Biological rhythms are ubiquitous in humans and regulate many biochemical and physiological endogenous processes . These rhythms dictate manifestation of several diseases and drug actions by altering kinetics and dynamics of medications . The rhythmic expression of diabetes mellitus is a consequence of circadian variation in glucose rhythm which peaks at the beginning of the activity period . Diabetes mellitus is a global life-threatening disorder that requires adequate management. However, most conventional regimens do not put into consideration the rhythm of the disorder hence there is increased risk of severe complications which includes reproductive toxicities in men . The effects on male fertility include impairment of spermatogenesis and low sperm count , reduced sperm motility and male sub-fertility , testicular and erectile dysfunction  and impairment of sperm DNA integrity .
Reactive oxygen species (ROS) mediated oxidative stress have been suggested to play a key role in the development of diabetic complications . There is also evidence that ROS, endogenous antioxidants levels, and some of the diseases resulting from their imbalance such as diabetes, are expressed in rhythms . Hence, management should employ therapy that will maintain a normal glucose level along with measures that will delay the progression or prevent complications and the use of antioxidants as complementary therapy. Furthermore, rhythms of the disease and that of the underlying mechanisms in addition to diurnal variations in drugs pharmacokinetics must be considered. Glimepiride has remained one of the drugs of choice used in the management of diabetes mellitus for over five decades [10, 11]. In addition to its cardiovascular benefits, nifedipine has shown promising antioxidant activity  and has also shown potential in ameliorating diabetic complications  Supplementation using alpha-lipoic acid (ALA) reduces glycated haemoglobin (HbA1c) in animal models  and also inhibits oxidative stress . Advances in recent research shows the beneficial outcomes of chronotherapy in some diabetic complications [16, 17]. Hence, knowledge of circadian variation in oxidative stress and related testicular toxicity in diabetes may provide basis for newer therapeutic options. This may be achieved by modulation of times of administration of ALA as an adjunct in the management of type two diabetes mellitus (T2DM). This study investigates the effect of chronomodulated alpha-lipoic acid and nifedipine combination with concurrent glimepiride treatment in oxidative stress mediated testicular toxicity in diabetic rats.
Male rats of Wistar strain numbering sixty three and weighing between 150-155 g were used. The rats were purchased from McTemmy Laboratory Concept, Ogbomoso and kept in the Departmental animal house of Pharmacology and Therapeutics. They were kept in clean aluminium cages with wood shavings as bedding, with ad libitum water and rodent feed. Ethical directives (2010/63/EU) proposed by the European parliament on use and care of animals which complies with that of the Ahmadu Bello University, Zaria were observed.
Alpha-lipoic acid (AO Pharmaceuticals, China), Nifedipine (Lek Pharmaceuticals, Slovenia), glimepiride (Sonafi pharmacueticals Germany), chloroform, streptozotocin (Sigma chemical, Germany), 10 % dextrose in water (Juhel Nigeria Ltd, Enugu), formaldehyde solution (Sigma chemical, Germany).
Induction of diabetes
Streptozotocin prepared in a citrate buffer solution of 0.1M concentration and a 4.5 pH was intraperitoneally administered at 50 mg/kg in 12 hours fasted rats. Water was replaced with 10 % dextrose in water for 24 hours to prevent hypoglycaemia resulting from pancreatic release of insulin . Seven days post induction, assessment of fasting blood glucose was ensured with a glucometer and animals with blood glucose above 200 mg/dL were selected for grouping.
Seven rat groups participated in the research. A group of non-diabetic and another group of diabetic rats received treatment with 1 mL/kg of water to serve as normal control and diabetic control respectively. All other groups were diabetic receiving 10 mg/kg glimepiride at 20:00h (14 HALO). Additionally, groups four to seven were treated with 20 mg/kg nifedipine at 08:00h (2 HALO) while groups five to seven received additional treatments with ALA at 08:00h (2 HALO), 14:00h (8 HALO) and 20:00h (14 HALO) respectively. All drugs were administered orally for four weeks after which rats were euthanized and epididymis and testis were excised for assessment of some fertility markers. Relative organ weight of the testis were calculated and the right testis were preserved in phosphate buffer for testicular cholesterol and antioxidant assay while all left testes were preserved in formalin for histological studies. Samples of blood were drawn after euthanasia and centrifuged to obtain serum from which testosterone levels were determined.
Sperm suspension was obtained by carefully isolating and chopping the caudal end of the epididymis in a ten milliliters Petri dish containing two milliliters of normal saline maintained at 37 °C immediately after euthanasia. Sperm motility and count were assessed using the method of Belsey et al.  while viability was measured in 1% tryphan blue reagent as described by Talbot and Chacon . Sperm count, motility and viability were presented in millions/mL and percentages respectively. Sperm morphology was determined by using the method of Linder et al. . A hundred sperm cells were investigated from each rat for deformities of tail and the head. The changes were determined by viewing under a light microscope and the percentage of head and tail defects were calculated. Sperm deformity index was calculated as follows:
Serum testosterone determination
Samples of blood were taken by means anticoagulant free vacutainers via the jugular veins and were spun in a centrifuge at 2000 g within 15 minutes. Levels of serum testosterone were determined using an enzyme linked free testosterone assay kit (DK0015 Diametra, Italy) according to the kit’s protocol.
Antioxidant enzyme assays
The right testes were homogenized thoroughly in isolation medium. Levels of testicular antioxidant reserve such as superoxide distumase (SOD), glutathione (GSH) and catalase (CAT) content and that of lipid peroxidation, malondialdehyde (MDA) were determined afterwards as described by Oraebosi et al. ; Obaji-Ogar et al.  and Mahadeshwara et al. .
Testicular cholesterol determination
This was carried out from the testicular homogenate by adopting the procedure of Zlatkis et al. . This was done in test tubes by preparing 0.3 mL of the homogenate in 6 mL of 0.05% ferric chloride. 3 mL of sulphuric acid was afterwards added to the test tube containing mixture and left undisturbed for 20 minutes. An incubation mixture without the addition of homogenate was used as blank. Using a UV-VIS spectrophotometer (752N), absorbance was read at 540 nm and levels of testicular cholesterol were expressed in mg/g tissue weight.
Relative testicular weight
This was determined as shown in the expression below:
Preserved left testes were passed in graded concentration of alcohol and xylene before they were blocked in paraffin wax. Sections of tissue samples were then discoloured with haematoxylin and eosin and were read under a microscope to check the histoarchitecture .
Levene’s test for homogeneity was done followed by Shapiro’s checks for normality. Analyses were carried out with one way analysis of variance and Hochberg’s post hoc with SPSS version 21. Values of P below or exactly 0.05 were measured as significant. The results are shown in tables as percentages or mean and standard error of mean,while histological results are in photomicrograph.
Time of day effects of ALA/nifedipine/glimepiride combination on sperm motility in diabetic rats
There was a significantly (p ≤ 0.01) lower percentage of active cells in untreated diabetic rats in comparison to normal, with a greater percentage (p ≤ 0.01) of sluggish and non-motile cells respectively in comparison to the normal group. Concurrent treatment with glimepiride at 20:00h alongside nifedipine at 08:00h or with ALA at 08:00h, 14:00h and 20:00h produced significantly (p ≤ 0.01) higher percentage active cells, lower sluggish and inactive cells in comparison to the untreated diabetic group. Drug treatment with 20:00h ALA however produced significantly (p ≤ 0.05) better sperm motility than other time points. Table 1 shows the results.
Time of day effects of ALA/nifedipine/glimepiride combination on sperm viability and count in diabetic rats
The number of viable cells and sperm count were significantly (p ≤ 0.01) higher in the group that received a combination of glimepiride at 20:00h and nifedipine at 08:00h or along with ALA at 08:00h 14:00h and 20:00h when compared to diabetic control. However the groups treated with a combination of glimepiride at 20:00h and nifedipine at 08:00h along with ALA at 20:00h produced better (p ≤ 0.05) prognosis than with ALA treatment at 08:00h and 14:00h. The result is shown in Table 2.
Time of day effects of ALA/nifedipine/glimepiride combination on sperm morphology in diabetic rats
The results for head and tail defects are shown in Table 3. The percentage of head and tail defects in the untreated diabetic group was significantly (p ≤ 0.01) higher in comparison to the normal rats. Various drug administration across all groups produced significantly (p ≤ 0.01) lower percentages for both head and tail defects. However, the groups treated with a combination of glimepiride at 20:00h and nifedipine at 08:00h along with ALA at 08:00h and 20:00h produced significantly (p ≤ 0.01) lower head and tail defects than treatment with a combination of glimepiride at 20:00h and nifedipine at 08:00h alone.
Time of day effects of ALA/nifedipine/glimepiride combination on testosterone, cholesterol and relative testes weight in diabetic rats
Mean levels of serum testosterone and testicular cholesterol with relative organ weight are shown in Table 4. These indices were significantly (p ≤ 0.01) different in untreated diabetic animals in comparison to normal values. However, values for these indices were shown to (p ≤ 0.01) produce significant amelioration in all groups receiving ALA in comparison to the diabetic control group. However, only the group that received ALA at 20:00h in combination with glimepiride at 20:00h and nifedipine at 08:00h showed values similar to those of the non-diabetic control group.
Time of day effects of ALA/nifedipine/glimepiride combination on testicular oxidative stress and antioxidant markers
The results of analyses revealed that marker for lipid peroxidation (MDA) was significantly (p ≤ 0.01) higher in the experimental group with significantly lower endogenous antioxidants reserve (SOD, CAT, GSH) in the untreated diabetic rats when compared to normal rats. All the groups treated with a combination of glimepiride at 20:00h and nifedipine at 08:00h along with ALA at 0800h and 2000h, respectively showed significantly (p ≤ 0.01) lower MDA levels and higher (p ≤ 0.01) endogenous antioxidant levels when compared to the untreated diabetic control group. In addition, rats which received ALA at 20:00h produced a better prognosis than other time periods. The results are shown in Table 5.
Time of day effects of ALA/nifedipine/glimepiride combination on testes histology in diabetic rats
Photomicrographic representation of diabetic rat with no drug treatment (Fig. 1B) showed testes without leydig cells (L), sertoli cells (arrows), and presence central spermatozoa (asterisk). Other treatment groups (Fig., 1C-1F) showed some histological distortion, while treatment with 20:00h+nifedipine 08:00h+ALA20:00h showed historachitecture similar to that of normal rats. This is shown in Figure 1.
A balance in testicular ROS and antioxidants is believed to be very essential for effective reproductive functions in men. Hence, oxidative stress in the testes resulting from an imbalance may be deleterious to reproductive health in men . This balance may be maintained through secretion of endogenous antioxidants such as CAT, GPx, and SOD (MnSOD, CuSOD, ZnSOD and Cu/Zn-SOD). Although Zinc is a nonreactive redox metal, its role as an antioxidant is due to the activities of copper/zinc-superoxide dismutase (ZnSOD and Cu/Zn-SOD) which helps in membrane stabilization and increased metallothionein activities. Deficiencies in these antioxidant activities may result in unwanted health issues via ROS-antioxidants imbalance. Such imbalances have been reported in diabetes mellitus and may lead to severe complications. The involvement of oxidative stress in diabetes and its resulting complications is due to high plasma glucose levels overtime . In diabetics, male fertility is affected by mechanisms that involve increased testicular ROS production , testicular testosterone deficiency caused by leydig cells destruction , and impaired spermatogenesis . In this study, similar abnormalities were observed in untreated diabetic male rats may be a consequence of disturbance in spermatogenesis and sperm maturation impairments. These changes in addition to alterations in sperm viability, morphology, motility, testicular weights, and increased testicular cholesterol are important indices for male fertility . They are often associated with increase in testicular oxidative stress. Hence therapies that reduce oxidative stress or improve endogenous antioxidants levels may be effective hindrances of the impairments of male fertility.
Although previous data have shown the benefits of ALA in mitigating testicular toxicity in rats by inhibiting oxidative stress , data from this study suggests that there is a time dependent variation in antioxidant activities of ALA in combination with nifedipine and glimepiride. This was identified after the mitigation of testicular toxicity in a group of rats was treated with ALA at night-time compared to other time points. This could be due to circadian variation in lipid peroxidation and endogenous antioxidant levels. The testes are known to possess antioxidant defence enzymes. However, their expression peaks during the dark phase in rats and it correlates with the acrophase of lipid peroxidation [29, 30]. In normal rats, these enzymes are able to cancel out the effects of emerging ROS, while oxidative stress results in diabetes due to imbalance. The values of lipid peroxidation and endogenous antioxidant as seen in the group treated with ALA at 20:00h appears to be similar to that of the non-diabetic control. This is an indication that ALA mitigated oxidative stress in this group of rats and it suggests that the drug’s approximate peak plasma concentration after 20:00h administration matches with the levels of peak lipid peroxidation to prevent oxidative stress. This could be of clinical importance because ROS mediated oxidative stress damages sperm in patients with fertility problems. This may explain the observed improvement in semen analysis profile, testosterone and cholesterol levels, and testicular weights for the group treated with ALA at 20:00h when compared to all other treatment groups.
The testis serves as a chief male reproductive organ whose function involves continuous production of spermatozoa in a process called spermatogenesis. Diurnal variation in spermatogenesis in rats has been reported to involve both cell division and cell differentiation . It is reported that spermatogenesis increases at early rest periods with a peak production of spermatocytes at 06:00h  and reverses at the dark phase. In addition, exposure to long photoperiods in rats are reported to stimulate the development of reproductive and gonadal functions (increase testes weight, testosterone and luteinizing hormone levels) in contrast to a decrease that occurs during long dark period . These reports imply that spermatogenesis peaks within the light phase with a trough at the dark phase. In this study, the restoration of reproductive markers followed the administration of ALA at 20:00h. This may suggest that ALA administration at this point (dark/rest phase) mitigated the processes responsible for a trough in reproductive markers in rats during the dark phase to keep approximate normal levels of reproductive parameters throughout the day. This is also supported by histological evidences where features from photomicrographs of the ALA 20:00h treated rats showed normal leydig cells, sertoli cells, spermatocytes, and spermatogonia. The role of these structures in reproduction has been reported. In rats, testosterone stimulates the release of prolactin . Also, an increase in prolactin levels mediate testosterone release through an upregulatory effects of luteinizing hormone receptors on leydig cells . ALA has been previously shown to enhance testosterone and prolactin levels in rats; hence the administration of ALA at 20:00h which falls within the time of their least expression  could have helped in regulating normal daily levels of these hormones and generating positive effects on reproductive parameters as seen in this study.
Time dependent triple therapy with alpha-lipoic acid, nifedipine, and glimepiride mitigates oxidative stress-mediated testicular damage in diabetic rats. The use of this combination in humans with approximate circadian timing may offer a new therapeutic approach for the treatment or prevention of testicular damage in diabetic males.
Conflict of interest
None to be declared