STUDY THE EFFECT OF ETHANOLIC EXTRACT OF CERATOINA SILIQUA , GLIMEPHAN AND METFORMIN ON SEMEN FLUID QUALITY IN DIABETIC MALE GUINEA PIG INDUCED BY ALLOXAN

This study was carried out in an animal house of the Collage of Veterinary Medicine/ University of Basrah, An attempt has been done to induce diabetic by alloxan in male guinea pigs and investigation of the effects of diabetic on semen fluid quality. Moreover, the present study aimed to evaluate the ameliorating effect of ethanolic extract of Ceratonia siliqua , metformin and Glimphan in experimentally induced in diabetic male guinea pigs. The study was applied on (30) adult male guinea pigs, their weight ranged between (800-1000g) and aged between 6-6.5 months. The male guinea pigs were divided randomly into five groups, each group consist of six guinea pig as the following: performance of male reproductive system as compared with other treated such as glimephan and metformin drugs. The results indicated significant and testosterone in serum and semen diabetic male (+ve) as compared with the control negative group and other groups while the results showed non-significant (P˃0.05) changes of FSH and LH concentrations in serum of diabetic male pigs treated with ethanolic extract of Ceratonia siliqua fruit as compared with control (-ve) group while the results were showed a significant(P≤0.05) rise of testosterone serum and semen diabetic male of Ceratonia siliqua fruit whencompared with the control (–ve) group and other groups.


INTRODUCTION
Diabetes mellitus(DM) is a syndrome of impaired carbohydrate, fat, and protein metabolism caused by either lack of insulin secretion or decreased sensitivity of the tissues to insulin. Programs by the world health organization (WHO) suggest that the number of people with diabetes may reach 366 million people in 2030 [1]. There are two general types of diabetes mellitus. Type 1 diabetes, also called insulin-dependent diabetes mellitus, is caused by lack of insulin secretion. Type 2 diabetes, also called non-insulin-dependent diabetes mellitus, is initially caused by decreased sensitivity of target tissues to the metabolic effect of insulin. This reduced sensitivity to insulin is often called insulin resistance.
In both types of diabetes mellitus, metabolism of all the main foodstuffs is altered.
The basic effect of insulin deficiency or insulin resistance on glucose metabolism is to prevent the efficient uptake and utilization of glucose by most cells of the body, except those of the brain. As a result, blood glucose concentration increases, cell utilization of glucose falls increasingly lower, and utilization of fats and proteins increases.
The diabetes mellitus leads to damages, impaired and failures of various organs. An important complication of diabetes is the disorder in the male reproductive system.
Glucose metabolism is an important event in spermatogenesis. Moreover, glucose metabolism is also important for maintaining basic cell activity, as well as specific functions, such as motility and fertilization ability in mature sperm. Diabetic disease and experimentally induced diabetes both demonstrated that either type 1 diabetes or type 2 diabetes could have detrimental effects on male fertility, especially on sperm quality, such as sperm motility [2]. One of these causes is the alarming elevate of the number of men developing DM during the reproductive age [3] and there is an elevating number of children and adolescents with type 2 DM (T2DM) [4].
When describe the treatment for DM is needed to find the most suitable antidiabetic drug [5]. Several of these compounds, if not all, have the ability to modulate cellular metabolism in a manner that may benefit some organs, but damage others.

Metformin hydrochlorid (Glucophage)
is an antidiabetic medicine that belongs to the group of biguanides, describe the first-line treatment for T2DM [6]. It is an insulin-sensitizing drug that exerts its anti-hyperglycemic effects by increasing the skeletal muscle uptake of glucose and reducing the absorption of glucose in the intestinal mucosa [7,8]. Metformin, the active ingredient in glucophage XR Moreover, it blocks liver gluconeogenesis through regulation of the gluconeogenic flux [9]. Nevertheless, although a common feature has already been attributed to this drug in promoting the activity of AMP-activated protein kinase (AMPK) [10], the exact molecular mechanisms of metformin action remain to be fully disclosed, despite several decades of research focused on this drug action [9].
Glimephan is a tablets active ingredient glimepiride, its an antidiabetic, it reduces blood glucose levels by stimulating insulin secretion [11].
Carob is medicine plant, native to mediterranean regions and is found in south of Syria, India and most of mediterranean areas as well as in California. The broken pod has a characteristic odor caused by its 1.3% isobutyric acid content [ [22,23,24].
This study aimed to investigate the hypoglycemic effect of ethanol extract of Ceratonia siliqua fruit by using an experimental animal model of alloxan-induced damage of β-cell of langerhans islet in male guinea pigs. To determine its effect on semen fluid quality and some biochemical parameters in diabetic male guinea pigs and it could ameliorate the diabetes mellitus and comparison the effects of ethanol extract with glimephan and metformin drugs.

Plant Material.
Ethanolic extract had been extracted from carob fruits (Ceratonia siliqua) that were used in this study. The carob was hand-picked from local market. It was washed with tap water. The fruits of the carob were turned to powder with the help of an electric grinder and kept in dark container at 25C°.

Preparation Ethanolic Extract from Carob Fruits(Ceratonia siliqua).
Fifty grams of dried carob fruits powder was defatted with (500 ml) of nhexane for 6hours by soxhlete. The combined n-hexane extract was concentrated below 50°C under reduced pressure in a rotary evaporator to get 7ml of brown oily mass [28].The remain of dried carob powder was (40g) refluxed in (500ml) of ethanol (70%) for 16 hours concentrated by rotary evaporator at 40˚C and dried at room temperature [25].

Experimental Animals.
Thirty adult male guinea pig weight ranged between (800-1000. gm) kept for an adaptation period for 1 month in the animal house of Veterinary Medicine College / University of Basrah. The experimental animals were kept in individual cages, provided with standard ration.

Experimental design.
The guinea pigs were divided into five groups of comprising 6 animals in each group as the following:

Induction of Diabetes Mellitus.
Diabetes mellitus were induced in twenty four starved male guinea pig by giving alloxan injected by one ml size syringe and in dose 150mg/kg for three days.

Collection of Blood Samples.
Blood samples (10ml) were collected from each animals at end of experiment by the heart (cardiac puncture). Blood was deposited into tube without anticoagulant and then the blood samples were centrifuged at (3000 rpm) for 15 minutes and serum samples stored in polyethylene eppendorff tubes at (-20ºC),which are used to study biochemical parameters (serum glucose and hormonal assay such as FSH,LH and testosterone ).
Study parameter:--Measurement of Body weight and body weight gain. ١ The animals were weighed in the 0 day, 15 th day and at the end period of the experiment.

3-Hormonal Assay.
Serum samples and plasma semen were estimated for FSH, LH and testosterone concentrations, using ELISA technique .

4.3-Sperm maturation by aniline-blue:-
The nuclear maturation was to evaluate by aniline-blue stain, according to [28].

4.4-Sperm Morphology:-
The changes in sperm morphology were to evaluate according to the method of [29]. At the end of experimental period the animals were sacrificed and the samples removing such as pancreas and testes were taken and fixed in 10% formalin according to [31].

Statistical Analysis:
The result were tabulated as mean values ± SD. and statistical analyze by using one-way analysis of variance. All statistical analyzes were performed using SPSS statistical version 23 software package [32].

Ceratonia siliqua Fruit, Glimephan and Metformin.
The results in Table (

Ceratonia siliqua Fruit, Glimphan and Metformin.
The results in Table (

3-Physical Properties of Semen Analysis in Diabetic Male Guinea Pig Treated
With Ceratonia siliqua Fruit, Glimephan and Metformin.
The results in Table (    Normal head without tail/tailless head:-The pigs in the control group and treated with extract had low number of spermatozoa with normal head without tail abnormality when compared with pigs in another treated groups. The percentage of this abnormality in the male guinea pigs (+ve control group) was higher than that of the control pigs and another groups.

4-Sperm morphology in Diabetic Male
Normal tail without head/headless tail:-The spermatozoa of diabetic pigs in (+ve control group) were observed much of the normal tail without head/ headless tail abnormality than those of the control guinea pigs and another treated groups.

5-FSH, LH and Testosterone Concentrations in Serum and Semen Male Guinea
Pig with Treated Ceratonia siliqua Fruit, Glimphan and Metformin Drugs.
The results in Table (  N=number of animals., Small letters denote differences between groups,P≤0.05 vs. control.

-Histological Examination:--Pancreas:-
The section of pancrease of negative control group guinea pig is composed of two major types of tissues, the acini, which secrete digestive juices into the duodenum, and, the islets of Langerhans, which secrete insulin and glucagon directly into the blood.  Fig.(6). While the section of testis untreated diabetic pigs  Fig. (7). Moreover, section of testis of pig treated with glimephan drug. The seminiferous tubules have mild vacuolation and shows normal association of spermatogenesis. as shown in Fig. (8). Also, section of testis of guinea pig treated with metformin drug showed normal seminiferous tubules and spermatogenesis, interstitial leydig cells and present sertoli cells as shown in Fig.(9).
But the testis of pig treated with ethanolic extract of Ceratonia siliqua showednormal seminiferous tubules, spermatogenesis, interstitial leydig cells between seminiferous tubules and present primary spermatocyte as shown in Fig.(10).

DISCUSSION
Hyperglycemia leads to dysfunction in target organs of diabetic animals [2].
Partial or complete insulin deficiency in diabetic animals appears to have adverse

Showing normal seminiferous tubules and spermatogenesis(Sp).(H&E stain,100X)
Sp effects on all organs, including reproductive systems [33] [34] [35]. This study confirms that diabetes have an adverse effect on male reproductive function as proposed by others [36] also this study demonstrates a protective effect of the ethanolic extract of Ceratoina siliqua recovery of diabetes and testicular dysfunctions eminent in diabetic state.
The body weight of diabetic guinea pigs was declined. This is associated with dehydration and catabolism of fats and proteins due to proteolysis on muscular tissues occurred in insulin deficiency states [37] [38], furthermore, our results also showed that treated with ethanolic extract of Ceratoina siliqua lead to improve the body weight gain in male guinea pigs, Through its contents which were flavonoids several flavonoid subclasses have shown decrease energy intake, increase glucose uptake in muscle in vivo, and decrease glucose uptake in adipose tissue in vitro.
In this study, glucose concentration of serum diabetic guinea pigs elevated above Also in this study the effect of diabetes on semen quality was investigatedand this is in agreement with the previous observations [42] [43] [44]. An induction of reduced fertility in the subjects [45]. This was attributed to damage of the secretory epithelia cells of the seminiferous tubules [46], probably due to oxidative damage from glucose auto-oxidation and excessive production of superoxide radicals and formation of advanced glycation end products associated with DM [33]. Our results showed that administration of ethanolic extract of Ceratoina siliqua enhanced the reproductive parameters of diabetic male guinea pigs. The extract was more prominent on the percentage motility and the live /dead ratio of the spermatozoa than the effects of the glimephan and metformin drugs, this result reported that the presence of flavonoid in the extract is potent antioxidant and able to scavenge free radicals, flavonoid also has an anti-hyperglycaemic activity, which may suppress glucose release from the liver as well as improving glucose uptake in peripheral tissues [43] .
In present study, the diabetic caused germ cells depletion in pigs. The spermatide and spermatozoa cells affected. The majority changes that occur on testicle tissue and spermatogenesis procedure following diabetic are probably due to the changes in testosterone concentration [44] while its treated with ethanolic extract of Ceratonia siliqua resulted stimulation on testicle tissue and spermatogenesis procedure, it increased the concentration testosterone by stimulating the gonad regulating cycle including the hypothalamo-pituitary axis. This in turn regulates the amount of testosterone in the organism if the testosterone concentration in the blood is low and the tests will signal the hypothalamus to release LHRH thus the hypophysis releases gonadotropin, LH and FSH consequently, the interstitial cell of leydig activate the production of testosterone [48,49].
The Ceratonia siliqua can ameliorative the spermatogenesis and steroidogenesis processes of leydic cells through the observation in this study we concluded that the ethanolic extract of Ceratoina siliqua induces anti-hyperglycemia and improves the male reproductive system was more than treated to glimephan and metformin drugs.
In addition, these findings suggest the possible utilization of the ethanolic extract of Ceratoina siliqua as a therapy.