: Pup with hidden humerus in right fore limb, bending of femur in left hind limb and broad paw in right hind limb in group II
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| | Fig. 26.: Pup showing normal growth plates with mild lateral deviation at the distal part of spine in group V
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| | Fig. 27.: Pup with improved intercostal space with developed growth plates and mild widening of proximal half of the vertebral column in group VII
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| | Fig. 28.: Pup with well developed growth plates improved intercostal space in group VIII.
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Tables | Table 1:: Serum thyroid profile in different groups of rats (Parent stock).
| | Groups (n=6) | T3 (ng/dl) concentration | T4 (μg/dl) concentration | TSH (μ IU/ml) concentration | | Group 1 | 115.68±4.46b | 4.71±0.14b | 0.05±0.01a | | Group 2 | 101.15±4.06a | 3.01±0.24a | 5.91±0.45c | | Group 3 | 110.73±4.84b | 4.36±0.18b | 0.87±0.03b | | Group 4 | 118.03±4.69b | 4.79±0.17b | 0.03±0.007a | | Group 5 | 111.74±4.17b | 4.24±0.17b | 1.04±0.03b | | Group 6 | 116.62±5.02b | 4.69±0.19b | 0.04±0.009a | | Group 7 | 106.78±4.71a | 3.45±0.12a | 1.91±0.16b | | Group 8 | 113.19±4.76b | 4.31±0.21b | 0.51±0.12b |
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| Means with different superscripts differ significantly (p<0.05), One way ANOVA (SPSS:15) | | | Table 2:: Serum estradiol (pg/ml) and progesterone concentration (ng/ml) concentration in different groups of rats (Parent stock).
| | Groups (n=6) | Estradiol concentration (pg/ml) | Progesterone concentration (ng/ml) | | Group 1 | 23.72±3.07a | 47.26±3.19a | | Group 2 | 37.43±3.78c | 57.67±5.46ab | | Group 3 | 29.57±3.23b | 54.12±4.12a | | Group 4 | 22.94±2.79a | 53.71±4.14a | | Group 5 | 31.66±3.07b | 49.22±3.67a | | Group 6 | 21.03±3.15a | 59.43±5.44ab | | Group 7 | 33.07±4.01b | 56.04±5.01ab | | Group 8 | 26.35±2.87ab | 61.72±5.01b |
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| Means with different superscripts differ significantly (p<0.05), One way ANOVA (SPSS:15) | |
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