1. INTRODUCTION
Several studies have linked declining reproduction especially male fertility to toxicants found in the environment, particularly endocrine-disrupting chemicals (EDCs), such as phthalates (Wong and Cheng, 2011; Nordkap et al., 2012). One of the phthalates, Di-n-butyl phthalate (DBP) has attracted special attention due to high production volume in millions of tons annually (Swan and Elkin, 1999; Guerra et al., 2010). As a result, human and animal exposure becomes inevitable with its attendant negative consequences on reproduction (Kolaric et al., 2008; Guerra et al., 2010; Zhou et al., 2011; Wang et al., 2012a, 2012b; Asghari et al., 2015; Hamdy et al., 2015; Rehani et al., 2015). In addition, DBP was reported to increase generation of ROS within the testes, concomitantly decreasing antioxidant concentration, resulting in impaired spermatogenesis (Lee et al., 2007; Zhou et al., 2011). DBP is metabolized into monoester, mono-butyl phthalate (MBP) which is a potent testicular toxicant (Oishi and Hiraga, 1980) this is considered to be the active agent in testicular toxicity (Sjoberg et al., 1986; Mylcreest et al., 2000). Approximately 3 million tons of phthalate are produced per annum around the globe. DBP is the most commonly used phthalates constitute about 40% of total phthalate use, blood storage bags usually have a high content of 20-40% DBP but primary source of exposure is through contaminated food (Shi et al., 2012; Liaqat, 2018). Phthalates are ubiquitous xenobiotics widely used in consumer products (Shea, 2003; Heudorf et al.,. 2007) with epidemiological studies revealing its detection in urine, blood and breast milk of humans (Swan et al., 2005, Main et al., 2006; Fromme et al., 2007). There is paucity of information on the use of antioxidants both as prophylactic and therapeutic measures in addressing oxidative stress (OS)-induced infertility in male animals. The aim of the study was to investigate the effects of melatonin and garlic on DBP induced oxidative stress on serum hormones and lipid profile and also evaluates the protective and therapeutic effects of melatonin and garlic on serum hormones and lipid profiles of rabbit bucks. The study also tried to assess the effect of DBP on serum hormone and lipid profile.
2. MATERIAL AND METHODS
2.1 Experimental Animals
Forty two (42) apparently healthy, New Zealand White rabbit bucks (Oryctolagus cuniculus), 10 - 12 month old with body weight of 1.80-2.00 kg were used for the study, water and feed were provided ad libitum.
2.2 Chemical and Allium sativum Acquisition and Preparation
Di (n-butyl) phthalate DBP (CAS Number 84- 74-2-technical grade-99% purity) was purchased from Sigma Aldrich USA. Dosage of 750 mg/kg was calculated and reconstituted in olive oil (Goya Extra Virgin Olive Oil, Sevilla, Spain) to form a solution of 50 %. Melatonin (5 mg/tablet, Nature made, USA), dissolved in 10 ml of distilled water to make 0.5 mg/ml suspension (Umosen et al., 2012). All preparations were administered to the animals using gastric tube. Allium sativum (garlic) bulb was sourced from Sabon Gari, Kaduna State, Nigeria.
2.3 Experimental Design
Forty two (42) rabbit bucks were randomly divided into seven (7) groups of six (6) bucks each, designated as groups A, B, C, D, E, F and G. All preparations were administered using rabbit canular. Group A: Olive oil 1.5 ml alone for 16 weeks. Group B: Olive oil 1.5 ml + DBP (750 mg/kg) for 16. Group C: Pretreated with melatonin @ 0.5 mg/ml for 8 weeks, then olive oil 1.5 ml + DBP (750 mg/kg) for another 8 weeks. Group D: Pretreated with A. sativum 5.0% for 8 weeks, then Olive oil 1.5 ml + DBP (750 mg/kg) for another 8 weeks. Group E: Predosed with (Olive oil 1.5 ml + DBP 750 mg/kg for 8 weeks), then Melatonin @ 0.5 mg/ml for another 8 weeks. Group F: Predosed with (Olive oil 1.5 ml + DBP 750 mg/kg for 8 weeks), then A. sativum 5.0% for 8 weeks. Group G: Predosed with (olive oil 1.5 ml + DBP 750 mg/kg for 8 weeks), then Melatonin @ 0.5 mg/ml + A. sativum 5.0% for another 8 weeks. All rabbits were fed diets corresponding to their groups as shown in Table 1. This study was carried out in accordance to guidelines and protocol approved by the Ahmadu Bello University Committee for Animal Use and Care with the approval number: ABUCAUC/2018/059.
2.4 Serum Hormone Profile
Blood samples were collected at 8th and 16th weeks of the study a total of eighty four (84) samples were collected for determination of serum follicle stimulating hormone (FSH), luteinizing hormone(LH) and testosterones (T) using competitive immunoassay technique {AccuBindTM ELISA Microwells (FSH product code: 425-300: LH product code: 625-300: T product code: 3725- 300)}according to the instruction manual provided by the manufacturer (Mono bind inc. 100 North pointe Drive, Ca 92630 U.S.A). The sensitivity of the FSH assay is 0.006 mlU/well, the intra and inter assay coefficient of variation (CV) is 5.4% and 9.0% respectively. The sensitivity of the LH assay is 0.003 mlU/well, the intra and inter assay coefficient of variation (CV) is 6.8% and 7.8% respectively. The sensitivity of the T assay is 0.0576 ng/ml, the intra and inter assay coefficient of variation (CV) is 9.8% and 9.1% respectively.
2.5 Serum lipid profile
Serum collected was analysed using semiautomated spectrophometer (Surecheme®) to determine the total cholesterol, triglycerides, high density lipids and low density lipids, according to the instruction manual provided by the manufacturer (Surecheme Products Limited, Ipswich, Suffolk, UK).
2.6 Semen evaluation
Ejaculates were obtained with the aid of artificial vagina and subjected to routine evaluation as described by Zemjanis (1970). This includes: Reaction time, volume, microscopic examination for motility, concentration, percentage live spermatozoa and morphological abnormalities.
2.6.1. Reaction time (libido)
A matured doe (teaser) was introduced to the buck prior to semen collection and observed for sex drive. The time in seconds it took the buck to sniff, groom and mount the female was recorded (Saleem, 2003).
2.6.2. Volume
Volume of semen was measured directly from the calibrated tube used for the collection.
2.6.3. Sperm motility
Gross (wave) motility: this was determined by examining a drop of raw undiluted semen on a prewarmed glass slide under light microscope at ×10 magnification. The estimate of the mass activity was based on the vigour of the wave motion. This was assessed on a 0-5 scoring system. Scores from least active (+0 = 10 - 20%) to most active +5 = 90 - 100%) was given to the wave motion of the spermatozoa according to the intensity of swirling bands. Individual motility: the percentage of spermatozoa with forward progressive motility was estimated by diluting a drop of semen with 4 drops of normal saline on a pre warmed glass slide and cover with a clean cover slip. Observation was done under high (×40) power magnification.
2.6.4 Spermatozoa concentration
This was determined using Neubauer haemocytometer as described by Azawi and Ismaeel (2012). Semen sample was sucked into the red cell diluting pipette up to the 0.1 mark (25μl) and the volume made up to the 101 mark (5ml) with 10% formal saline which ensured thorough mixing by capillary action, the mixture was allowed to spread under the cover slip, placed tightly on the haemocytometer after few drops were discarded. The cells were allowed to settle before counting under ×40 power magnification. Sperm cells were counted in 5 smaller squares of the improved Neubauer haemocytometer and the concentration determined using the following formula: Number of sperm cells/ml = number of sperm cells counted in 5 smaller squares × 5 × 104 × dilution factor(5000) (Bearden and Fuquay, 1992; Azawi and Ismaeel, 2012).
2.6.5. Percentage live sperm cells
This was determined as described by Esteso et al. (2006). A thin smear of semen was made on a clean grease free slide and stained with 2 drops EosinNigrosin stain. This technique was based on the principle that Eosin-nigrosin penetrates and stains dead sperm cells, while live sperm cells repel the stain. Dead spermatozoa stained pinkish or reddish while live spermatozoa remained colourless. Two hundred (200) stained and unstained sperm cells were counted when the slide dried, using light microscopy at ×40 magnification and percentage of each estimated (Esteso et al., 2006).
2.6.6 Sperm abnormalities
This was determined by making a thin smear of the semen sample, on clean grease-free glass slide and stained with 2 drops of Eosin-negrosin. Two hundred sperm cells were counted per slide using hand counter under light microscopy at ×100 magnification using oil immersion. All abnormal cell types were counted and recorded (Rekwot et al., 1987).
2.7 Statistical analysis
Data were expressed as mean ± standard deviation (SD) and subjected to repeated measures one-way analysis of variance (ANOVA) for repeated sampling, while one-way analysis of variance was used for single sampling, followed by Tukey’s multiple comparison test. Graph Pad prism version 5.0 for windows 2003 from Graph pad prism software, San Diego, California (www.graphpad.com) was used. Values of P < 0.05 was considered significant.
3. RESULTS
3.1 Serum Hormonal Assay
The mean ± SD of testosterone, luteinizing hormone and follicle stimulating hormone of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 8 and week 16 of the study are presented in Figures 1-3.
3.1.1 Serum testosterone
There was significant difference (P ≤ 0.0001) in the mean testosterone concentration (ng/ml) between group C (3.23 ± 0.14) and groups (B, 0.43 ± 0.12; E, 0.37 ± 0.13; F, 0.47 ± 0.03 and G, 0.42 ± 0.06) at week 8 (Fig 1). There was significant difference (P ≤ 0.0001) in the mean testosterone concentration (ng/ml) in group B (0.40 ± 0.06) with other groups (A,3.13 ± 0.19; C,2.56 ± 0.05; D,2.28 ± 0.15; E,1.57 ± 0.23; F,1.43 ± 0.15 and G,1.83 ± 0.33) and also, group C (2.56 ± 0.05) differed significantly with groups (E, 1.57 ± 0.23; F, 1.43 ± 0.15 and G, 1.83 ± 0.33) at week 16 of the study (Fig 1).
3.1.2 Serum luteinizing hormone
There were significant differences in the mean luteinizing hormone concentration (pg/ml) between groups (C, 4.60 ± 0.26) and groups (B, 1.00 ± 0.26; E, 1.10 ± 0.32; F, 1.13 ± 0.09 and G, 1.13 ± 0.21) at week 8. There was significant difference (P = 0.0261) in the mean luteinizing hormone concentration (pg/ml) in group B (1.50 ± 0.29) with all the other groups (A, 4.33 ± 0.67; C, 4.00 ± 0.23; D,4.07 ± 0. 55; E,3.17 ± 0.93; F, 2.6 ± 0.21 and G,3.33 ± 0.33) and also group (C, 4.00 ± 0.23) differed significantly with groups (E, 3.17 ± 0.93; F, 2.6 ± 0.21 and G, 3.33 ± 0.33) at week 16 of the study (Fig 2).
3.1.3 Serum follicle stimulating hormone
There were significant differences in the mean serum follicle stimulating hormone (pg/ml) between groups (C, 7.70 ± 0.27) and groups (B, 2.03 ± 0.29; E, 2.50 ± 0.64; F, 2.53 ± 0.43 and G, 1.67 ± 0.33) at week 8. There was significant difference in the mean serum follicle stimulating hormone concentration (pg/ml) in group B (2.50 ± 0.50) with all the other groups (A, 7.8 ± 0.89; C, 6.73 ± 0.96; D,6.93 ± 1.15; E,3.67 ± 0.88; F, 4.00 ± 0.58 and G,4.33 ± 0.67) and also group (C, 6.73 ± 0.96) differed significantly with groups (E, 3.67 ± 0.88; F, 4.00 ± 0.58 and G,4.33 ± 0.67) at week 16 of the study (P ≤ 0.01; Fig 3).
Fig 1. Mean serum testosterone (ng/ml) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 8 and 16 of the study. Values are expressed as mean ± SD. Different alphabet (a, b, c, and d) shows different levels of significance. T: testosterone
Fig 2. Mean serum luteinizing hormone (pg/ml) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 8 and 16 of the study. Values are expressed as mean ± SD. Different alphabet (a, b, c, and d) shows different levels of significance. LH: luteinizing hormone
Fig 3. Mean serum follicle stimulating hormone (ng/ml) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 8 and 16 of the study. Values are expressed as mean ± SD. Different alphabet (a, b, c, and d) shows different levels of significance. FSH: follicle stimulating hormone.
3.2 Serum Lipid Profile
The mean ± SD of serum total cholesterol, triglycerides, high density lipid and low density lipid of rabbit bucks of the seven treatment groups (A, B, C, D, E, F and G) sampled at week 16 of the study are presented in Fig 4-7.
3.2.1 Total cholesterol
There were significant difference (P = 0.0086), in the mean serum total cholesterol concentration (mmol/L) between group B (4.08 ± 0.09) and groups (C, 2.5 ± 0.39; D, 2.35 ± 0.22; E, 3.2 ± 0.50; G, 2.9 ± 0.29) Also there was significant difference (P< 0.05) between groups (C, 2.5 ± 0.39; D, 2.35 ± 0.22 and G, 2.9 ± 0.29) and groups (E, 3.2 ± 0.50 and F, 3.6 ± 0.57), (Fig 4).
3.2.2 Triglycerides
There were significant difference (P = 0.0066) in the mean serum triglycerides concentration (mmol/L) between group B (1.83 ± 0.21) and all other groups (A, 0.88 ± 0.14; C, 1.15 ± 0.10; D, 1.28 ± 0.17; E, 1.1 ± 0.12; F,1.35 ± 0.06 and G, 1.2 ± 0.15), (Fig 5).
3.2.3 High density lipid
There were no significant difference (P = 0.7338), in the mean serum high density lipid concentration (mmol/L), in all the groups (A, 1.8±0.23; B, 1.98±0.23; C, 1.45±0.23; D, 1.48±0.23; E, 1.93±0.53; F, 1.9±0.34; G, 2.20±0.48), (Fig 6).
3.2.4 Low density lipid
There were no significant difference (P = 0.3837) in the mean serum low density lipid concentration (mmol/L) in all the groups (A, 0.65±0.09; B, 1.45±0.49; C, 0.55±0.24; D, 0.38±0.11; E, 0.75±0.25; F, 1.33±0.58; G, 0.83±0.53), (Fig 7).
Fig 4. Total cholesterol (mmol/L) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 16 of the study. Values are expressed as mean ± SD. Different alphabet (a, b, and c) shows different levels of significance.
Fig 5. Triglycerides (mmol/L) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 16 of the study. Values are expressed as mean ± SD. Different alphabet (a, and b) shows different levels of significance.
Fig 6. High density lipid (mmol/L) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 16 of the study.
Fig 7. Low density lipid (mmol/L) of rabbit bucks of the treatment groups A, B, C, D, E, F and G sampled at week 16 of the stud
3.3 Semen Parameters
There were significant differences in mean reaction time, sperm motility, concentration and percentage abnormal spermatozoa between group B and other treatment groups (Table 2).
4. DISCUSSION
The “United States environmental protection agency” defined endocrine-disrupting chemicals (EDCs) as exogenous agents that affect the synthesis, transport, binding action, metabolism and elimination of hormones required for homeostasis, development and reproduction (DiamantiKandarakisc et al., 2009). Phthalates are known for their weak estrogen properties and acts as EDCs due to their capability to contest with steroid hormone binding its receptors (Shelby, 2006). Exposure to DBP in this study decreased the levels of serum FSH (Fig 2) and LH (Fig 3), this may be an indication that pituitary function may have been affected by DBP exposure. Lee et al. (2004) reported that DBP affects pituitary hormone-producing cells at both prepubertal and adult stages in males, LH is the primary regulator of T synthesis in the leydig’s cell of the testis. The observed changes in the levels of serum T in DBP exposed groups is that the decrease T production in the testes may have resulted from the decrease LH secreted from the anterior pituitary as observed in our study (Fig 1). Likewise, the decrease FSH concentration in DBP exposed groups, may have caused the decreased semen parameters observed in the DBP exposure groups (Table 2), since FSH plays a strategic role in spermatogenesis. In the testes, the antiandrogenic effect of phthalate is mainly triggered by interfering with T synthesis (Oda and Waheeb, 2017). Furthermore, phthalate, down-regulate the expression of genes required in cholesterol conveyance which is critical in synthesis of T (Thompson et al., 2005; Borch et al., 2006). Melatonin is reported to affect the hypothalamic pituitary-gonadal (HPG) axis and modifies the secretion of hormones such as FSH, LH and T (Sanchez-Hidalgo et al., 2009). Our study corroborates the above study as melatonin treatment improved the hormone profile (FSH, LH and T) of groups exposed earlier to DBP before treatment (E, F and G). For groups (C and D) pretreated before exposure to DBP maintained a fairly stable levels of the serum hormones, it could be that the melatonin has an antioxidant effect of longer duration of time or that the concentration attained in the testes before exposure to DBP resisted the effect of DBP in the testes. Also the observed improvement in serum hormone levels in garlic treated groups could be due to its reported role as a potent antioxidant (Chen et al., 2013; Adaki et al., 2014; Shinkut, 2015).
Lipids play multiple roles that either individually or collectively influence many cell processes. Fatty acids and cholesterol are important substrates for reproductive hormone synthesis, increase fats in diet may lead to increase level of reproductive hormones (Ibtisham et al., 2018). Cholesterol is one of the most important biomolecules in animals and has significant role in cellular function and integrity. It is also a precursor of all sexual hormones (Simons and Ikonen, 2000; Parton and Hancock, 2004; Agarwal and Jain, 2015). Study has shown that phthalates disrupts the expression of several genes involved in cholesterol transport and steroidogenesis (Euling et al., 2013). Before cholesterol can be up-taken in the cell for steroidogenesis, double bonds are removed by the enzyme 7-dehydrocholesterol reductase (DHCR7). The expression of dhcr7 is found to be reduced in rats exposed to a single dose of mono-(2-ethylhexyl) phthalate (MEHP) (Lahousse et al., 2006). Similarly, the expression of the scavenger receptor class B-1 (SRB1), which is responsible for transporting high density lipoprotein cholesteryl esters into the cell was down regulated by DBP in rats (Barlow et al., 2003; Lehmann et al., 2004; Thompson et al., 2004 Borch et al., 2006). Once the cholesterol has cross the cellular membrane, the steroidogenic acute regulatory protein (stAR) and the peripheral benzodiazepine receptor (PBR) transport it to the inner mitochondrial membrane (Agarwal and Jain, 2015). The first steps of steroidogenesis involve the transformation of cholesterol into pregnenolone by the enzyme cytochrome P450 side chain cleavage (CYPIIAI). DBP was reported to reduce the expression of CYPIIAI in rats (Barlow et al., 2003; Thompson et al., 2004; Borch et al., 2006). Relating the above findings with the result of our study, this perhaps explains decreased in serum hormones levels in group B of our study (Fig 1, 2 & 3), despite corresponding increase in cholesterol in the same group B (Fig 4). These findings is in parallel with other studies (Agarwal and Jain, 2015; MathieuDenoncourt et al., 2015). There are few studies that link serum triglycerides with sperm parameters independent of hypercholesterolemia. Zmuda et al. (1997) reported a decrease in endogenous testosterone associated with an increase in triglycerides level. Alqubaty. (2013) also reported significant negative correlation between serum total testosterone and triglycerides. The above findings corroborate the observed increased in triglycerides in DBP exposed group B (Fig 5) in this study with a corresponding decreased serum testosterone. Furthermore, Ergun et al. (2007) observed that increased triglycerides may have deleterious effect on spermatogenesis correlating with decrease sperm motility and T.
5. CONCLUSION
DBP causes decrease in FSH, LH, T and semen quality. Melatonin and garlic protect and also had ameliorating effect against DBP on serum hormonal profile and semen parameters of rabbit bucks. DBP causes increase in total cholesterol and triglycerides levels.
ACKNOWLEDGEMENT
We are grateful to Dr. Ya’uYahuza, Mr. Bolaji and Mr. Yohanna Lekwot for their technical assistance.
This article was originally pùblished in Alexandria Journal of Veterinary Sciences. Vol. 66 (2): 1-10 Jul 2020. DOI: 10.5455/ajvs.70467.
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