INTRODUCTION:

The uterine and in specific the endometrium had a vital roles in usual reproductive cycles, placentation, implantation and supportive a healthy fetus till parturition (1). Pyometra is factually demarcated as “puss in the uterine.” This fatal infectious disease is categorized by intraluminal accretion and inflammation of purulent materials in the uterine. Pyometra is practically greatest well-known to veterinary medicinal science, (2).

Considering that the medicinal treatment of pyometra (surgical and antibiotics) has several undesirable effects. Ceftriaxone is an antibacterial beneficial for the management a number of bacterial diseases, this included meningitis, middle ear infections, pneumonia, endocarditis, joint infections, skin infections, intra-abdominal infections, gonorrhea, pelvic inflammatory diseases, urinary tract infections and pyometra. Mutual side effects included seizures, allergic reactions, gall bladder disease, hemolytic anemia, and pain at the site of injection (3). An alternative treatment was an herbal preparation appeared to possess pharmacological properties favorable to the treatment of pyometra in animals, such as Thymus vulgaris had been believed to be antiseptic, astringent, medication, anthelmintic, antimicrobial, disinfectant, carminative, tonic is energetic against cocci bacteria and enterobacteria in addition, it considered a source of normal antibiotics and antioxidants. Thymus vulgaris had the ability to restore and restart a ordinary ovulation, in addition to the necessary oil of the thymus had an influence on cystitis, urethritis and pymetra which they considered the most common causes of infertility in animals (4,5), the drive of the recent study was to estimate the role of ceftriaxone and Thymus vulgaris extract on induced pyometra.

MATERIALS AND METHODS:

Thymus vulgaris leaves extraction:

Thymus vulgaris leaves were grounded and kept until use, for the preparation of Alcohol extraction by putting (50)g of Thyme powder in a extraction unit (Soxhelt) and added (350) ml of ethanol (70%), extraction continued for (12) hours at (40)°C by using Vacuum Rotary Evaporator (6).

Doses and administration of Thymus vulgaris leaves and ceftriaxone:

The extract of Thymus vulgaris was administrated to mice at dose (500mg/kg) B.W orally, which primed by dissolving five gram of extract in distilled water till talented the volume to 100ml agreeing to Shaban et al., (7). Ceftriaxone was administered to mice at dose 50 mg/kg and the concentration was 5mg/ml according to Moine et al., (8).

Pyometra induction:

Forty five female mice were injected with 15µg/kg/day of 17α-ethinyl estradiol given subcutaneous for two weeks, after end the period of induction, five mice were taken randomly and scarified to prepare the histopathological section to ensure the pyometra was induced (9).

Experimental design:

female mice were dispersed into five groups each group had ten animals, the period of treatment was two weeks, the 1st group (G1) was induced and treated with a daily dose of Thymus vulgaris leaves extract (500mg/kg) administered orally, the 2ed group (G2) was induced and given I/M daily dose of Ceftriaxone (5mg/kg), the 3ed group (G3) was induced and treated daily with Thymus vulgaris leaves extract (250mg/kg) and given I/M daily dose of Ceftriaxone (2.5mg/kg), while 4rth group (G4) was induced and treated with distilled water as +ve control group, with the 5rth group (G5) was without any treatment (–ve control group).

Haematological parameters:

The procedure of Hemoglobin was measured according to the method mentioned by (10). While Packed Cell Volume (PCV) was measured depending on the method revealed by (11). In addition, the White Blood Cells counting and Differential Counting of WBCs were calculated according to the method mentioned by colse, (12).

Fertility index:

Fertility index % was calculated according to (13): Fertility index % = whole sums of females gravid ÷ whole sums of females coupled x 100.

Follicles stimulating hormone (FSH) and luteal hormone (LH):

The Hormonal magnitudes were equipped rendering to steps instituted in commercial Radio immunoassay (RIA) kits, the assessment was completed in scientific laboratory of radioactive isotopes.

Total serums of albumin and globulin g/dl:

Total albumin and globulin were measured according to standard Biochemical assays methods depending on albumin and globulin kit (14).

Histopathological changes:

After the end of period of treatment, mice were sacrificed, uterus was impassive and vacant from the fat and connective tissues and then kept in 10% formalin until done histological sections which made according to (15).

Statistical analysis:

Program of SAS has been customized to do statistical analysis in this study; the statistical between averages was likened with less a significant differences LSD (16).

RESULTS:

The PCV and Hb levels counts in G1 revealed a significant upsurge (P<0.05) as compared with G2 and G4, while the Hb and PCV levels in G2 displayed a statistical decline (P<0.05) as compared with G3 and G5. The Hb and PCV levels in G3 revealed a significant increase (P<0.05) as compared with G4 with no significant change (P<0.05) as compared with G5.

The values of WBCs, N%, L% and M% in G1 exhibited a statistical rise (P<0.05) as compared with G2, G3 and G5. As well as the values of WBCs, N%, M% in G2 exhibited a statistical decline (P<0.05) as compared with G3, G4 and G5. While the values of WBCs, N% and M% in G3 revealed a statistical surge (P<0.05) as compared with G5 and in the same time revealed a statistical decline (P<0.05) as compared with G4. Furthermore, the WBCs, N% and M% in G4 appeared a significant increase (P<0.05) as compared with G5 and totally others treated groups, except L% revealed a statistical decline (P<0.05) as compared with G1 and G5 with no remarkable difference (P<0.05) as compared with G2 and G3 (Table 1). The E% in all treated groups revealed a statistical decline (P< 0.05) as compared with G5.

The level of albumin in G1 and G3 indicated a statistical surge (P<0.05) as compared with G2 and G4 with no momentous differences (P<0.05) as compared with G5. Whereas the albumin in G2 and G4 displayed a significant decline (P<0.05) as compared with G5. As well as, the G2 appeared a significant surge (P<0.05) in albumin level as albumin with G4. The level of globulin in G1 exhibited a significant rise (P<0.05) as compared with G2, G3 and G5, in addition the globulin level in G3 showed no remarkable differences (P<0.05) as compared with G5, while the globulin level in G4 group reveled a statistical surges (P<0.05) as compared with G5 and wholly others treated group, as in (Table 2).

Table (2): Effect of Thymus vulgaris leaves extract and Ceftriaxone on total albumin and globulin levels (g/ml) of Females mice.

Parameters, groups	albumin level (g/ml)	globulin levels (g/ml)
G1	2.5± 0.13 a	5.4 ± 0.27 b
G2	2.3 ±0.17 b	4.4± 0.24 d
G3	2.7 ± 0.14 a	5.0± 0.29 c
G4	1.8 ± 0.09 c	5.8 ± 0.21 a
G5	2.6± 0.10 a	4.9 ±0.26 c

* Means with a different letter in the same column statistically different (P< 0.05).

The levels of FSH and LH in G1 and G3 indicated a statistical surge (P<0.05) as matched with G2 and G4 with no statistical differences (P<0.05) as matched with G5 as in (Table 3).

Table (3): Effect of Thymus vulgaris leaves extract and Ceftriaxone on FSH (U/L) and LH (U/L) levels of Females mice.

Parameters groups	FSH (U/L)	LH (U/L)
G1	1.29 ± 0.13 a	1.89 ± 0.15 a
G2	0.89± 0.06 b	1.22± 0.16 b
G3	1.31± 0.10 a	2.00 ± 0.09 a
G4	0.86± 0.09 b	0.98± 0.08 b
G5	1.26± 0.12 a	1.99 ± 0.14 a

Means with a different letter in the same column statistically different (P< 0.05).

The Fertility index% in G3 revealed an improvement of fertility more than other groups (G1, G2 and G4). Furthermore, the G4 displayed a clear decline in fertility as compared with all other groups as in (Table 4).

Table (4): Effect of Thymus vulgaris leaves extract and Ceftriaxone on Fertility index % of female mice.

Parameter Groups	Fertility index%
G1	80%
G2	70%
G3	90%
G4	5%
G5	100%

Histopathological changes:

The histopathological sector in the uterus of mouse induced pyometra and preserved with distilled water displayed severe hemorrhage with neutrophils and mononuclear cells penetration in subepithelial layer with hyperplasia of epithelial cells as in (Figure 1), while the histopathological in the uterus of mouse induced pyometra and treated with a daily dose of Thymus vulgaris leaves extract (500mg/kg) revealed a squamous metaplasia of epithelial cells of uterine gland with keratin in their lumen and mono-nuclear cells infiltrations in all layers of the uterus as in (Figure 2), also the histopathological section in the uterus of mouse induced pyometra and treated with daily dose of Ceftriaxone (5mg/kg) showed marked neutrophils and mononuclear cells infiltrations in subepithelial and between uterine gland as in (Figure 3). Whereas, the best results were appeared in uterus section of animal induced pyometra and treated with daily dose of Thymus vulgaris leaves extract (250mg/kg) and Ceftriaxone (2.5 mg/kg) which showed mononuclear cell infiltration in all layers of the uterus as in (Figure 4).

Figure (1): Histopathological section in the uterus of animal induced pyometra and treated with distilled water as positive control group showed severe hemorrhage with neutrophils and mononuclear cells infiltration in subepithelial layer with hyperplasia of epithelial cells (H&E stain 40X).

Figure (2): Histopathological in the uterus of animal induced pyometra and treated with a daily dose of Thymus vulgaris leaves extract (500 mg / kg) showed squamous metaplasia of epithelial cells of uterine gland with keratin in their lumen and mononuclear cells infiltration in all layers of the uterus (H&E stain 40X)

Figure (3): Histopathological section in the uterus of animal induced pyometra and treated with daily dose of Ceftriaxone (5mg/kg) showed marked neutrophils and mononuclear cells infiltration in subepithelial and between uterine gland (H&E stain 40X)

Figure (4): Histopathological in the uterus of animal induced pyometra and treated with daily dose of Thymus vulgaris leaves extract (250 mg / kg) and Ceftriaxone (2.5 mg/kg) showed mononuclear cell infiltration in all layers of the uterus (H&E stain 40X).

DISCUSSION:

The decrease in Hb, PCV, L% total albumin levels and increase in WBCs, N%, M%, E% and globulin levels in G4 perhaps resulted from increasing tension on the body resistance mechanisms lead to produce an increase in leucocytes to fight the infections lead to sever inflammation, in addition, monocytosis, lymphopenia, eosinophilia and neutrophilia may be resulted from retaining of infected exudates in the uterine which employs a chemotactic effects to neutrophils resultant into enhanced lymphopenia and granulopoiesis may be occurred from severe stress, current results are supported by Basanti et al., (17), as well as, total monocytosis, lymphopenia and neutrophilia were usually occurred in pyometra. In addition to damage of red blood cells by harmful influences of pyometra on the bones marrow lead to loss of erythrocytes on the uterus lumen and reduced erythropoiesis, as well as, pymetra may be lead to reduced whole iron binding capacities is related with chronic and acute inflammatory illnesses and may be also prompt iron shortage and consequent anemia. While the decrease in PCV which might be due to concomitant dehydration with pyometra which is supported with the past studies recorded by (18). The hypoalbuminemia and hyperglobulinemia in G4 may be resulted from dehydration, chronic antigen stimulation, an acute inflammatory disorders and sever infection, this results were in agreement with findings were observed by Verstegen et al., (19). The decrease in FSH, LH and fertility index % in G4 may be attributed to 17α-Ethinylestradiol reduced fertility and prevented of an ovulation, influence on estrous cyclist, ovarian impairment and augmented pregnancy length, in addition to its ability to disrupt the endocrine system, this result was supported by the findings recorded by (20). The drop in LH-surge and uneven FSH may be related to the deteriorated GnRH neurons function and abridged GnRH secretions by down-articulated kisspeptin neurons in the anteroventral periventricular nucleus in preoptic area of 17α-Ethinylestradiol treated animals. (21).

The increase levels of PCV and Hb in G1 as compared with G2 and G4 can be explained by Thymus vulgaris have decrease level of saponins can be augmented intestinal villi diameters which is penetrable to big molecules such as ferritin. Thus, ferritin attentiveness may be accountable for the augmented Hb and RBCs totals. Furthermore, The plant is similarly a gorgeous source of different vital vitamins such as folic acid, B-complex, vitamin A, K, E, C, beta carotene and vitamin B-6 that important in increase Hb and PCV, Thymus vulgaris essence exhibited a positive effect on changing of hematological characteristics, the same result reported by Seirafy and Sobhanirad, (22), who illustrated that different components of herbal extracts due to nutritive and anti-oxidative effects could stimulate the blood cells producer organs. The increase WBCs, N%, L%, M% and E% in G1 as compared with G5 with an improvement in hematological and histopathological characteristics as compared with G4 can be clarified by carvacrol (CVL) and thymol had the ability in inhibiting inﬂammatory edema, CVL acted as a inhibitor of COX-2, inhibitor to cytokines and leukotrienes, stimulator to peroxisome proliferators activated receptor and inhibitor to prostanoid release, in addition, to Thymus vulgaris phenolic compounds which exhibited considerable antimicrobial, antioxidants and anti-inﬂammatory eﬀects in vivo, Thymus vulgaris had an irritative eﬀect that lead to histamine release and then increase eosinophil's (23). Animals trials have been also established the actions of Thymus vulgaris essential oils in contradiction of the proliferations and colonizations of Escherichia coli via altering cells walls bacterial penetrability lead to pore creation and osmotic shocks and leakages of active contents of cytoplasm in outside the cells lead to deaths of them. Furthermore, an elevated percentage of lymphocytes and WBC differential values may be due to Thymus vulgaris acted as an antidote to the oxidant therefore stimulated the immune response and thus strengthen the immune system and due to this tissue changes, this result agree with (22). Our result was in link with (24) who found that the albumin and globulin ratio in rainbow treated with Thymus vulgaris were enhanced may be due to effects of plant to stimulate protein synthesis in relative organs. In addition, the increase in the globulin fraction indicated the effective role of Thymus vulgaris in increasing immunity resulted from its character in rising, defensive cells and constraining non enzymatic oxidation. Similar findings were reported by (24). The enhancement in LH, FSH and fertility in G1 may be belonged to Thymus vulgaris had a stimulating effect inovogenesis and a positive effect on the maturity by its antioxidative effects and its important constituent vitamins C and E lead to increase fertility, Thymus vulgaris plant are single of the vital foundations of iron, manganese, potassium, magnesium, calcium and selenium, vitamin A and antioxidants that is essential for keeping well mucus membranes and tissues and thus may be had an effect on pyometra which considered one of the greatest mutual causes of infertility in females so the Thymus vulgaris worked against female infertility, these results agree with (5,25).

The decrease in the PCV, Hb, WBCs, N%, L%, M% and increase in E% with an improvement in histopathological section as compared with G4 in G2 may be attributed to Cefotaxime is a broad spectrum had effect for fighting Gram-positive and Gram-negative aerobics and anaerobic bacteria and it's extremely firm in the attendance of beta-lactamases. These hematological alterations could be explained on basis that cefotaxime may be caused some sort of bone marrow depression in addition to hypersensitivity reaction (26). The decrease in LH, FSH and fertility in T2 group can be explained by antibiotic may influence in fertility; most antibiotic B- lactium included cephalosporin may have toxic effect on fertility by decrease ovarian sex hormones (27). Furthermore, the most common adverse effects associated with cefotaxime and other cephalosporins are induction of chromosomal aberrations in germ cells, several studies reported that Cefotaxime induced a variety of fertility troubles in animals. Moreover intramuscular administration of cefotaxime sodium induced a significant decrease in serum total protein and albumen, these results may be due to cefotaxime enhanced liver damage and liver enzymes serum level (26).

While the G3 revealed an improvement in all parameters of hematological and biochemical recorded in current study in addition to histopathological section of uterus as compared with G2 which may be attributed to Thymus vulgaris extract had the ability in decreasing and preventing side effect cefotaxime during period of treatment pyometra through several an important constituents containing Thymus vulgaris which acted as antioxidative effects such as vitamins C and E, in addition to other vital foundations of Ca+2, iron, manganese, potassium, magnesium and selenium, Vitamin A (5,25).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 24.03.2019 Modified on 25.04.2019

Research J. Pharm. and Tech. 2019; 12(10):4683-4688.

DOI: 10.5958/0974-360X.2019.00806.0

Param groups	P.C.V.	Hb	WBCs	N%	L%	M%	E%
G1	33.9± 3.51 b	12.0± 1.33 a	8042± 12.12b	60.9± 13.50b	40.7±8.00 a	5.6± 0.25 b	3.8± 0.11 a
G2	30.0± 2.48 c	9.8± 2.73 b	5140± 13.11e	51.4± 12.22e	30.9±8.50 c	4.2± 0.72 d	3.9± 0.32 a
G3	35.8± 2.09 a	11.9 ± 1.81a	7009± 12.09d	59.9± 11.91c	31.1±9.13 c	4.9± 0.57 c	3.6± 0.31 a
G4	27.3± 3.99 d	9.1 ± 1.66 b	9001± 13.01a	63.0± 12.11a	29.0±8.99 c	6.9± 0.21 a	3.7± 0.19 a
G5	36.1± 1.63 a	12.2 ± 1.01 a	6099± 12.56c	56.7± 13.07d	34.9±9.12 b	4.4± 0.40 d	3.2± 0.30 b