Adejumo B. I. (Medical Laboratory Sciences Department, University of Benin, Benin City, Nigeria.)

Nwobu G. O. (Medical Laboratory Sciences Department, Igbinedion University, Okada, Nigeria.)

Ewenighi C. O. (Medical Laboratory Sciences Department, Ebonyi State University, Abakaliki, Nigeria)

Dimkpa U. (Physiology Department, Nnewi Campus, Nnamdi Azikiwe University, Awka, Nigeria)

Odionyema U. C. (Department of Medical Laboratory Science, College of Health Technology, Akure, Nigeria)

Ezimokhai T. J. P . (Lulli Diagnostics Limited, Benin City, Nigeria.) 

All correspondence to: Adejumo B.I., Medical Laboratory Sciences Department, University of Benin,
Benin City, Nigeria. Email: babatunde.adejumo@uniben.edu, bigadejumo@yahoo.com

ABSTRACT
Aim: This study aimed at determining the prevalence of Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) in Edo State, Nigeria. Method: Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) levels were estimated using ELISA reagents from Biomerica Inc. Other parameters such as fasting blood sugar, urine glucose, and urine protein were assessed using standard colorimetric and biochemical techniques. A total number of four hundred and fifty five (455) subjects, 211 (46%) males, and 244 (54%) females with ages ranging between 5 – 76 years were assessed. Subjects were free from long standing illness, endocrine disorder and infectious diseases. Most of the subjects were those attending diabetes clinics in secondary and tertiary Institutions in Edo State, relatives of type I diabetics and volunteered controls. A structured questionnaire was used to collect sociodemographic data. Sociodemographic data included age at diagnosis, age at inclusion, gender, family history of diabetes, drinking and smoking habits, and the duration of diabetes. In cases of patient uncertainty the duration was calculated from the documented date of diagnosis. Other information was obtained from the patients’ files, including the mode of presentation, type of treatment, and course of the disease.
RESULTS: The prevalence of IAA in the study population is 100% in Type I diabetics, 10% in Type II, 26.7% in diabetic relatives, 1.6% in non-diabetic control, while 11. 9% represents the prevalence of IAA in the general population. ICA also has 100% prevalence in Type I diabetics, 10% in Type II, 26.7% in diabetic relatives, 2% in non-diabetic controls, and 12.1% in the general population.
CONCLUSION: The seroprevalence of ICA and IAA is high in this study, public enlightenment, change of life style and early routine check is advocated to prevent diabetes development, complications and reduce mortality rate.

Key words: Diabetes mellitus, IAA, ICA, Prevalence, Edo state

INTRODUCTION
Diabetes mellitus is a genetically and clinically heterogeneous group of disorders (1). It is a disease of ß cells of the Islets of Langerhans or disease resulting from defective secretion and utilization of insulin (2). The hyperglycaemia of diabetes mellitus results from abnormalities of insulin secretion (2). The secretion can be delayed, deficient or inappropriate for the blood glucose homeostasis, or its action being affected by circulating antagonists, e.g. antibody or abnormalities of its tissue receptor (3). Diabetes mellitus is broadly classified into two main groups, Insulin Dependent Diabetes Mellitus (IDDM), Type 1 and Non Insulin Dependent Diabetes Mellitus (NIDDM), Type 2. Type 1 is caused by auto destruction of ß cells of Islets of Langerhans

while the Type 2 results from non-production of insulin or insufficient circulating insulin.

Insulin autoantibodies (IAA) are one of several markers for Type 1 (autoimmune) diabetes. Unlike the other markers, their legend is unique to the beta cell (4). IAAs are the first markers to appear during the symptomless period which precedes diabetes and they are present in the vast majority of young children destined to develop diabetes (4). They are present at titres which relates inversely to age at onset of IDDM (5) (6), and are believed to mark for the rapidity of progression to the disease. Thus, IAA are rare in IDDM of adult onset, which is generally of slow progression, but present almost universally in children who develop diabetes below the age of five years (6), where progression is typically rapid. The origins of IAA, and the timing of their appearance in the circulation, are however unknown. Nevertheless, the rare description of congenital IDDM where the infant, but not the mother, is seropositive for islet related autoantibodies including IAA (7) indicate that insulitis can begin during gestation.

Recent studies in humans and in animal models of spontaneous insulin-dependent diabetes have demonstrated the presence of islet cell cytoplasmic antibodies (ICA), islet cell surface antibodies (ICSA) and insulin autoantibodies (IAA) during a prolonged but relatively asymptomatic pre-diabetic period (8)(9)(10)(11)(12)(13)(14). Most studies evaluating immune markers for prediction of Type 1 diabetes mellitus have focused on first degree relatives, although only 10% of newly diagnosed patients have affected first degree relatives. A previous study (15) has reported a significant association of IgG IAA with ICA – positive in first-degree relatives of Type 1 diabetics. Several attempts have been made to determine the prevalence of diabetes by surveys of community. Most surveys have shown that there are about as many unknown diabetes as known cases.
There is paucity of studies in the prevalence of IAA and ICA in Nigerian population. We therefore aimed at determining the prevalence of both IAA and ICA in diabetics compared with their non-diabetic controls. In addition, we determined the prevalence of IAA and ICA in the general population and the relatives of type 1 diabetics. An early detection of circulating IAA and ICA is important in order to identify the
individuals in the general population, the siblings, and families of IDDM patients, who are at a high risk of developing this disease because of their genetic predisposition to diabetes hence the need for this study.

Materials and Methods
Subjects
This study was conducted at Central Hospital, Benin City. The subjects included 55 diabetic patients on appointment visit to diabetic clinic and those on admission; 150 relations of the type 1 diabetic patients; 250 volunteer controls and a general population of 455 participants. Participants’ consents were obtained before participation in the study. Ethical clearance was obtained from the Ethical Committee of Central Hospital, Benin City, Edo State, Nigeria.

Classification of diabetes
Patients were classified as Type 1 or Type 2 according to the clinical criteria recommended in the 1997 World Health Organisation(16). Classifications were based on age at diagnosis, mode of onset (acute versus insidious presentation), duration of disease, current treatment, BMI, waist-to-hip ratio, blood pressure, random or fasting glucose, HbA1c and urine ketones. A classification of Type 1 diabetes was defined by the following criteria: onset in patients aged 30 years or less, presentation of acute classical symptoms, and that required insulin therapy to control hyperglycaemia. A classification of Type 1 diabetes was also designated to patients older than 30 years that required insulin treatment, lacked metabolic control, and were determined to be underweight. A classification of Type 2 diabetes was designated to patients who were older than 30 years at diagnosis and did not need insulin for metabolic control. A Type 2 classification was also designated to patients younger than 30 years who were obese and had diabetes for a long duration without requiring insulin treatment. Patients that did not clearly exhibit the clinical features of either Type were classified as undetermined and excluded from the present analysis.

Sample Collection
Ten millilitres of blood was collected intravenously, five millilitres was dispensed into a plain container, and the other five millilitres glucose and protein were immediately detected qualitatively using Combi – 9 strip.

Data Analysis
Statistics was done using Statistical Package for Social Sciences program (SPSS) version 16.0. Values obtained in this research were represented as mean ± Standard error of mean (SEM) for continuous variables and percentages for categorical variables. Analysis of variance (ANOVA) was used to compare data at 95% confidence interval (p<0.05).
was dispensed into a fluoride oxalate container. The blood samples were spun at 1500rpm for 10minutes and the supernatant serum/plasma were separated into separate tubes. . The serum/plasma samples were stored at -200C for up to 2weeks prior to the analysis of fasting blood glucose and antibodies. IAA and ICA were determined using ELISA reagents from Biomerica Inc., U.S.A. Blood glucose was determined colorimetrically using Randox kits from United Kingdom. Fresh urine samples were voided into clean sterile containers, glucose and protein were immediately detected qualitatively using Combi – 9 strip.

Tables 1 and 2 show the general prevalence of IAA (n = 54) in the general population (n = 455) to be 11.9% while the prevalence of ICA (n = 55) in the general population is (n = 455) is 12.1%. However, in diabetic Type 1 population (n = 5), the prevalence of ICA and IAA is 100%. In Type 2 diabetic population, the prevalence of IAA (n = 50) is 10%, while ICA (n = 50), is 10% (n = 50) respectively. Diabetic relatives population of IAA and ICA (n = 150) have a combined general prevalence of 26.7% (n = 40). However, the general population prevalence of IAA (n = 250) and ICA (n = 250), are 1.6% (n = 4) and 2% (n = 5) respectively.

DISCUSSION
Most studies on the role of autoimmune markers in the pathogenesis of type 1 (insulin – dependent) diabetes mellitus have focused on first degree relative. Although, the risk for type-1 diabetes is increased among relatives, it is likely many of them may have a reduced acute insulin response to glucose (17, 18). The present study indicated 26.7% prevalence of both IAA and ICA in relatives of type 1 diabetic patients. Another study, (19) in London among English school children found prevalence of 2.8% and 6.8% respectively in the population and siblings of the diabetic patients. The work carried out by a researcher (20), at Malmon, Sweden in a study of a heterogenous group of high risk subjects (non-diabetic patients genetically at higher risk for IDDM including discordant monozygotic twins and ICA positive first degree relatives), IAA were detected in 31% of ICA positive individuals.
The present study indicated 100% prevalence of IAA and ICA in the diabetic type 1 patients and 10% prevalence in type 2 diabetics for the two antibodies respectively. In addition, we observed 1.6% prevalence of IAA and 2.0% of ICA in non-diabetic controls. Our finding is not in agreement with some workers (21), who found the prevalence of IAA in 18% of newly diagnosed IDDM patients. But with an improved radiometry antibody assay, they found approximately 40% of fresh onset, untreated IDDM patients. The reason may be due difference in the methodology. Similarly, our study did not agree with a previous study (22) which found 2 out of 68 diabetics and 1 out of 68 controls among Nigerian diabetics have ICA. Some workers (23), (24), (25), (26), (27), (28), have also reported prevalence of 20 – 50% among newly diagnosed IDDM patients in Pasco Country, Florida, USA. However, with an improved radiometry antibody assay, they found approximately 40% of fresh onset, untreated IDDM patients. The reason may be due differences in methodologies. The 100% prevalence observed in IAA and ICA in this study may be due to the fact that the patients have all commence the administration of exogenous insulin.
Our data also indicated 11.9% prevalence of IAA in the general population. This is at variance with the work of some researchers (29), who reported the prevalence of IAA to be 4% among 1031 populace in Sweden. Also the prevalence of ICA in the general population (n = 455) is 12.1%. This is at variance with the work of (30) who found the prevalence of ICA to be 6.8% among Tanzanian Africans. Another previous study (29) also found 1.4% prevalence of ICA in a Swedish population.
It is believed that the variations in prevalence in this work relative to other previous works may be due to differences between the Africans and Caucasians in terms of diet, climate, method of diagnosis and other etiological factors that trigger the onset of the diabetes mellitus. However, to the best of our knowledge, this is the first documented work to evaluate combined IAA and ICA measurements among African populations either with diabetes or without diabetes.

Conclusion
The seroprevalence of ICA and IAA are high in this study and their occurrence are almost at the same rate in all the groups, it shows that categorising diabetes into type 1 or 2 on the basis of ICA may generally not fit the pattern of diabetes as seen in Nigeria and possibly other parts of the tropics. Though there is no plan for follow up on this work, if those positive IAA and ICA individuals are followed up, majority of them may eventually develop the disease, which brings us to the need to advocate for early routine check up and vigorous public enlightenment to avoid development of diabetes mellitus, which may lead to complications and subsequently mortality.

REFERENCES
1. Fajans S.S., Cloutier M.C. and Crowther R.I. Clinical and Etiologic Heterogeneity of idiopathic Diabetes Mellitus. Diabetes; (1978); 27:11- 12.

2. Lynch M.J., Raphael S.S., Mellor L.D., Spare D.D. and Inwood J.H.M. Medical Laboratory Technology and Clinical Pathology 2ndEd. W.B Saunders Co. (Philiadephia), (1969); pp367.

3. Robert H.W. and Daniel P. (Jnr). The Pancreas. In Robert, HW (Ed.).textbook of Endocrinology, 6th Edi,

W.B. Saunders Co.(Philadelphia); (1974):pp 553.

4. Eisenbarth G.S.T. Type 1 diabetes mellitus.A chronic autoimmune disease. N.Engl J. Med. (1986); 313: 1360 – 1368.

5. Vardi P., Ziegler A.G., Matthews J.H., Dib S., Keller R.J., Ricker A.T., Wolsdrof J.H., Herskowitz R.D., Rabizadeh A., and Eisenbarth G.S. The concentration of insulin autoantibodies at onset of Type 1 diabetes; inverse log-linear correlation with age.Diab.Care; (1988); 11:736-739.

6. Karjalainen J., Salmela P., Ilonen J., Surcel H.M. andKmp M.A comparism of childhood and adult Type 1 diabetes mellitus.N.Engl J. Med. (1988); 320: 881-886.

7. Dotta F., Gillio C., Fanilla C., Moretti A., Bosco N., Sulli P., Colarizi C., Tiberti E., Anastasi G. and Di Mario U. Autoimmunity to pancreatic ß cells in congenital diabetes mellitus.Diabetologia. (1996); 39 (suppl 1): A97.

8. Gorsuch A.N., Spencer K.M., and Lister J.The natural history of Type 1 diabetes mellitus: evidence for a long prediabetic period. Lancet. (1981);2: 1363-1365.

9. Betterie C., Zanette F., Tienoo and Trevban A. Five year follow-up of non-diabetes with islet cell antibodies.Lancet II;(1982): 284.

10. Beakkoskov S., Dyrberg T. and Learnmark A. Autoantibodies to a 64-kilodalton islet cell protein precede the onset of spontaneous diabetes in the BB rat. Science (1984); 224:1348.

11. Dyrberg T., Poussier P., Nakhooda F., Marliss E.B. and Lernmark A. Islet cell surface and lymphocyte antibodies often precede the spontaneous diabetes in the BB rat. Diabetologen, (1984); 26: 159.

12. Srikanta S., Ricker A.T., McCulloch D.K., Soeldner J.S., Eisenbarth G.S., and Palmer J.P. Autoimmunity to insulin, beta cell dysfunction, and development of insulin-dependent diabetes mellitus. Diabetes; (1986); 35:139-142.

13. Takei I., Maruyama T., Taniyama M. and Kataoka K. Humoral immunity in the NOD mouse. In insulitis and type 1 diabetes Lesson from the NOD mouse (ed by Tarui Y. Tochino and K. Nonaka). Academic Press. Tokyo. (1986); pp 101.

14. Reddy S., Bibby N.J. and Elliot R.B. Ontogeny of islet cell antibodies, insulin autoantibodies, and insulitis in the non-obese diabetic mouse. Diabetologia, (1988);31:322.

15. Dean B.M., Becker F., McNally J.M., Tarn A.C., Schwartz G., Gale E.A.M. and Bottazzo G.F. Insulin autoantibodies in the pre-diabetic period correlation with islet antibodies and development of diabetes. Diabetologia.(1986);29:339-342.

16. W.H.O. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus: Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 20: 1183–1197, 1997.

17. Srikanta S., Ganda O.P., Jackson R.E., Gleason M.E., Kaldany M.R., Garovoy E.I., Milford C.B., Carpenter B., Soeldner J.S., and Eisenbarth G. S. Type 1 diabetes mellitus in monozygotic twins: chronic progressive beta cell dysfunction. Annals Int. Med. (1983); 99:320-326.

18. McCulloch D.K., Klaff L.J. and Kahn S.E. Non progression of subclinical ß-cell dysfunction among first degree relatives of IDDM patients 5 years follow up of the Seatle family study. Diabetes, (1990); 39:549 – 556.

19. Bingley P.J., Bonifacio E., Shattock M., Gillmor H.A., Sawtell P.A., Dunger D.B., Scott R.D.M., Bottazzo G.F., and Gale E.A.M.Can islet cell antibodyies predict IDDM in the general population? Diabetes Care, (1993); 16:45-50.

20. Osson M. L. Sundkvist G. And Lernmark A. Prolonged incubation in the two colourimmuno fluorescence Test increases the prevalence and titres of islet cell antibodies in Type 1 (insulin-dependent) diabetes mellitus. Diabetologia, (1987); 30(5): 327-332.

21. Palmer J.P., Asplin C.M., Clemons P., Lyen K., Tatpati O., Raghu P.K. and Paquette T.L. Insulin antibodies in insulin dependent diabetics before insulin treatment. Science (Wash DC), (1983); 222:1337-1339.

22. Oli J.M., Bottazzo G.F., and Doniach D. Islet cell antibodies and diabetes in Nigerians.Trop Geogr Med. 1981; 33:161–164.

23. Arslanian S.A., Becker D.J., Rabin B., Atchison R., Eberhardt M., Cavender D., Durman J. and Draish

A.L. Correlates of insulin antibodies in newly diagnosed children with insulin-dependent diabetes before insulin therapy. Diabetes, (1985); 34:926-930.

24. Bergman S., Ludvigsson J., Binder C. and Mandrup – Poulsen T. Insulin antibodies before treatment in ICA – positive children with IDDM. Diabetes Res. Clin Practice Suppl. (1985); 1:45.

25. Wilkin T., Armitage M., Casey C., Pyke D.A., Hoskins P.J., Rodier M., Diaz J.L., and Leslie R.D.G.Value of insulin autoantibodies as serum markers for insulin dependent diabetes mellitus. Lancet. (1985); 1:480-482.

26. Atkinson M.A., Maclaren N.K., Riley W.J., Winter W.E., Fisk D.D. and Spillar R.P. Are insulin autoantibodies markers for insulin-dependent diabetes mellitus? Diabetes.(1986);35:894.

27. Karjalainen J., Kmp M., Mustonen A., Ilonen J. and Akerblom H.K. Relation between antibody and complement fixing islet cell antibody at clinical diagnosis of IDDM. Diabetes, (1986); 35:620.

28. McEvoy R.C., Witt M.E., Ginsberg-Fellner F. and Rubinstein P. Anti-insulin antibodies in children with Type 1 diabetes mellitus: genetic regulation of production and presence at diagnosis before insulin replacement. Diabetes, (1986); 35:634-641.

29. Samuelsson U., Ludivigeon J., and Sundikvist G. Islet cell antibodies (ICA), insulin antibodies (IAA), islet cell surface (ICSA) and C- peptide in 1031 school children in a population with a background incidence of IDDM. Diabetes Res. Clin.Pract. (1994); 26 (3) : 155-162.

30. McLarty D.G., Athaide I., Bottazzo G.F., Swai A.M., and Alberti K.G. Islet cell antibodies are not specifically associated with insulin-dependent diabetes in Tanzanian Africans. Diabetes Res ClinPract.1990; 9:219–224.

 

A.A Sadeeq; A.O Ibegbu; J. A Timbuak; M, Tanko; S.P Akpulu; H.R Bello; S.A Musa
Department of Human Anatomy; Faculty of Medicine. Ahmadu Bello University, Zaria. Nigeria

L. H. Adamu: Department of Anatomy; Faculty of Medicine. Bayero University, Kano. Nigeria 

I.S El-Ladan: Department of Anatomy; Faculty of Medicine. Umaru Musa Yar’addua University, Katsina Nigeria

H.O Kwanashie: Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences. Ahmadu Bello University, Zaria. Nigeria.

All correspondence to: Abubakar Sadeeq Adamu; aasadeeq@abu.edu.ng

ABSTRACT
Mercury is a wide spread environmental pollutant that occurs naturally and has been reported to cause some health problems worldwide. Calcium is an essential neurotrace elements that is required for nerve cells to release neurotransmitters. The present work study the effects of mercury toxicity on memory and level of calcium (ca2+) using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats. Twenty four (24) adults Wistar of both sexes were used for the studies and randomly divided into four groups of six rats per group. Group 1 was the Control and received normal saline, while Groups 2, 3 and 4 received 12.45mg/kg, 28.9mg/kg and 49.8mg/kg body weight of mercuric Chloride respectively for twenty one (21) days through oral route. Animals were trained for spatial learning and memory using Morris water maze method and latency time to find the flat form was recorded. The animals were anesthetized and humanely sacrificed using chloroform. The brain was fixed in Bouin’s fluid and processed histologically using H and E stain. Moreover, some of the tissues were crushed by using mortar and pestle after oven drying at 100oC for short-live irradiation method which was exposed to characteristic Gamma-ray to reveal the presence of Ca2+ ion. The photomicrograph reveals that, there was distortion of the pyramidal cells, congestion, necrosis and sparse distribution of hippocampal cells. The spatial memory test showed a decreased in latency time among the control group while the treated groups showed an increased in latency time that was statistically significant (P=0.05). NAA-1 shows an increased level of calcium concentration among mercury treated animals (P=0.01) when compared to the control animals in group I. It was concluded from this studies that; mercuric chloride exposure has effects on the cyto-archetecture of the hippocampus and alters the level of neurotrace element (calcium ion) which in turn affects spatial learning and memory in adults Wistar rats.

Keywords: NAA-1, short-live analysis, Mercury, memory, calcium ion.

INTRODUCTION
Mercury, a heavy metal is a highly deleterious environmental pollutant that can lead to many health problems in the world (WHO, 2003). Man in his environment is exposed to much potential hazards by heavy metals via bioaccumulation and biodegradation which are transferred to man via food chain due to anthropogenic activities (Wang et al., 2007). Mercury can exist either as elemental, organic and inorganic mercury (Burger et al., 2011). Sources of Mercuric compounds are mostly from Industrial sources, gas, fumes, battery disposals, broken mercury thermometer and coal combustion (Akagi, 1995; Bjomberg et al., 2011). Natural source of Mercury such as Mercury chloride that is found in higher densities in rocks and volcanic activities can give half off of the mercury present in nature (Park, 2000; Booth, 2005). There are many routes of exposure to mercury which include: Oral exposure, inhalational exposure and dermal exposure (Vupputuri et al., 2005: Berlin, 2006). Mercury and its compounds have been shown to also have effects on the growth, weight, renal system, liver, enzymes, memory  and psychological disturbances to mention but a few (Kosan et al., 2001; Rao, 2001; Valera et al., 2008;). Signs and symptoms of mercury poisoning include; Irritability, excitability, restlessness of the skin and eyes, headache, dizziness, difficulty in breathing and frequent urination (ATDRS, 2011). Mercury has no known nutritional or biomedical importance but has various applications and uses such as preservation, employed by pharmaceutical company, agriculture and in cosmetic production (W.H.O, 2005). Calcium is especially important element for signal transduction in cells. For this reason, it is sometimes called a second messenger (Khachaturiazn et al., 1989). Coupled with the fact that calcium is necessary for neurons to release neurotransmitters in the brain, calcium plays an important role in the areas of the brain responsible for storing and retrieving memory (Khachaturiazn et al., 1989). The present work, study the effects of mercury toxicity on memory and level of ca2+ using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats.

MATERIALS AND METHOD
Experimental Animals
Twenty four Wistar rats of average weight of 210g was used for this study and were acclimatized for three weeks in the animal house of the Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria. The animals were fed with Grower’s mesh brand of the Animal feed which was prepared in pellet form to reduce spillages. The animals were fed 3 times daily while clean water was provided in plastic drinking bottle and animals were allowed to feed and drink ad-bilitum. The animals were divided into four (4) groups with six animals per group.
Chemical substances
Mercuric chloride (May and Bakers limited, Dagenham England) with batch number XN202 was obtained from Steve Moore chemicals limited Samaru, Zaria. Kaduna State, Nigeria.
Administration of Mercury Chloride
The LD50 of Mercuric chloride was adopted from the Manufacturer (as 166mg/kg body weight. The Concentration of Mercury chloride used was determined using 30%, 15% and 7.5% of the LD50 per kg body weight according to the manufacturer’s instruction. Animals in Group I served as the Control and were given normal saline, while animals in Group II, III and IV were orally administered with mercuric chloride at 12.45 mg/kg, 24.9 mg/kg and 49.8 mg/kg body weight respectively of the LD50 per kg body weight. The administration of mercuric chloride lasted for 21 days through oral route.

Neurobehavioral test
Spatial learning and memory test using Morris water maze
Morris water maze test was used to develop and test spatial learning and memory in animals according to the methods of Morris (1981) which was further developed by Liu et al (2011). Animals were placed in a circular pool of clear transparent water which was partition into four quadrants. According to this method, a platform was submerged beneath the surface of the maze pool; the animal task is to find the hidden platform. The animal starting point was changed from time to time so as to build a cohesive spatial representation of the pool in order to find the platform and the latency time was recorded both during the training and experimental periods.
Animal Sacrifices
After the administration, the animals were humanely sacrificed and incision was made through the skin and muscle of the skull. The skull was opened through a mid-sagittal incision and the brain was removed and fixed in Bouin’s fluid. The tissues were routinely processed and stained using haematoxylin and eosin. And some tissue were processed for short-lived irradiation for neurotrace elements detection.

Tissues Preparation for Neuro-trace Element Analysis Method
Trace element analysis of the brain tissue was carried out with the aid of Nuclear Research Reactor (NNIR-1) using NAA-1 method at the Center for Energy Research and Training Ahmadu Bello University, Zaria, Kaduna, Nigeria according to IAEA (2004). The brain tissue samples were Oven dried at 100oC using Memmert DINI ovum-280 model-KA. After successful drying, tissues were crushed in Agate mortar and piston at zero contaminant level in order to have the tissues in powdered form which were then put in sealed polyethene bag and weighed using Metter AE 240 Electronic Digital balance to prepare tissue for nuclear irradiation. In NAA-1 short-lived irradiation method used, each sample was irradiated for 5 minutes and exposed to a semi-conductor detector for characteristic gamma-rays detection for 10 minutes followed by another 10 minutes for 3 hours after the first count. This is to allow safe handling of the radioactive nuclei formed (Akaho and Nyako, 2002).The short lived analysis revealed the presence of neurotrace element calcium (Ca2+). The samples were counted for short –lived irradiation using Maestro software attached to an Analog to Digital Converter (ADC) card and Multi-channel Analyzer card (MCA) according to method of Jonah (2005).

Statistical analysis
Data obtained was expressed as Mean ± SEM (Standard error of Mean). One Way Analysis of Variance (ANOVA) was used to compare the Means between and within the groups. P-value less than 0.05 was considered statistically significant. Statistical analysis was performed using EZanalyze v3.0 and a post hoc test of Bonferroni was applied. Chart was produce using Microsoft(R) Excel 2007 for windows. The Trace elements present in the tissue were analyzed using WIN SPAN 2004 software for peak analysis and efficient calibration and calculation of elements detected.

RESULTS
The effect of mercury chloride exposure on Morris water maze test.
The results on mercuric chloride exposure on spatial learning and memory test, showed a decreased in the meantime taken for the animals to complete Morris water maze task among the control group throughout the period of administration as shown in Table 1. While animals in group II and III had an increased in latency time to find an escape route during Morris water maze activity, though the increase is between weeks 3 and weeks 2 respectively which was significant (P=0.05). And animals in group IV showed an increased that was statistically significant (P=0.01) throughout the period of administration as shown in table 1 below.

Table 1: Effect of mercury chloride ingestion on spatial learning and memory using Morris water maze test

the hippocampus of group III animals, with degeneration of the pyramidal cell layer, loss of some of the pyramidal cells and clumping of pyramidal cell nuclei. Group IV animals show disorientation of the pyramidal cell layer and degeneration of some pyramidal cells with the pyramidal cells appearing to be smaller than normal as shown in Plate D.

Short-lived analysis for calcium (Ca2+) level in the brain
The results of Neuro-trace element analyses indicates the presence of Calcium (ca2+) in the brain tissues of the animals which showed a significant increase (P =0.01) in concentration of Calcium in Group III and IV animals when compared to the control. While Groups II animals showed an increased in calcium concentration that is not significant as shown in figure 1

DISCUSSION
The pyramidal cells in the hippocampus manifested some changes like necrosis degeneration and loss of neuronal cell fiber compared to the control group which could be as a result of the exposure of mercuric chloride. This implies that the activity of the hippocampus in memory formation and learning will be impaired and the role of the hippocampus that involved storage and retrieval of information will also be lost. The findings in this study agree with the studies of Wolf et al., (2003), who reported that rats exposed to high concentration of mercury vapor, showed neurodegenerative changes in the hippocampus which was responsible for memory deficit in such animals.
Result from the present study shows that, there was an increased in time taken by the experimental rats to find the hidden platform in Morris water maze test which was significant. Conversely, the pyramidal cell layer of the hippocampus appears to be damaged with dead cells, and vacuolated spaces and distortion in the general morphology of the pyramidal cells. These alterations can consequently result to memory impairments which could be as a result of neuronal degeneration. The destruction of the pyramidal cells implies that activity from the brain region that projects into the pyramidal layer of the hippocampus will also be lost such as memory and learning ability (Wolf, 2009; Quirino, 2012). Mutter (2010) had reported that short term occupational exposure to high levels of mercury induced slight cognitive deficits. A memory deficit among animals exposed to methyl mercury was not significant in latency time or swim length between the different groups of animals according to Olson (2005).
The findings of the present study revealed a high level of calcium in the brain due to mercury intoxication. Increased in calcium level among groups that receive medium and high doses of mercuric chloride implies that, the role of calcium as a second messenger in signal transmission between cells will be loss, which is required for nerve cells to release neurotransmitters in the central nervous system. This finding agrees with Bandtlowc (1993) which explained that excess calcium at the surface of nerve cells causes them to ‘fire’ spontaneously sending messages around the brain for no purpose except confusion and cause a ‘short circuit’ allowing messages sent to one destination to get misrouted to another which causes memory loss.

CONCLUSION
It was concluded from the present study that oral administration of mercuric chloride has effects on spatial learning and memory and can cause histopathological assault in the hippocampus like necrosis, loss of nerve fibers of the pyramidal cells and neuronal clumping. The short-lived analysis for neurotrace elements revealed the presence of calcium in the brain which was high in concentration due to mercury exposure.

ACKNOWLEDGMENT
I appreciate the support of Mal. Shehu Shika of Center for Energy Research and Training. Ahmadu Bello University Zaria, Nigeria for a bench space in his Laboratory and Mal Ado Garba of A. B.U for his financial support.

REFERENCES
1. Akagi, A. (1995). Human exposure to mercury due to gold mining in the Tapajo river basin, amazon, Brazil: Specification of mercury in human hair, blood, and urine. Water, Air and Soil Pollution. 80: 85–94.

2. Akaho.E.H and Nyako B.J. (2002). Characteristic of neutron flux in irradiation site of MNSR reactor using Westcott formalism for the neutron activation analysis. Apply radiation isotopy 57,265-273

3. Agency for Toxic Substance Diseases Registry (2011). Exposure to hazardous substances and reproductive health. American Family Physician 48(8):1441-1448.

4. Bandtlowc. E., M. F. Schmidtt,. D. Hassingemr., E. Schwab & S. B. Kater (1993). Role of Intracellular Calcium In Ni-35-Evoked Collapse Of Neuronal Growth Cones. Science 259: Pp80-83

5. Berlin M. (2005).Mercury. In: Friberg L, Nordberg G.R, Vouk V.B, eds. Handbook on the toxicology of metals. 2nd ed. New York, NY: Elsevier Press.

6. Bjornberg.A., M. Vahter, B., Berglund, B., Niklasson, M., Blennow., and G. Sandborgh-Englund, (2011). “Transport of methylmercury and inorganic mercury to the fetus and breast-fed infant,” Environmental Health Perspectives, vol. 113, no. 10, pp. 1381–13

7. Burger, J.,C. Jeitner, and M. Gochfeld, (2001)“Locational differences in mercury and selenium levels in 19 species of saltwater fish from New Jersey,” Journal of oxicology and Environmental Health, vol. 74, no. 13, pp. 863–874.

8. Booth S and D. Zeller. (2005). “Mercury, food webs, and marine mammals: implications of diet and climate change for human health,” Environmental Health Perspectives, vol. 113, no, pp. 521–526
9. IAEA (2004) WINSPAN 2006 a multipurpose gamma ray spectrum analysis software CIAE, Beijing china.

10. Jonah S.A; Balogun G. I.,Umar., I Maiyaki .M.C. (2005). Neutron spectrum parameters in irradiation channels of Nigerian research reactor -1(NNIR-1) for NAA standardization Journal of radio anal nuclear chemistry 266(1) 83-88.

11. Khachaturiazn. S. 1989. The Role Of Calcium Regulation In Brain Aging: Reexamination Of A Hypothesis. Aging 1: 17-34.

12. Kosan, C, A. K. Topaloglu, and B. Ozkan, (2001)“Chronic mercury intoxication simulating pheochromocytoma: effect of captopril on urinary mercury excretion,” Pediatrics International, vol. 43, no. 4, pp. 429–430.

13. Liu. Li., Jiong D., Charles M., Junying G., Gang., Hu, M., Xiao.K (2011). Pretraining affects Morris water maze performance with different patterns between control and ovariectomized plus d-galactose-injected mice. Behavioural Brain Research 217:1, 244-247

14. Morris W (1981) Test For Spatial Learning And Memory.Science.136,

15. Mutter J, Curth A, Naumann J, Deth R, Walach H (2010). Does inorganic mercury play a role in Alzheimer’s disease? A systematic review and an integrated molecular mechanism Journal Alzheimers Disease. 22(2):357-74.

16. Olson K, Boush G.M (2005). Decreased learning capacity in rats exposed prenatally and postnatally to low doses of mercury. Bulletin of Environmental Contaminant Toxicology 13:73-79.

17. Park, S.H, S. Araki, A. Nakata (2000). “Effects of occupational metallic mercury vapor exposure on suppressor-inducer (CD4+CD45RA+) T lymphocytes and CD57+CD16+ natural killer cells,” International Archives of Occupational and Environmental Health, vol. 73, no. 8, pp. 537–542,

18. Quirino Cordeiro Júnior, M.D.; Marcília de Araújo Medrado Faria, M.D.; Renério Fráguas Júnior, M.D. (2012) Depression, Insomnia, and Memory Loss in a Patient With Chronic Intoxication by Inorganic Mercury. The Journal of Neuropsychiatry and Clinical Neurosciences 191-205

19. Rao M.V and P. S. N. Sharma ( 2001). “Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice,” Reproductive Toxicology, vol. 15, no. 6, pp. 705–712,.

20. Valera B, Dewailly E, Poirier P (2008). Cardiac autonomic activity and blood pressure among Nunavik Inuit adults exposed to environmental mercury: a cross-sectional study. Environmental Health. 28:924.

21. W.H.O (World Health Organisation) (2003). Elemental mercury and inorganic mercury compounds: Human health aspects. Concise International Chemical Assessment Document. CICAD 50. Geneva.2003

22. Wang J.S, Huang PM, Liaw WK, (2007). Kinetics of the desorption of mercury from selected fresh water sediments as influenced by chloride. Water, Air, Soil Pollution 56:533-542.

23. Wolf U, Rapoport M.J, Schweizer TA (2009). “Evaluating the affective component of the cerebellar cognitive affective syndrome”. Journal Neuropsychiatry Clinical .Neuroscience. 21 (3) 245–53.

24. Vupputuri S, Longnecker MP, Daniels JL, Guo X, Sandler DP (2000). Blood mercury level and blood pressure among US women: results from the National Health and Nutrition Examination Environmental research. 97:195–200

Toryila J.E, Amadi K, Odeh S.A, Egesie U.G.: Human Physiology Department, Faculty of Medicine, ABU Zaria.

Adelaiye A.B. Achie L.N: Human Physiology Department, Faculty of Medical Sciences Unijos.

All correspondents to:  Toryila J.E Human Physiology Department, Faculty of Medicine, ABU Zaria. 

ABSTRACT
BACKGROUND: Contraception is an important health issue in preventive medicine because it protects women globally from the effects of unwanted pregnancy and allows them to integrate into society. This research priority included efforts to discover the short and long term effect of combined oral contraceptive (DUOFEM) on some Physiological parameters and possible mechanism of actions in female wistar rats.
Method: Eighty (80) female wistar rats aged 10-12 weeks weighing 180-250 g were used for the study. They were divided into four groups of 20 rats each comprising 10 treated and 10 control rats. The treated rats received 0.6mg/kg body weight of COC intragastically for 36, 48, 60 and 72 days in five-day cycles (four days treatment with one-day break. A haematology analyzer was used to perform a complete blood count (CBC or FBC) .An enzyme-linked immunosorbent assay (ELISA) was used for the quantitative determination of alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT). Prothrombin time (PT) and Activated partial thromboplastin time (APTT) were performed using Sysmex CA-6000 Coagulation Analyzer. Liver function tests, Erythropoietin, interleukin-6 (IL-6) and interleukin-11 (IL-11) were determined using rat ELISA kit (Karmiya Biomedical company, USA).
Results: There were significant decrease in Hb, PCV, RBC and WBC counts, lymphocytes (L), PT, IL-6 and IL-11 in all treated groups compared to controls (P<0.005). There was significant increase in alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), sodium and potassium in all treated groups compared to controls(P<0.05). There were however no significant changes in activated thromboplastin time (APTT) and erythropoietin (EPO). The lowest values of Hb, PCV, RBC count, Lymphocytes, PT, IL-6 and Fibrinogen and the highest values of ALT,ALP and AST were observed in long
term used (72 days).Long term used of combined oral contraceptives may leads to more complications than short time use. Combined oral contraceptives users should be monitored for some Physiological parameters.

KEY WORDS: Combined Oral Contraceptives, Physiological Parameters, Wistar rats.

INTRODUCTION
Birth control is a major factor in public health and welfare, preserving the general and reproductive health of women and allowing them to choose the movement of a planned pregnancy

[1]. The World Health organization (WHO) and other global organizations are seeking ways to increase the amount of information and access people have to contraception and other resources related to family planning all round the world.
Oral contraceptives had been reported to have beneficial effects in reducing the incidence of pelvic inflammatory disease, decrease risk of ectopic pregnancy, benign breast lesions, ovarian and endometrial cancers, protection against osteoporosis and rheumatoid arthritis among the users

[2]. Oral contraceptives are sometimes used to treat heavy or irregular menstruation and endometriosis. Oral contraceptive agents can also be used in hormonal replacement therapy, and in the emergency post-coital contraception

[3]. Oral contraceptive decreases the risk of ectopic pregnancy, benign breast lesions, ovarian and endometrial cancers, and offer protection against osteoporosis and rheumatoid arthritis

[4]. Despite the general acceptability and the obvious advantages that have been attributed to oral contraceptives use, some serious side effects have been reported in women taking them. Studies have indicated a relationship of oral contraceptives use and cardiovascular disease, altered levels of coagulation factors, thrombosis, platelet changes, atherosclerosis and multiple sclerosis. Estrogen has been known to have prothrombin effects and elevates cardiovascular and venous thromboembolism risk

[5]. There is little or no data on the effects of Combined Oral Contraceptives (DUOFEM) on Haematological parameters, especially the growth factors such as the cytokines or interlukin-11 and 6, and erythropoietin. Also little or no data have been established in PT, APTT and Liver Function Test using animal model. It is hoped that the result might throw some light on the need of monitoring coagulopathy in women taking these pills. The world population is now seven billion. Nigeria population is estimated to be 176 million and will reach 400 million by the year 2050 [6]. Rapid population growths would have a detrimental effect on socioeconomic development of Nigeria.
There is a concern over population explosion in Nigeria and the drive to control it is leading to indiscriminate used of oral contraceptives. Unintended pregnancy leads to induced abortion which is not legalized in Nigeria, except to save the woman`s life. This research priority included efforts to discover the short and long term effect of combined oral contraceptive (DUOFEM) on some Physiological parameters and possible mechanism of actions in female wistar rats.The knowledge might provide useful interventions towards solving the problem(s). Thus the safety in the contraceptives use and improvement in the health of the user are assured.

Materials and methods
DRUGS: The combined oral contraceptive used is DUEFEM®. They were obtained from family clinic, Ahmadu Bello University Teaching Hospital, Shika-Zaria, and from the Society for Family Health (SFH) Abuja, Nigeria. COCs DUOFEM® tablets which combined ethinyl estradiol and Norgestrel were manufactured by Wyeth Ayerst (USA) and packed and marketed by the Society for Family Health, Lagos, Nigeria. DUOFEM® is a child spacing pill containing ferrous fumarate tablets. Each DUOFEM cycle contains 28 pills; each white tablet contains 0.3mg Norgestrel and 0.03mg Ethinglestradiol and each brown tablet contains 75mg ferrous fumarate. DUOFEM® has a molecular weight of 312.4458g/mol [7].
Eighty (80) female wistar rats aged 10-12 weeks weighing 180-250 g were used for the study. They were divided into four groups of 20 rats each comprising 10 treated and 10 control rats. The treated rats received 0.6mg/kg body weight of COC intragastically for 36, 48, 60 and 72 days in five-day cycles (four-days treatment with one-day break). The COC was given intragastically in 5-day cycles (4-day treatment with 1-day break). All controls were given fresh water ad libitum daily for the period of the experiment. Experimental animals in the study were treated in accordance with the National Protection Laws of Animal Welfare [8]. Ethical clearance was obtained from the Ahmadu Bello University Animal Ethical Committee.
Haematology analyzer was used to perform a complete blood count (CBC or FBC) .An enzyme-linked immunosorbent assay (ELISA) was used for the quantitative determination of alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) . Prothrombin time (PT) andActivated partial thromboplastin time (APTT) were performed using Sysmex CA-6000 Coagulation Analyzer. Liver function tests, Erythropoietin, interleukin-6 (IL-6) and interleukin-11 (IL-11) were determined using rat ELISA kit ( Karmiya Biomedical company, USA). Fibrinogen was estimated by Clauss Assay [9]

Statistical Analysis: The result obtained from this study was analyzed using SPSS version 20 for windows. Analysis of Variance (ANOVA) was used to compare means, and values were compared at P < 0.05. Post Hoc multiple comparisons for significant differences between groups were established by Turkey’s HSD. All the data are expressed as Mean ± Standard Error of Mean (SEM).

RESULTS:
Table 1: The effects of combined oral contraceptives on some haematological parameters in female wistar rats

A, b, c, and d- significant, N- No significant

Lowest values of Hb, PCV and RBC count were observed in treated group D (72days)

Table 2: The effects of combined oral contraceptives on some haemostatic parameters and cytokines in female wistar rats

A, b, c, and d – significant, N- No significant

The lowest values of PT, IL-6 and Fibrinogen were observed in the treated group D (72 Days).

Figure 1: The effects of combined oral contraceptives on AST in female wistar rats 

 Short and Long-Term Effects of Combined Oral…

DISCUSSION
In this study the effect of COC on haematological and biochemical parameters in female wistar rats were investigated. The result shows a reduction in haemoglobin(Hb), Packed cell volume(PCV), Red blood cell (RBC), White blood cell (WBC) counts and lymphocytes in all the groups that were given COC, compared to the controls in each group (P<0-05). The finding of lower Hb, PCV, RBC, WBC and Lymphocytes agrees with the finding of [10].The findings were however, contrary to that of other investigators like Bulur [11], Babatunde [12], Abdalla [13], who reported no changes in full blood count in women on COC. The least values of Hb, PCV and RBC were found in group D (72 days). It could be that the use of COC for a longer period may lead to anaemia. The finding of lower Hb, PCV and RBC counts in COC treated groups compared to controls may be as a result of the haemodilution effect of estrogen and the effect of estrogen on the cytokines like IL-11 and IL-6. The findings of lower values of WBC counts in COC treated groups suggest that the use of COC may alter immune response.
The findings of lower WBC count in COC treated groups was in agreement with that of Sajida [10], and contrary to the findings of Surasak et al [14] who observed no changes in WBC count and that of Araz et al[15] who observed increase in WBC count in COC treated female wistar rats. The difference may be as a result of the use of different COC with different concentration of estrogen and progesterone and the duration of use. There was no significant changes observed in COC treated groups in platelet counts compared to the controls. This finding agrees with that of Surasak et al. [14], Peter [16] Bulur et al [11] and Babatunde et al [12]. In general, the use of COC in female wistar rats suppressed haemopoiesis in this study resulting in lowering of Hb, PCV, RBC and WBC counts.
There was no significant change in serum erythropoietin level in all the controls. This finding is contrary to that of Prechile et al.[17] who found that estradiol benzoate inhibited the production of erythropoietin (EPO). Erythropoietin promotes the survival, proliferation and differentiation of erythrocytic progenitors. It appears COC has little or no effect in EPO production. Decreased RBC count in this study may not be as a result of COC effect on EPO. There was decrease fibrinogen levels in all the COC treated groups compared to the control. This finding differs from that of Eliana et al [18] and Peter et al. [16] who reported increase in fibrinogen level in women taking COC. The finding is also contrary to that of Akhigbe et al [19] who found no significant change in fibrinogen level in female wistar rats treated with COC. Fibrinogen functions as a messenger molecule that coordinates and regulates the body response to inflammation. The association between increase in plasma fibrinogen and thrombosis and the risk of myocardial infarction, atherosclerosis and other cardiovascular diseases are well established. Higher level of fibrinogen raises the risk of stroke [13] .Low fibrinogen levels is associated with low risk of cardiovascular diseases. Effect of COC on the liver as observed in this study may be the cause of decreased in fibrinogen level by a third generation COC (DUOFEM).
Prothrombin Time (PT) significantly reduce in all the COC treated groups compare to the controls (P<0.001). There was no significant reduction in Activated Prothrombin Time (APTT). This finding agrees with that of Abdalla 13] ,in Sudan found significant reduction in PT and APTT in women taking COC. Babatunde [12] found that there was no significant change in the level of APTT in Nigerian women taking COC for three months. This is in agreement with the finding of this study. Also Eliana [18] found reduced PT and APTT in Albanian women taking COC. Reduction in prothrombin time may be as a result of reduced serum level of fibrinogen. Estrogen is also said to increase coagulation factors leading to decreased prothrombin time. The finding of this study is also in agreement with that of Ahmed [20] and Nasir [21] who reported a significant reduction in APTT and PT in women taking COC. The exogenous estrogen in the COC has been shown to accumulate in the liver stimulate the release of procoagulants (Factors II, VII and X) from the degenerating liver cells. Significant decreased in PT observed in this study is possible due to enhanced coagulation process as a result of increased plasma levels of coagulation factors of extrinsic pathway.
There was a significant decrease in serum level of antithrombin (AT), protein C (PC) and protein S (PS) in COC treated groups compared to the controls. Proteins C and S and antithrombin are components of the anticoagulant system. In haemostasis, the procoagulant
system is in balance with the anticoagulant and fibrinolytic system [22]. It is important to emphasize that a disturbance in normal hemostasis may result in thrombosis. This finding is in agreement with that of Soare [23] and John [24] who observed lower values of PC in women using oral contraceptives. Decreased AT, PC and PS may be responsible for the changes observed in haemostasis. AT, PC and PS are vitamin K-dependent. Effect of COC on the liver may be responsible for the reduction of serum level of AT, PC and PS in COC treated groups.
There was a significant decrease in serum level of interleukin-6 (IL-6) and a slight decrease in serum interleukin-11 (IL-11). Estrogen is able to decrease IL-6 expression by blocking the estroblast’s synthesis of IL-6 receptors [25]. IL-11 and IL-6 are haemopoiesis-promoting factors capable of enhancing the growth of myeloid, erythroid and megakaryocytic progenitor cells. They are capable of mediating a complex array of pro- and anti-inflammatory effects. Reduction in IL-11 and IL-6 may be responsible for decreased RBC, PCV, WBC and platelet counts in this study. IL-6 produces C – reactive protein (CRP) which leads to cardiovascular risk. Experimental studies have shown strong correlations between the risk of cardiovascular diseases and inflammatory markers such as CRP and tissue neurosis factor-a (TNF a) [26].
IL-6 is a pleotropic cytokine which stimulates B-lymphocyte and T-lymphocyte differentiation, and activates macrophages and natural killer cells (NK). IL-6 promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions [27]. The finding of reduced serum IL-6 and11 is in agreement with other finding [28] who reported decrease serum level of IL-6 and 11 in menopausal women taking hormonal replacement therapy.
There were increased values of alkaline phosphate (ALP), Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) in all COC treated groups compared to controls (P<0.001). The liver plays a central role in the metabolism of estrogens and progesterones. COC acts directly or indirectly on the liver to produce a variety of biological effects which have both physiological and pathological significance [28]. Higher levels of liver enzymes in the blood streams are prime indicators of liver damage.
The use of COCs has been rarely associated with liver tumours, both benign (hepaticadeomas and nodular hyperplasia) and malignant (hepatocellular carcinoma). The finding of increased liver enzymes is in agreement with that of Dickerson [29] and contrary to that of Surasak [14] who found no changes in liver enzymes in women on COC. Raised serum level of ALP, AST and ALT in treated groups may be due to functional alterations involving the hepatic excretory mechanism. The effect of the COC (DUOFEM) on the liver may be the cause for haemostatic disorder, since PC, PS and AT were reduced.
There was a significant increase in serum sodium level in group D (72 days, P<0.044) compared to the control. Potassium was significantly increased in groups C (60 days) and D (72 days). Sodium retention has been reported in women taking COC [19]. The result in this study is contrary to that of Taneepanichskul [30] who reported no significant changes in electrolytes in women taking COC.
Hypertension occasionally occurs in women receiving estrogen-progesterone combination for contraception. Estrogen raises the plasma level of rennin and increases the production rate of aldosterone which leads to water and sodium retention [31, 32] reported reduced potassium level and increased sodium level in female rats. There was no significant change in bicarbonate and chloride in treated groups compared to the controls. Body electrolyte balances are critical for normal cellular function and maintaining adequate blood and plasma volume and osmolality.

CONCLUSION
This study confirms findings of previous studies which also reported an increased risk for venous thromboembolism with third generation COC. This research work has confirmed the effect of COC (DUOFEM) on haemopoiesis. There is a potential risk of long term immune suppression in female taking COC (Duofem). Estrogen has shown to have long term effect on liver cell which may affect liver enzymes production and functions. There is the tendency for COC to be used earlier in life and for longer period of time, the number of women entering the high risk group for venous thromboembolism and liver cell adenomas maybe on the increase. Therefore, we recommend that women on COC should be regularly monitored for Physiological parameters.

ACKNOWLEDGMENT
I want to appreciate all staff of Haematology, Immunology, Chemical Pathology departments and Histopatology Dept. of ABUTH Zaria for their assistance in the analysis of the samples. Dr. Olayemi of Society for Family Health Abuja, for supplying the COC (DUOFEM) and all staff of Human Physiology Department, University of Jos.

REFERENCES

1. World Health Organization (2009). Hormonal Contraception and Liver Disease. Contraception 80; 325 –326.

3. Vessy, M. P. (1995). Endometrial and ovarian cancer and oral contraceptives: Findings in a large cohort study. British Journal of Cancer 71, 1340-1342.

4. Rosing, J., and Tans, G. (1999). Effect of oral contraceptives on haemostasis and thrombosis. American Journal of Obstetrics and Gynecology 180(6), 375-197.

5. Margolis, K. L., Adami, H. O., Luo, J., Ye, W., and Weiderpass, E. (2007). A prospective study of oral contraceptive use and risk of myocardial infarction among Swedish women. Fertility and Sterility 88, 310-316..

7. Chitturi S, Farrell GC 2013). Adverse effects of hormones and hormone antagonists on the liver. In, Kaplowitz N, DeLeve LD, eds. Drug-induced liver disease. 3rd ed. Amsterdam: Elsevier, , pp. 605-20. (Review of hepatotoxicity of oral contraceptive steroids including cholestasis, vascular disorders, benign tumors and hepatocellular carcinoma).

8. Akinsanya M A, Adeniyi T T, Ajai GO, Oyedele MA (2010). Effect of Vitamin E and folic acid on some antioxidant activities of female wistar rats administered combined oral contraceptives. African journal of Biochemistry Research , 4 (10):pp 238-242.

9. Clauss, A. (1957). Gerinnung Sphysiologieche schnelinethode zur bestimmung des fibrinogen. Acta Haematol, 17:237. Cited by: Beutler, E., Lichtman, M. A., and Colle .E

10. Sajida, S. H., Al-Chalaby, S. M. T., and Amjad, F. A. (2006). Effect of oral contraceptive pills on haematological indices. Tikrit Medical Journal 12(1), 65-69.

11. Bulur, S., Albayrak, M., Bulur, S., Keskin, F., Köse, S.A., Aslantas, Y., Türker, Y., and Ozhan, H. (2012). Effect of combined oral contraceptive use on platelet volume in women at reproductive age. Clinical. Experimental. Obstetrics and Gynecology 39(3), 314-316.

12. Babatunde, A. S., and Olatunji, P. O. (2004). Short-term effect of oral contraceptive pills on some haemostatic parameters in healthy Nigerian women. Nigerian. Postgraduate Journal 11(4), 246-250.

13. Abdalla, T. M., Kordofani, A. A. Y., and Nimir, A. A. H. (2008). Haemostatic studies in Sudanese women on oral contraceptive pills. Khartoum Medical Journal 1(3), 116-118.

14. Surasak, T. (2007). Effect of a new oral contraceptive with drospirenone on vital signs, complete blood count, glucose, electrolytes, renal and liver function. Journal of Medical Association Thailand 90(3), 426-431.

16. Peter, U. O. (2013). Women use of oral contraceptives – does it have any effect on haematological parameters? Annals of the College of Medicine 33(1 & 2).

17. Prechile, C., Ira, A., Rappaport, L. F. S., Mario, C., and Albert, S. G. (1972). The role of Estrogen in the regulation of Erythropoietin. Endocrinology. Obstetrics and Gynecology 180(6), 5375-5382.renal and liver function. J Med Assoc Thai.; 90: 426 – 31.

19. Akhigbe, R. E., Azeez, M. O., Ige, S. F., Oyeyipo, L. P., Ajao, F. O., and Alade A. O. (2008). Haematological effects of long-term administration of combined oral contraceptive in rats. International Journal of Pharmacology 4(5), 403-406..

20. Ahmed J. Al-Husaynee and Muna A. Kashmona (2007). Effect of combined oral contraceptive pills on some haemostatic parameters. Annals of the College of Medicine Mosul, 33(1 & 2): 66-69.

21. Nasir A, Qamaruddin B, Salman NA. Effect of low dose oral pill on haemostatic parameters in a set of Pakistani population. J. Pakistan Med. Assoc. 2008: 58: 229

22. Karl, M., Bjorn, A., and Bjorn, D. (2008). Human activated protein C variants in a rat model of arterial thrombosis. Thrombosis Journal 6(16), 1477-1560..

23. Soare, A. M., and Popac (2010). Deficiencies of proteins C, S and antithrombin III and activated Protein C Resistance. Their involvement in the occurrence of Arterial thromobosis. Journal of Medical Life 3(4), 412-415.

24. John, E. G., and Emmanuel, C. B. (2012). Protein S deficiency. http://emedicine.medscape .com/article/205582-overview 5-8-2014

25. Subhadeep, C., Olga, L., and Sandra, T. D. (2008). Estrogen is a modulator of vascular inflammation. IUBMB Life 60(6), 376-382.

26. Trussell, J. (2007). “Contraceptive Efficacy”. In Hatcher, Robert, A., et al. Contraceptive Technology (19th rev. ed.). New York: Ardent Media.

27. Dickerson J, Bressler R, Christian CD (1992). Liver function tests and low-dose estrogen oral contraceptives. Contraception; 22(6): 597-603.

28. Taneepanichskul S, Jaisamrarn U, Phupong V (2007). Effect of a new oral contraceptive with drospirenone on vital signs, complete blood count, glucose, electrolytes,

30. Kang, J. H., Wiggs, J. L., Rosner, B. A., Hankinson, S. E., Abdrabou, W., and Fan, B. J. (2010). Endothelial nitric oxide synthase gene variants and primary open-angle glaucoma: Interactions with gender and postmenopausal hormone use. Investigation Ophthalmological Sciences 51, 971–979.

Oyewopo AO., Lawal IA., Olawepo A., Enaibe BU., Ajao MS

Department of Anatomy, Faculty of Basic Medical Sciences, Colleges of Health Sciences, University of Ilorin, Kwara State, Nigeria.

Oyewopo C.I.: Department of Anaesthesia, University of Ilorin, Teaching Hospital (UITH), Ilorin Kwara State.

Afolabi O.O.: Department of Morbid Anatomy, University of Ilorin, Teaching Hospital (UITH), Ilorin Kwara State.

All correspondence to: Dr. Oyewopo AO, E-mail: wolesake@yahoo.com.

ABSTRACT
Introduction: The objectives of this study is to determine the effects of cryptorchidism and hyperthermia on the testicular cytoarchitecture of adult male rats, quality and quantity of semen and the level of oxidative stress in the testes before and after intervention of Alpha-tocopherol (Vitamin E).
Methods: Twenty four (24) adult Wistar rats were used for this experiment and they were divided into seven groups (n=4). Experimental cryptorchidism was carried out in some animals while others were subjected to hyperthermia from two sources (hot oven and light bulb) for 28 days; followed by intervention of 100mg/kg alpha-tocopherol for fourteen (14) days.
All animals were then euthanized under anaethesia. The testes were excised and fixed and processed using haematoxylin and eosin technique. Plasma was assayed for glutathione, superoxide dismutase (SOD) and malondialdehyde (MDA).
Discussion: Cryptorchidism and hyperthermia caused in cycto-architectural distortion in the testicular structures of exposed animals and significant reductions in caudal epididymal sperm count and motility, morphology and life and death ratio. Plasma SOD and GLU levels also reduced significantly in exposed animals while MDA levels increased significantly, indicating peroxidation of testicular tissue. Alpha-tocopherol due to its anti-oidative properties impeded lipoperoxidation of the membrane, conserving cell union and increasing the amount of germ cells in the seminiferous epithelium.
Conclusion: At doses and durations tested, alpha-tocopherol (aT) reverted to a significant extent the effects of cryptorchidism and hyperthermia by reducing apoptosis in germ cells, promoting cell survival and reducing histological alterations to the seminiferous epithelium.

Keywords: Cryptorchidism, hyperthermia and Alpha-tocopherol.

INTRODUCTION
The testes function in production of the male gametes or spermatozoa and male sexual hormone, (hence referred to as double glands) which stimulates the accessory male sexual organs and causes the development of the male sex characteristics (Mescher, 2010; Lutz, 2009). In most mammals, the temperature around the testicular region is lower than the core body temperature. In man, scrotal temperature is 2-30C lower than rectal temperature and the optimum temperature. Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility (Thundathi et al., 2012). Oyewopo and Togun in 2005 reported that a small increase in the temperature of the testis does not destroy the germinal epithelium; however, it reduces testis weight and sperm production and brings a greater incidence of morphologically abnormal spermatids and spermatozoa (Oyewopo and Togun, 2005; Bedford, 1991). Occupational exposure to high temperatures adversely affects testicular function causing partial or complete spermatogenic arrest, also causes deterioration in sperm morphology and impairs motility. This leads to oligoasthenoteratozoospermia (OAT) and azoospermia (Dada et al., 2001). Cryptorchidism (undescended testes); the absence of one or both testes from the scrotum happens to be the most common birth defect of the male genitalia, which if persists could result in reduced fertility and increased risk of testicular germ cell tumors (Wood and Elder, 2009). Cryptorchidism has been implicated to cause oxidative stress in animals that the process was performed on, it was reported that Cryptorchid rats had lower testicular weight, sperm count, germ cell count, testicular superoxide dismutase (SOD) concentration, testicular total protein and higher testicular malondialdehyde (MDA) concentration compared to animals in control group (Ayobami et al., 2013; Dutta et al., 2013). Alpha-Tocopherol (aT) is an important antioxidant that localizes to cell membranes. The primary function of vitamin E is as an antioxidant; because it helps reduce oxidation of lipid membranes and the unsaturated fatty acids and prevents the breakdown of other nutrients by oxygen. This protective, nutritional antioxidant function is also performed and enhanced by other antioxidants, such as vitamin C, beta-carotene, glutathione (L-cysteine), and the mineral selenium (Laila and Sahar 2009; Aybek et al., 2008; Traber and Kayden 1987). A significant reduction of lipoperoxidation was observed in the cryptorchid group treated with a-Tocopherol compared to the untreated cryptorchid group, at long term, an increase in the area and maturation of the seminiferous epithelium, a decrease in apoptosis and histological alterations and an increase in fertility was observed in animals treated with alpha-Tocopherol (Saalu et al., 2014; Rosa et al., 2011).
The broad aim of this study investigate the protective properties of alpha tocopherol (vitamin E) on cryptorchidic and heat (hyperthermia) induced testicular injury.

MATERIALS AND METHODS
The experiment was performed in conformity with the Rules and Guidelines of the Animal Ethics Committee of University of Ilorin. The experiments were conducted at the Department of Anatomy, College of Health Science, University of Ilorin.
Animals
Twenty four (24) male Wistar rats (150-210g) were purchased from Banky livestock company, Oke-Ose, Ilorin, Kwara State. The animals where kept in the animal house of the Faculty of Clinical Science, University of Ilorin, under light and dark cycle at room temperature. Proper aeration maintained by the use of wire gauze wooden cage. The rats were fed on growers’ feed from Bendel feeds Nigeria Limited, Ilorin, and distilled water throughout the duration of the experiment. The rats were arranged randomly into groups A-F, kept in different compartments of the cage. The rats were left under the above stated condition for two weeks so as to acclimatize with the condition of their new environment.
Grouping of animals
Group A- (n=4) control animals; these experimental animals were given pelleted feed and tap water ad libitum throughout the period of the experiment;
Group B- (n=4) experimental cryptorchidism was carried out this group;
Group C- (n=4) experimental cryptorchidism was carried out and then 100mg alpha tocopherol for 14days;
Group D- (n=4) animals were exposed to heat from hot oven for 28 days;
Group E- (n=4) animals were exposed to heat from hot oven for 28 days then 100mg/kg vitamin E for 14days;
group F- (n=4) animals were exposed to heat from light bulb for 28 days, group G- animals were exposed to heat from light bulb for 28 days then 100mg/kg vitamin E for 14days.
Animal sacrifice and tissue collection
At the end of the substance administration, the rats were sacrificed 24hrs after the last day of the exposure using ketamine (0.2ml). The animals were pinned on the dissecting board. Dissection was done on the anterior abdominal wall (thoraco-abdominal sagittal incision) using the necessary surgical equipment/instruments. Blood was first collected via the apex of the heart with a hypodermic syringe, and the blood was centrifuged, the epididymis was also excised and placed in normal saline for semen analysis. The testis was removed and then fixed Bouin’s fluid.
Semen analysis
The testes from each rat were carefully exposed and removed. They were trimmed free of the epididymides and adjoining tissues. Epididymal sperm concentration: Spermatozoa in the right epididymis were counted by a modified method of Yokoi and Mayi, (2003). Briefly, the epididymis was minced with anatomic scizzors in 5mL physiologic saline, placed in a rocker
for 10 minutes, and allowed to incubate at room temperature for 2 minutes. After incubation, the supernatant fluid was diluted 1:100 with solution containing 5 g sodium bicarbonate and 1mL formalin (35%). Total sperm number was determined by using the new improved Neuber`s counting chamber (haemocytometer). Approximately 10µL of the diluted sperm suspension was transferred to each counting chamber of the haemocytometer and was allowed to stand for 5 minutes. This chamber was then placed under a binocular light microscope using an adjustable light source. The ruled part of the chamber was then focused and the number of spermatozoa counted in five 16-celled squares. The sperm concentration was the calculated multiplied by 5 and expressed as [X] x 106 /ml, where [X] is the number of spermatozoa in a 16-celled square (Oyewopo et al., 2010).
Sperm progressive motility: This was evaluated by an earlier method by Sonmez et al, (2005). The fluid obtained from the left cauda epididymis with a pipette was diluted to 0.5 mL with Tris buffer solution. A slide was placed on light microscope with heater table, an aliqout of this solution was on the slide, and percentage motility was evaluated visually at a magnification of x 400. Motility estimates were performed from three different fields in each sample. The mean of the three estimations was used as the final motility score. Samples for motility evaluation were stored at 350c. Sperm morphology: The sperm cells were evaluated with the aid of light microscope at x 400 magnification. Caudal sperm were taken from the original dilution for motility and diluted 1:20 with 10% neutral buffered formalin (Sigma-Aldrich, Oakville, ON, Canada). Five hundred sperm from the sample were scored for morphological abnormalities (Atessahin et al., 2006). Briefly, in wet preparations using phase-contrast optics, spermatozoa were categorized. In this study a spermatozoon was considered abnormal morphologically if it had one or more of the following features: rudimentary tail, round head and detached head and was expressed as a percentage of morphologically normal sperm (Oyewopo et al., 2010).

Estimation of lipid peroxidation (Malondialdehyde)
Lipid peroxidation in the testicular tissue was estimated calorimetrically by thiobarbituric acid reactive substances TBARS method of Buege and Aust (1 978). A principle component of TBARS being malondialdehyde (MDA), a product of lipid peroxidation. In brief, 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25 N HCl and 1 5% TCA) and placed in water bath for 1 5 min, cooled. The absorbance of clear supernatant was measured against reference blank at 535nm. Concentration was calculated using the molar absorptivity of malondialdehyde which is 1 .56 x1 05 M-1 cm-1 and expressed as nmol/mg protein (Oyewopo et al., 2010).

Estimation of Glutathione level
Total GSH was estimated in various tissues by the method of Sedlak and Lindsay. Briefly, 5% tissue homogenates were prepared in 20 mM EDTA, pH 4.7, and 100 µl of the homogenate or pure GSH was added to 0.2 M Tris-EDTA buffer (1.0 ml, pH 8.2) and 20 mM EDTA, pH 4.7 (0.9 ml) followed by 20 µl of Ellman’s reagent (10 mmol/l DTNB in methanol). After 30 min of incubation at room temperature, absorbance was read at 412 nm in a Beckman DU-640 spectrophotometer. Samples were centrifuged before the absorbance of the supernatants was measured (Sedlak and Lindsay, 1968).

Estimation of Superoxide dismutase (SOD) level
Testicular tissues were transferred into 5ml ice-cold sucrose solution (0.25M) and homogenized. The homogenates were further centrifuged at 3000 rpm for 15 min to obtain the supernatant, which was then aspirated with Pasteur pipette into sample bottle, stored overnight at 4°C before being used for assays. Tissue activities of superoxide dismutase (SOD) were determined by the method of Marklund and Marklund, (1974).
Statistical analysis
Data collected were analysed using Microsoft Excel and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (HSD) with the aid of SPSS V20. Data were presented as means ± SEM (standard error of mean) P value less than 0.05 (p<0.05) was considered statistically significant.

RESULTS
HISTOLOGICAL OBSERVATION
The micrograph from control group (group A) showed normal population of germinal epithelium, intact structure and arrangement of seminiferous tubules, presence of leydig cell, and well vascularized. Micrograph from group B (cryptorchidic animals) showed degeneration of spermatogenic cells and absence of spermatozoa in the lumen. Micrograph from Group C (cryptorchidic animals that were given alpha tocopherol) showed regeneration of spermatogenic cells and evidence of spermatogenesis due to presence of spermatozoa in the lumen. Micrograph from animals exposed to heat from hot oven (Group D), showed degeneration of basement membrane and clustering of spermatogonia cells, but Regeneration of of spermatogenic cells and normal spermatogenesis was evident in animals exposed to heat from hot oven followed by alpha-tocopherol (group E). Micrograph of animals exposed to heat from light bulb (Group D), showed distorted interstitium and cellular hyperplasia, which was reversed in animals that was given alpha-tocopherol.

Figure (A-G): photomicrographs showing the cyto-architecture of the testes across all experimental groups. Stain- Haematoxylin and Eosin (H & E). Mag- 400.
A- Control, B- cryptorchidism, C- crypt + vit E, D- hot oven, E- hot oven + vit E, F- light bulb, G- light bulb + vit E.
L-Lumen, White arrows- Spermatogenic cells, Black arrow- Basement membrane

Semen analysis
Table 1: percentage sperm count, sperm motility, life and ratio and sperm morphology across all groups.

Biochemical analysis
Table 2: malondialdehyde (MDA), glutathione (GLU) and superoxide dismutase (SOD) levels across all groups.

DISCUSSION
Cryptorchidism and hyperthermia caused desquamation and degeneration of germ cells which might be due to the destabilization of Sertoli cell membranes caused by lipoperoxidation, irregular shape of the seminiferous tubule, and degeneration of the interstitial tissues; this was in accordance to the report of Dutta et al., in 2013; Oyewopo and Togun in 2005 and Lee and Coughlin, in 2001. In cryptorchidic and hyperthermic animals exposed to alpha tocopherol (aT), lipoperoxidation of the membrane was impeded,
conserving cell union and increasing the amount of germ cells in the seminiferous epithelium (Shikone et al., 1994).
The reduction in sperm parameters analysed as seen in figures 4.2 to 4.6 (i.e. sperm counts, motility, life and death ratio and morphology) in both cryptorchidic and heat treated animals might be due oxidative stress action that cryptorchidism and hyperthermia cause to testicular tissues. Oyewopo & Togun in 2005 and Lee & Coughlin in 2001 also reported a significant reduction in sperm parameters of cryptorchidic and heat treated animals. But the effects of cryptorchidism and hyperthermia (heat) was reversed in animals treated with alpha tocopherol (Vitamin E) by not only returning sperm parameters to normalcy but also enhance the rate of spermatogenesis in the animals. My finding was corroborated by works of Saalu et al., in 2014 and Rosa et al., in 2011.
Cryptorchidism and hyperthermia cause a decrease in antioxidant enzyme activity or an increase in the production of ROS, (i.e. superoxide anion, hydroxyl radical, nitric oxide and hydrogen peroxide) which stimulates lipoperoxidation which results in increase in malondialdehyde and reduced glutathione and superoxide dismutase levels in exposed animals. The work of Saalu et al., 2014; Rosa et al., 2011; Ishii et al., 2005 corroborate my findings. However administration of alpha tocopherol to the treated groups showed increase in cell’s endogenous antioxidant defence system, inhibiting ROS production and impede lipoperoxidation (as seen in figures 4.8 to 4.10). This result was corroborated by work of Saalu et al., 2014; Ayobami et al., 2013; Rosa et al., 2011 and Laila and Sahar in 2009.
CONCLUSION
Cryptorchidism and hyperthermia resulted in oxidative stress in exposed animals which is evident in distortion seen in the histological slides, reduced sperm parameters and antioxidant activities. This work showed that aT reduced apoptosis in germ cells, promoting cell survival and reducing histological alterations to the seminiferous epithelium.

REFERENCES
1. Aybek H., Aybek Z., Rota S., Sen N. and Akbulut M., (2008). The effects of diabetes mellitus, age, and vitamin E on testicular oxidative stress. Fertil Steril., 90(3): 755-760.

2. Ayobami O. A., Hameed A. A., and Isiaka A. A. (2013). Effects of methanolic extract of moringa oleifera leaves on semen and Biochemical parameters in cryptorchid rats. Afr J Tradit Complement Altern Med. (2013) 10(5):230-235.

3. Bedford J.M. (1991). Effects of elevated temperature on the epididymis and testis: experimental studies. Adv Exp Med Biol. 1991; 286:19-32.

4. Dada R., Gupta, N.P., Kucheria K. (2001). Deterioration of Sperm Morphology In Men Exposed To High Temperature. J Anat. Soc. India 50(2) 107-111 (2001).

5. Dutta S., Joshi K.R., Sengupta P., Bhattacharya K. (2013). Unilateral and bilateral cryptorchidism and its effect on the testicular morphology, histology, accessory sex organs, and sperm count in laboratory mice. J Hum Reprod Sci. 2013 Apr; 6 (2):106-10.

6. Ishii T., Matsuki S., Iuchi Y., Okada F., Toyosaki S., Tomita Y., Ikeda Y., Fujii J., (2005). Accelerated impairment of spermatogenic cells in SOD1-knockout mice under heat stress. Free Radic. Res. 39, 697–705.

7. Laila Sabik M.E. and Sahar S. A. (2009).Alpha-tocopherol and ginger are protective on Cyclophosphamide-induced gonadal toxicity in adult male albino rats. Basic and Applied Pathology 2009; 2: 21–29.

8. Lee P.A. and Coughlin M.T. (2001).Fertility after Bilateral Cryptorchidism Evaluation by Paternity, Hormone, and Semen Data. Horm Res 2001; 55:28–32.

9. Lutz Slomianka (2009). Blue Histology- Male Reproductive System. School of Anatomy and Human Biology – The University of Western Australia.

10. Marklund S. and Marklund G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 47: 469-474.

11. Mescher A.L. (2009). Junquiera’s Basic Histology: Text and Atas. 12th Ed. Pg- ISBN 978-0-07-160431-4; MHID 0-07-160431-6.

12. Oyewopo AO, Saalu LC, Osinubi AA, Imosemi IO, Omotoso GO, Adefolaju GA. (2010). The attenuating effects of zinc on Propoxur-induced oxidative stress, impaired spermatogenesis and deranged steroidogenesis in Wistar rats. Journal of Medicine and Medical Sciences, Vol. 1(5) pp. 178-184. June 2010.

13. Oyewopo O. A. and Togun V. A. (2005) Effect of temperature on motility and concentration on male Sprague Dawley rats epididymal spermatozoa. Sci. Focus. 10: 35-40.

14. Rosa Vigueras-Villaseñor M., Idahue O., Oscar Gutierrez-Pérez, Margarita Chavez-Saldaña, Osvaldo C., Daniel S.M., and Julio Rojas-Castañeda C. (2011). Protective effect of a-tocopherol on damage to rat testes by experimental

cryptorchidism. Int J Exp Pathol. 2011 Apr; 92(2): 131–139.

15. Saalu, L.C., Oluyemi, K.A. and Omotuyi, I.O. (2014). Alpha-Tocopherol (vitamin E) attenuates the testicular toxicity associated with experimental cryptorchidism in rats. African Journal of Anatomy Vol. 1 (1), pp. 001 -005, January, 2014.

16. Sedlak J. and Lindsay R.H. (1968). Estimation of total, protein-bound and non-protein sulfhydryl groups in tissues with Ellman’s reagent. Analytical Biochemistry, 25: 192-205.

17. Shikone T., Billig H. and Hsueh A.J. (1994). Experimentally induced cryptorchidism increases apoptosis in rat testis. Biol Reprod. 1994 Nov; 51(5):865-72.

18 Thundathi J.C., Rajamanickam G.D., Kastelic J.P., Newton L.D. (2012). The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function. Reprod Domest Anim. 2012 Aug; 47 Suppl 4:170-7.

19 Traber M.G. and Kayden H.J. (1987). Tocopherol distribution and intracellular localization in human adipose tissue. The American journal of clinical nutrition.

20. Wood H.M., Elder J.S. (2009). “Cryptorchidism and testicular cancer: separating fact from fiction”. The Journal of urology 181 (2): 452–61.