Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
232
IMPACT OF AQUEOUS EXTRACT OF ROSEMARY ON
TESTICULAR TISSUE IN MALE RATS WITH
HYPERTHYROIDISM
Fouad Ziedan Hamza*, Sumayah Faruq Kasim **, Sarah Qahtan M. Salih**
*Faculty of Veterinary Medicine, University of Kufa, Kufa, Iraq
**College of Health and Medical Technology, Middle Technical University, Baghdad, Iraq.
Corresponding Author: fouadz.alquraishi@uokufa.edu.iq
Keywords: Hyperthyroidism, Rosemary, Levothyroxine.
ABSTRACT
The aim of the study to evaluate the enhanced effect of aqueous extract of
rosemary on testis tissue in male rats after inducing hyperthyroidism by L–thyroxin and
compare with propylthiouracil effect. The animals (32 male rats) were divided into two
groups. The first group was a control group consists of 8 male rats that were treated with
distilled water for 12 days and the rest of the animals induced with hyperthyroidism by
levothyroxine 0.5 mg / kg subcutaneously for 12 days. After the induction, the animals
were divided into four groups each consists of eight male rats. The first group was the
control group and they were treated with distilled water for another 12 days, the second
group included male rats induced hyperthyroidism were treated with distilled water for 12
days orally, while the third group included male rats induced hyperthyroidism that were
treated with propylthiouracil at a dose of 10 mg / kg for 12 days orally, and the fourth
group included male rats induced hyperthyroidism that were treated with rosemary extract
at a dose of 12 mg / kg for 10 days, orally. The results showed that rosemary extract led to
complete healing of testicular tissue, unlike propylthiouracil.
INTRODUCTION
The testes are chiefly involved in tightly twisted seminiferous tubules, which are
provided by the loose interstitial connective tissue wherever the steroidogenic Leydig
cells are lying (1). Every tubule made up of a basement membrane, peritubular myoid
cells, and elastic fibers. inside the basement membrane, seminiferous tubules are filled by
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
233
a columnar epithelium that consists of somatic Sertoli cells and germ cells. the close
Sertoli cells are linked by tight specialized junctions to form the diffusion barrier, the socalled
blood-testis barrier, that divides the seminiferous tubule into two functional
sections, the basal and the adluminal.The basal compartment composed of spermatogonia,
Sertoli cells, and preleptotene/leptotene spermatocytes (2).
Hyperthyroidism is characterized by increased circulating T4 levels, compromised
responsiveness of LH and follicle-stimulating hormone, altered endocrine profile, all of
which result in impaired testicular functions, morphology, reduced seminiferous tubule
diameter, delayed spermatogenesis, stunted sperm development and reduced sperm
motility (3).
Thyroid hormone is a major metabolic regulator of testicular development and
function that could influence spermatogenesis (4).
R. officinalis L., publicly known as rosemary, is a herbal plant belonging to the
Lamiaceae family and originated from the Mediterranean zone. However, it can be found
everywhere in the world. It is an aromatic and perennial plant, bush-shaped with full
leaves branches, having up to two meters height and green leaves that exude a
characteristic aroma. R. officinalis might be applied as a spice in cooking, as food
industry natural preservative, and as an ornamental and medicinal herbal plant (5, 6).
Administration of rosemary orally reduced fertility in male albino rats. The
weights of reproductive organs were markedly decreased. The weight, size, and secretory
function of testes, epididymides, seminal vesicles, ventral prostates, and vasa deferentia
are closely regulated by androgens (7).
Propylthiouracil (PTU) is an anti-thyroid drug which prohibits both the synthesis
and release of thyroid hormones in thyroid gland (8). Previous studies have shown that
transient neonatal hypothyroidism, induced by treatment with PTU, increases testicular
size, Sertoli cell numbers, and daily sperm production in the adult rat and mouse (9). This
study revealed the effect of aqueous leaves extract of rosemary and Propylthiouracil on
testicular tissue in male rats with hyperthyroidism.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
234
MATERIALS AND METHODS
1. Chemicals:-propylthiouracil (PTU) and-L-Thyroxine (T4)
2.Preparation of extracts: The leaves of Rosemary were brought from a market in Kufa,
the leaves were ground and stored in the dark. 10 g of the powder was stirred in 100 ml of
distilled water at 50 °C for half an hour. Then through a rough cloth and a filter paper, the
extract was filtered, and then the filter was dried completely by freezing and then it was
used (10).
3.Histopathological analysis: The animals were sacrificed under light anesthesia and testis
were immediately removed and saved in formalin 10%, and then it was prepared for
standard methods of tissue segmentation and examined under a light microscope.
4. Experimental animal design:
Induction of hyperthyroidism by L-thyroxin:
Thirty-two male rats were divided into two groups. The first group included 8 rats, the
control group was received 0.1 ml/day distilled water orally by gavage. The second group
included 24 rats that were treated with Levothyroxine for 12 days 0.5mg/kg
subcutaneously for the purpose of induction of hyperthyroidism (11).
After the induction of hyperthyroidism we divided the animals into 4 groups, each
group containing 8 male rats that treated for 24 days as a negative control group, animals
were received 0.1 ml/day distilled water orally by gavage for 24 days.
A positive control group (hyperthyroidism), animals were treated subcutaneously
for successive 12 days with Levothyroxine 0.5 mg/kg. From the 13th to 24th day the
animals were received 0.1 ml/day distilled water orally by gavage. Hyperthyroidism
treated with PTU, animals were treated subcutaneously for successive 12 days with
Levothyroxine 0.5 mg/kg. From the 13th to 24th day the animals were received 10 mg/kg
propylthiouracil orally by gavage. Hyperthyroidism treated with rosemary, animals were
treated subcutaneously for successive 12 days with Levothyroxine 0.5 mg/kg. From the
13th to 24th day the animals were received 10 mg/kg aqueous leaves extract of rosemary
(Rosemarinus officinalis) orally by gavage.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
235
RESULTS
Histological Findings:
The testicular tissue sections findings were different among the experimental
groups and the negative control group. In negative control group, the histological section
for testis showed normal seminiferous tubular architecture, spermatogenetic activity, and
normal interstitial cells and no significant occupied lesion whereas the histopathological
section of positive control group showed amyloid accumulation between seminiferous
tubules with blood vessels congestion and damage in the some connective tissue septa of
seminiferous tubules as presented in (Fig. 1.)Histological section of testis treated with 10
mg/kg propylthiouracil showed sever blood vessels congestion with necrotizing cells of
seminiferous tubules including spermatogonia cells, sertoli cells and primary
spermatocytes, seminiferous tubules flattened and elongated with damage of connective
tissue of septa between seminiferous tubules to give strong attachment between these
tubules as presented in (Fig. 2.). Finally in (Fig. 3.) The histological section of testis that
treated with 10 mg/kg aqueous leaves extract of rosemary (Rosemarinus officinalis)
showed the testicular texture back to normal structure after successive twelve days of
treatment.
Fig.1. (A) Negative control group. Histological section for testis is showing
normal seminiferous tubular architecture, spermatogenetic activity, and
normal interstitial cells and no significant occupied lesion in the testis
section, H&E , 20X.
.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
236
Fig 1(B) Positive control group given Levothyroxine 0.5 mg/kg s.c. the histopathological
section of testis is showing amyloid accumulation between seminiferous tubules
(black arrow) with blood vessels congestion (yellow arrow) and damage in the some
connective tissue septa of seminiferous tubules (blue arrow),
Fig.2. Group treated with 10 mg/kg propylthiouracil (A) Histological section for testis is
showing sever blood vessels congestion (black arrow) with necrotizing cells of
seminiferous tubules including spermatogonia cells, sertoli cells and primary
spermatocytes (blue arrow) H&E , 20X.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
237
Fig 2 : Group treated with 10 mg/kg propylthiouracil (B)Histopathological section for
testis is showing seminiferous tubules flattened and elongated (blue arrow) with
damage of connective tissue of septa (yellow arrow) between seminiferous tubules to
give strong attachment between these tubules, H&E, 20X
Fig.3. Group treated with 10 mg/kg aqueous leaves extract of rosemary (Rosemarinus
officinalis). The histological section of testis treated is showing the testicular
texture back to normal structure after (12) days of treatment, H&E, 20X .
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
238
DISCUSSION
Thyrotoxicosis in rats is dealt with late Leydig cell maturation and alterations of
spermatogenetic (12). Hypothyroidism and thyrotoxicosis are dealt with changes affected
functions of the endocrine, sexual, or reproduction (13). The propylthiouracil treatment,
an antithyroid remedy, caused a maturation to cease of spermatogenesis, lowering the
number of Leydig cells, reduce the tubular diameter, lead to interstitial edema and
condensation of the basement membrane (14).
In contrast, rosemary showed a substantial beneficial effect in comparison of PTU
on the testicular tissue; in which the latter caused many defectives. A previous study
showed that oral administration of rosemary reduced fertility in male albino rats (15).
Rosemary (Rosemarinus officinalis) is one of the household herbs that include a number
of phytochemicals, including camphor, betulinic acid, caffeic acid, ursolic acid, the
antioxidants carnosic acid and rosmarinic acid, it used in traditional medicine to treat a set
of troubles (16). Extracts of rosemary leaves contain flavonoids and phenols which
possess a diversity of bioactivities in vitro including antibacterial, antitumor,
antinociceptive, antioxidant, antidiabetic, antithrombotic, antiulcerogenic, antiinflammatory
agents, and antidiuretic (17).
Therefore, this paper established scientifically the use of the aqueous leaf extract
of rosemary as an ameliorator in recovering the testicular changes that had induced by
levothyroxine, rosemary showed full recovery in these changes represented in normal
testes tissue with no significant occupied lesion in comparison to the effect of PTU. The
reason for the improvement in the testicle using the aqueous extract of rosemary can be
attributed to the fact that it contains many antioxidants.
Acknowledgements:
Many thank to Dr. Saif Sattar Rasheed for his help and all the facilities in reading the
histopathological sections.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
239
REFERENCES
1-Riley, S. C., Leask, R., Denison, F. C., Wisely, K., Calder, A. A., & Howe, D. C. (1999).
Secretion of tissue inhibitors of matrix metalloproteinases by human fetal membranes,
decidua and placenta at parturition. Journal of Endocrinology, 162(3), 351-359.
2-Cheng, C. Y., & Mruk, D. D. (2002). Cell junction dynamics in the testis: Sertoli-germ cell
interactions and male contraceptive development. Physiological reviews, 82(4), 825-
874.
3-Wortsman, J., Rosner, W., & Dufau, M. L. (1987). Abnormal testicular function in men
with primary hypothyroidism. The American journal of medicine, 82(2), 207-212.
4-Wagner, M. S., Wajner, S. M., & Maia, A. L. (2008). The role of thyroid hormone in
testicular development and function. The Journal of endocrinology, 199(3), 351.
5-González-Trujano, M. E., Peña, E. I., Martínez, A. L., Moreno, J., Guevara-Fefer, P.,
Deciga-Campos, M., & López-Muñoz, F. J. (2007). Evaluation of the
antinociceptive effect of Rosmarinus officinalis L. using three different experimental
models in rodents. Journal of ethnopharmacology, 111(3), 476-482.
6- Rašković, A., Milanović, I., Pavlović, N., Ćebović, T., Vukmirović, S., & Mikov, M.
(2014). Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and
its hepatoprotective potential. BMC complementary and alternative medicine, 14(1),
225.
7-Agrawal, S., Chauhan, S., & Mathur, R. (1986). Antifertility effects of embelin in male
rats. Andrologia, 18(2), 125-131.
8-Moriyama, K., Tagami, T., Usui, T., Naruse, M., Nambu, T., Hataya, Y., ... & Nakao, K.
(2007). Antithyroid drugs inhibit thyroid hormone receptor-mediated
transcription. The Journal of Clinical Endocrinology & Metabolism, 92(3), 1066-
1072.
9- Hess, R. A., Cooke, P. S., Bunick, D. A. V. I. D., & Kirby, J. D. (1993). Adult testicular
enlargement induced by neonatal hypothyroidism is accompanied by increased Sertoli
and germ cell numbers. Endocrinology, 132(6), 2607-2613.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq.
240
10-Amin, A., & Hamza, A. A. (2005). Hepatoprotective effects of Hibiscus, Rosmarinus and
Salvia on azathioprine-induced toxicity in rats. Life sciences, 77(3), 266-278.
11- Panda, S., & Kar, A. (2014). Antithyroid effects of naringin, hesperidin and rutin in l-T4
induced hyperthyroid rats: Possible mediation through 5′ DI activity. Pharmacological
Reports, 66(6), 1092-1099.
12-Rijntjes, E., Wientjes, A. T., Swarts, H. J., de Rooij, D. G., & Teerds, K. J. (2008).
Dietaryinduced hyperthyroidism marginally affects neonatal testicular
development. Journal of andrology, 29(6), 643-653.
13-La Vignera, S., Vita, R., Condorelli, R. A., Mongioì, L. M., Presti, S., Benvenga, S., &
Calogero, A. E. (2017). Impact of thyroid disease on testicular
function. Endocrine, 58(3), 397-407.
14-Tahmaz, L., Gökalp, A., Kibar, Y., Kocak, I., Yalçın, O., & Özercan, Ý. (2000). Effect
of hypothyroidism on the testes in mature rats and treatment with levothyroxine and
zinc. Andrologia, 32(2), 85-89.
15- Nusier, M. K., Bataineh, H. N., & Daradka, H. M. (2007). Adverse effects of
propranolol on reproductive function in adult male mice. Pakistan journal of
biological sciences: PJBS, 10(16), 2728-2731.
16- Ahmed, N. A., Nahed, M., & El-Aal, R. A. A. (2015). Assessing the effect of Thyme and
Rosemary as antiaflatoxicosis on fertility in male rats. J. Am. Sci, 11(12), 294-302.
17-Razavi-Azarkhiavi, K., Ali-Omrani, M., Solgi, R., Bagheri, P., Haji-Noormohammadi,
M., Amani, N., & Sepand, M. R. (2014). Silymarin alleviates bleomycin-induced
pulmonary toxicity and lipid peroxidation in mice. Pharmaceutical biology, 52(10),
1267-1271.
