Body weight and mortality rate in control and experimental rats
As presented in Table 1, at the end of the experiment, results revealed that IP injection of CP significantly (P < 0.05) decreased body weight and increased (P < 0.05) mortality rate than in control or CP + L-GFequina groups. Meanwhile, IP injection of L-GFequina alone significantly (P < 0.05) increased body weight than control or CP + L-GFequina groups.
Table 1
Effect of CP, L-GFequina or CP + L-GFequina on body weights and mortality rate in rats (Mean ± SE).
Groups | Body Weight before (g) | Body weight after (g) | Mortality rate (%) |
Control | 156.6 ± 1.2a | 175 .0 ± 6.0b | 0.0b |
CP | 159.6 ± 1.7a | 146.2 ± 6.2c | 30.0a |
L-GFequina | 158.9a ± 1.4a | 194.3 ± 4.8a | 0.0b |
CP + L-GFequina | 154.8 ± 1.1a | 176.7b ± 3.6b | 10.0b |
a,b within the same column differ significantly at P < 0.05 |
b,c within the same column differ significantly at P < 0.05 |
Effect of treatment on reproductive organs weight
Data illustrated in Table 2 and Fig. 2 represent the effect of treatment with CP with or without L-GFequina on reproductive organs weight in rats. Results revealed that there was significant (P < 0.05) decrease in reproductive tract, left ovary and uterine weights in CP group (Fig. 2B) compared with the control animals (Fig. 2A). In rats injected with PRP alone, reproductive tract weight, and uterine weight were higher (P < 0.05) than in the other groups (Fig. 2C)). While, no significant difference was detected for reproductive tract, ovarian and uterine weights for control and CP + L-GFequina group (Fig. 2D).
Table 2
Effect of IP injection of CP, L-GFequina or CP + L-GFequina on ovarian and uterine weight in rats (Mean ± SE).
Groups | Reproductive tract weight (g) | Ovarian weight (g) | Uterus weight (g) |
Right | Left |
Control | 0.42 ± 0.04b | 0.05 ± 0.01 | 0.05 ± .005 a | 0.3 ± 0.03,b |
CP | 0.27 ± 0.01 c | 0.04 ± .002 | 0.03 ± .003b | 0.2 ± 0.01 c |
L-GFequina | 0.60 ± 0.01 a | 0.05 ± .004 | 0.05 ± .005 a | 0.5 ± 0.01 a |
CP + L-GFequina | 0.40 ± 0.02 b | 0.05 ± .003 | 0.04 ± .003 | 0.3 ± 0.02 b |
a,b within the same column differ significantly at P < 0.05 |
b,c within the same column differ significantly at P < 0.05 |
Histopathological changes
Ovarian tissue in control rats showed normal archticture, and multiple follicles were seen at various stages of development and maturation. These included primordial, primary, and secondary follicles and multiple mature Graafian follicles. Corpora lutea were also observed formed of sheets of lutein cells with ample acidophilic cytoplasm and basophilic nuclei (Fig. 3A). Ovarian tissue of rats treated with CP showed degenerated tissues with depleted follicles; primordial, primary, and mature follicles compared to the control animals (Fig. 3B). Graafian follicles showed separation of cells and loss of the cumulus oophorous, shrunken granulosa cells, and several atretic follicles were observed. Ovarian tissue in rats treated with L-GFequina showed normal archticture with multiple follicles at various stages of development and maturation, together with multiple corpora lutea (Fig. 3C). Ovarian tissue of CP + L-GFequina showed ovarian architecture similar to control. Multiple follicles at all stages of development and maturation, corpora lutea occupied the ovarian cortex and no atretic follicles were detected (Fig. 3D).
Regarding the effect of IP injection of CP, CP + L-GFequina, or L-GFequina on histology of the uterus in rats: The control and the L-GFequina treated groups showed normal endometrium with numerous, proliferating endometrial glands exhibiting non-dilated lumina. Lining epithelium consisted of active, high columnar epithelium. Stroma was an active form of proliferating spindle cells (Fig. 4A). In CP-treated animals, histopathological changes were noticed in the uterus such as increased epithelial degeneration, inactive low cuboidal to the flattened epithelial lining, vacuoles within the epithelial lining, and focally dilated endometrial glands, and densely fibrotic stroma. Decreased glandular proliferation was observed, and vascular proliferation and numerous blood vessels were also seen (Fig. 4B). The uterus of the L-GFequina treated groups showed active endometrium with numerous, proliferating endometrial glands exhibiting non-dilated lumina. The lining epithelium was active and consisted of high columnar cells. Stroma was an active form of proliferating spindle cells (Fig. 4C). The groups treated with CP + L-GFequina showed signs of fibrosis, increased cellular proliferation of the endometrium, decreased vacuoles within the epithelial lining, and a decrease in the vascular proliferation compared to the CP-treated group (Fig. 4D).
Effect of treatment on ovarian morphology
Table 3 represent the effect of the treatment of rats with CP, L-GFequina or CP + L-GFequina on ovarian morphology. In CP-treated rats, the number of follicles and CL in both the right and left ovary and the total number of follicles and CL per ovary were significantly (P < 0.05) lower than in the other groups. Meanwhile, in L-GFequina injected rats, the number of ovarian follicles and CL was significantly (P < 0.05) higher than in control or CP + L-GFequina. In CP + L-GFequina-treated rats, the number of ovarian follicles and CL was similar to that in the control group.
Table 3
Effect of CP, L-GFequina or CP + L-GFequina on follicles and CL number (Mean ± SE).
Groups | No follicles/ovary | Total no follicles /animal | No CL/ovary | Total no CL/animal |
Right | Left | Right | Left |
Control | 3.2 ± 0.8a | 1.6 ± 0.4b | 4.8 ± 0.6b | 4.0 ± 0.5b | 4.9 ± 0.7b | 8.6 ± 0.6b |
CP | 0.0c | 0.4 ± 0.2c | 0.4 ± 0.1c | 1.1 ± 0.3c | 0.0c | 1.1 ± 0.2c |
L-GFequina | 5.9 ± 0.6b | 3 ± 0.3a | 8.9 ± 0.4a | 7.7 ± 0.9a | 7.2 ± 0.9a | 14.9 ± 0.9a |
CP + L-GFequina | 3.0 ± 0.3a | 1.7 ± 0.3b | 4.7 ± 0.3 | 5.2 ± 0.5b | 5.6 ± 0.6b | 10.8 ± 0.5b |
a,b within the same column differ significantly at P < 0.05 |
b,c within the same column differ significantly at P < 0.05 |
Ovarian and uterine morphometric analysis
The effect of treatment with CP, L-GFequina or CP + L-GFequina on ovarian and uterine morphometric measures is presented in Table 4. Results showed that treatment with CP significantly (P < 0.05) decrease the number of primordial, primary, secondary, and antral follicles, and increase (P < 0.05) the number of atretic follicles compared with the control group. A significantly (P < 0.05) higher number of primordial, primary, secondary, and antral follicles and CL was recorded in the L-GFequina-treated group than in the other groups. In CP + L-GFequina-treated rats the number of primordial, primary, secondary, and antral follicles was similar to the control group.
Table 4
Morphometric changes in ovaries of control, CP, L-GFequina and CP + L-GFequina treated rats (Mean ± SE).
Group | Primordial fol. | Primary fol. | Secondary fol. | Antral fol. | CL | Atretic foll |
Control | 34b | 33b | 12a | 4b | 1b | 4c |
CP | 23c | 19c | 9b | 2b | 1b | 37a |
L-GFequina | 55a | 40a | 15a | 13a | 3a | 15b |
CP + L-GFequina | 30b | 29b | 13a | 3b | 2b | 5c |
a,b within the same column differ significantly at P < 0.05 |
b,c within the same column differ significantly at P < 0.05 |
Regarding the uterine tissue samples, the present work revealed that IP injection of CP in female rats produced a significant (P < 0.05) decrease in the glandular area and an increase (P < 0.05) in the stroma and dilated glands area compared to the control rats. In the meantime, IP injection of L-GFequina significantly (P < 0.05) increased the glandular area and dilated glands compared with the control or CP + L-GFequina groups. Meanwhile, the glandular and stromal areas were similar between the CP + L-GFequina and the control group (Table 5).
Table 5
Morphometric changes in uterus of control, CP, L-GFequina and CP + L-GFequina treated rats (Mean ± SE).
Group | Glands | Stroma | Dilated glands |
Control | 2.184 ± 0.133b | 18.701 ± 1.121a | 1.825 ± 0.053c |
CP | 0.858 ± 0.121c | 45.439 ± 1.866b | 8.097 ± 1.929a |
L-GFequina | 6.488 ± 0.205a | 18.446 ± 1.567a | 4.643 ± 0.235b |
CP + L-GFequina | 2.327 ± 0.169b | 16.938 ± 0.798a | 3.051 ± 0.357b |
a,b within the same column differ significantly at P < 0.05 |
b,c within the same column differ significantly at P < 0.05 |
Blood profile
Data demonstrated that IP injection of CP significantly (P < 0.05) decreased RBCs, HB, hematocrit, WBCs, lymphocytes number, percentage, granulocytes number and percentage, and platelet distribution width. While CP treatment significantly (P < 0.05) increased procalcitonin, platelet distribution width, and platelet large cell ratio compared with the other groups. In CP + L-GFequina and L-GFequina treated groups, RBCs, HB, and hematocrit values were significantly (P < 0.05) higher than in other groups. IP injection of CP + L-GFequina significantly (P < 0.05) increased platelet distribution width and red cells distribution width compared with the other groups (Table 6).
Table 6
Effect of treatment with CP, L-GFequina or CP + L-GFequina on blood parameters in female rats (Mean ± SE).
Item Group | Control | CP | Equine PRP | CP + L-GFequina |
Red blood cell count (106/µl) | 6.4 ± 0.1a | 5.6 ± 0.4b | 6.7 ± 0.2a | 6.9 ± 0.2a |
Hemoglobin (g/dl) | 13.8 ± 0.3b | 12.1 ± 0.5b | 15.3 ± 0.6a | 15.1 ± 0.5a |
Hematocrit (%) | 38.8 ± 0.9b | 36.4 ± 1.9b | 46.3 ± 1.4a | 46.3 ± 1.4a |
MCV (fL) | 60.8 ± 0.8b | 64.4 ± 1.5a | 65.1 ± 0.4a | 65.1 ± 0.4a |
Mean Corpuscular hemoglobin | 21.9 ± 0.3 | 21.7 ± 0.5 | 22.6 ± 0.1 | 22.6 ± 0.1 |
MCHC (pg) | 34.9 ± 1.5 | 34.2 ± 0.8 | 36.3 ± 0.4 | 35.1 ± 0.3 |
Procalcitonin (µg/L) | 0.3 ± 0.03c | 0.9 ± 0.01a | 0.2 ± 0.03c | 0.5 ± 0.04b |
White blood cell count (103/µl) | 4.9 ± 0. 4a | 3.7 ± 0.3b | 5.9 ± 0.4a | 5.5 ± 0.1a |
Lymphocyte percentage (%) | 74.8 ± 0.9a | 68.1 ± 1.8b | 76.4 ± 2.0a | 75.5 ± 1.6a |
Lymphocyte number(103/µl) | 3.5 ± 0.6a | 2.7 ± 0.4b | 3.7a ± 0.1a | 3.6 ± 0.3a |
Granulocyte number (103/µl) | 0.5 ± 0.1a | 0.2 ± 0.1b | 0.5 ± 0.1a | 0.4 ± 0.03a |
Granulocyte percentage (%) | 12.9 ± 0.6a | 9.6 ± 1.1b | 12.4 ± 0.7a | 13.2 ± 0.4a |
Platelet large cell ratio (%) | 7.0 ± 0.2a | 10.6 ± 0.4b | 6.7 ± 0.4a | 7.1 ± 0.4a |
Platelet count (fL) | 456.5 ± 42.9b | 159.4 ± 8.2c | 340.4 ± 19.3c | 611.6 ± 46.1a |
a,b within the same row differ significantly at P < 0.05 |
b,c within the same row differ significantly at P < 0.05 |
Antioxidant capacity
Results showed a significant (P < 0.05) increase in serum nitric oxide and MDA levels among CP-treated groups in comparison with the other groups. There was no significant difference between the control and CP + L-GFequina or L-GFequina groups in terms of MDA and NO levels (Fig. 5).