Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 5 (may 2020) : 543-548

+(-)catechin raises body temperature, changes blood parameters, improves oocyte quality and reproductive performance of female mice

A.A. Mohammed1,*, A.A. Al-Hozab1
1Department of Animal and Fish Production, College of Agriculture and Food Sciences, King Faisal University, Saudi Arabia.
Cite article:- Mohammed A.A., Al-Hozab A.A. (2019). +(-)catechin raises body temperature, changes blood parameters, improves oocyte quality and reproductive performance of female mice . Indian Journal of Animal Research. 54(5): 543-548. doi: 10.18805/ijar.B-981.

ABSTRACT

The present study aimed to explore changes in physiological and reproductive performances upon supplementation of +(-) cactechin (200 vs. 2000 mg/kg BW) in mice. In addition, body temperature, heart rate, blood oxygen and glucose were explored after general anesthesia of +(-)cactechin animals. Sixty albino female mice were classified into three groups; control group (G1) versus two +(-) cactechin groups [G2 (200 mg/kg BW) and G3 (2000 mg/kg BW)] for 15 days in aqueous solution. Changes of body temperature, heart rate, blood parameters, oocyte quality and reproductive performances were determined and recorded. In addition, changes of body temperature, heart rate, blood oxygen and glucose were measured after anesthesia. The results indicated that +(-)catechin supplementation resulted in significant increase in body temperature, blood oxygen and WBCs in addition to significant hypoglycemia. The oocytes quality and reproductive performances were improved upon +(-)catechin supplementation. +(-)catechin supplementation did not improve the negative side effects of anesthesia. In conclusion, supplementation of +(-)catechin could benefit physiological and reproductive performances in mice. 

INTRODUCTION

Flavanols are a type of polyphenol found in plants and products such as fruits, green tea, wine and cocoa. Presence of flavanols in food affects quality parameters such as bitterness, sourness, astringency, sweet-ness and aroma. There are different types of flavanols as catechins, which in particular are thought to be responsible for the amazing health benefits. (+)-catechin and (-)-epicatechin are universal components of vascular plants and they are frequent components of traditional herbal remedies. The catechin  isomers are mostly found in cacao and tea. It is a plant secondary metabolite, natural phenol and antioxidant substance. Several in vitro and in vivo studies on catechins have been reported to possess multiple properties.
        
Effects of nutrition and supplements on physiological and reproductive performance have been reported (Mohammed and Attaai 2011; Mohammed and Al-Suwaiegh 2016; Chaudhari et al., 2018; Dar et al., 2018; Sheoran et al., 2018). Flavanols play protective and antioxidant effective role for plants and the living organisms that consume the plants. Green tea catechins contain various kinds of functional elements having healthy effects as anti-inflammatory, antioxidative, neuroprotective, cholesterol-lowering and anti-obesity (Wolfram et al., 2006). Wang et al., (2009) found improvements of body composition and reduction of abdominal fat in moderately over weights due to consumption of high-catechin green tea. There have been several studies in recent years indicating the beneficial effects of green tea in treating obesity and improving metabolism of glucose and lipid (Wang et al., 2009; Suzuki et al., 2012).
        
There are conflicting results were obtained due to the effects of catechins, which might be due to different reasons as experimental dosages and periods, sample sizes and others (Wang et al., 2009). While there is considerable interest in the potential health benefits of flavonoids intake, the potential adverse effects of consuming very large amounts of these compounds have not been intensively studied. Results of epidemiological studies support the concept that the chronic consumption of flavonoid-rich diets can be associated with a reduced risk for certain chronic diseases, including neurodegenerative, cardiovascular and select cancers (Katz et al., 2011; Kim et al., 2014). Roychoudhury et al., (2017) mentioned that results from in vivo studies are eagerly awaited. Because maternal nutrition has a significant effect on developmental competence of oocytes and the resulting embryos and fetus during pregnancy, the present work carried out to explore oocytes viability and the resulting reproductive performances upon supplementing albino female mice with 200 and 2000 mg/kg/day.
        
General anesthesia is imperative in several scientific studies and resulting in transient negative side effects (Mohammed et al., 2018). Because of the potential health benefits of flavonoids in treating obesity and improving metabolism of glucose and lipid; the question arose; is + (-)cactechin treatment alleviate such transient negative side effects due to general anesthesia. Therefore, the second aim in this study is to explore the changes in body temperature, PO2, heart rate and blood glucose levels due to general anesthesia of +(-) cactechin (200 and 2000 mg/kg/day) treated mice at different time.

MATERIALS AND METHODS

Animals’ feeding and management
 
The experiment was carried out on College of Agriculture and Food Sciences, King Faisal University. Sixty female albino mice (22.34 ± 0.08g body weight) were kept in controlled room set to maintain a temperature of 25 ± 3°C and a relative humidity of 50 ± 10%. The animals were randomly assigned to three groups. The control group (N=20; 22.58 ± 0.60 g) was given basal control diet obtained from Arasco (KSA). The second (N=20; 22.4 ± 0.42) and third groups (N=20; 22.04 ± 0.55) were given basal control diet and supplemented with +(-)catechin (Sigma) with 200 and 2000 mg/kg body weight per day. +(-) catechin supplementation for females lasted 15 days. The doses of +(-)catechin were dissolved in water and drenched daily. The control animals were drenched water only.
 
Experiment I
 
Effect of +(-)catechin on changes of physiological and reproductive performances.
 
Body temperature, partial pressure of oxygen and heart rate
 
Body temperatures were recorded using Digital Thermometer (Cofoe Portable Digital Termomete Infrared Thermometer). The pulse oximeter and heart rate monitor was used (CMS60D-VET) to measure partial pressure of oxygen (PO2) and heart rate (Mohammed et al., 2018).
 
Blood samples’ collections and analyses
 
Blood samples were obtained from orbital sinus of animals in each group according to Hoff (2000). Hemocytometer was used to determine red (million/mm3) and white (thousand/mm3) blood cells in addition to micro-hematocrit centrifuge and micro-capillary reader were used to determine hematocrit (%). Blood glucose recorded using blood glucose meter (iCare advanced Medical). Thereafter, the remaining blood sample was centrifuged to obtain plasma for determination of total protein using clinical refractometer (SCHUCO, Japan).
 
Collections of germinal vesicle oocytes and grading
 
The females of each group were injected with 7.5 IU of PMSG (Folligon, Intervet). The injected females were sacrified 44-48 h after PMSG injection. Ovaries were removed and oocytes were released by puncturing of ovarian follicles with 30-G sterile needles under a stereomicroscope. Germinal vesicle (GV) stage oocytes released into TCM 199 supplemented with 5% FBS. Oocytes were collected immediately and categorized into two class; denuded GV and cumulus-enclosed GV oocytes.
 
Brilliant cresyl blue staining and oocytes diameter
 
The collected cumulus-enclosed GV oocytes were washed three times and incubated for 90 minutes at 37°C in humidified air atmosphere in KSOM medium supplemented with 4% BSA and 26 mM brilliant cresyl blue (BCB). The oocytes were observed under microscope after the incubation time and classified according to BCB staining into dark blue cytoplasm (BCB+) and colorless cytoplasm (BCB–). The diameters of denuded GV oocytes and the resulting cumulus-free GV oocytes were recorded to the nearest micron using 0.01 mm eyepiece for compound biological microscope eyepiece.
 
Reproductive performance
 
The females of each group were used for evaluating reproductive performance. Females with vaginal plug after insemination were considered pregnant and checked for parturition 4 times/day since day 18 of vaginal plug. The numbers and weight of pups per female were counted and weighed.
 
Experiment II
Effects of +(-)catechin on physiological changes after general anesthesia
 
Body temperature, heart rate, blood oxygen and glucose after general anesthesia (13.3 mg/kg BW diazepam and 26.6 mg/kg BW xylazine) at 0, 20 min, 40 min, 1h, 2h, 3h and 4h of G1 control and G2 and G3 +(-)catechin groups were recorded.
 
Statistical analyses
 
Statistical analyses was done according to general linear model of SAS program (2008). Differences between control and (+)-catechins treated groups were evaluated in physiological and reproductive characters by one-way ANOVA. Duncan Multiple Range Test was used to test the effect of treatments. Level of significance was set at P<0.05.

RESULTS AND DISCUSSION

In this study, the effects of low (200 mg/kg BW/day) and high +(-)catechin (2000 mg/kg BW/day) supplementation to female mice were explored on body temperature, heart rate, blood oxygen, red and white blood cells, total protein and glucose, oocyte quality and reproductive performances (Table 1-2).
 

Table 1: Effects of +(-)catechin on physiological parameters in mice.


 

Table 2: Effect of +(-)catechin on oocyte quality and litter size and weight in mice.

+(-)catechin supplementation resulted in significant (P < 0.05) increase in body temperature, blood oxygen and WBCs in addition to significant hypoglycemia (200 mg/kg/day) group. Furthermore, the quality of oocytes and reproductive performances were improved un/significantly due to +(-)catechin supplementation. Because the information from the previous studies (Wu et al., 2007; Mohammed et al., 2011; Mohammed et al., 2012) indicated hypothermia and hyperglycemia upon injection of diazepam and xylazine drugs in mice,  rat and rabbit. Therefore, the effects of +(-)catechin on values of body temperature, heart rate, blood oxygen and glucose were explored after general anesthesia using diazepam and xylazine drugs. The results indicated that low and high supplementation of +(-)cactechin did not improve the transient negative effects of general anesthesia.

        
There are several recent studies carried on catechin for their biologically significant functions (Cao et al., 2016; Chanphai and Tajmir-Riahi 2018; Santangelo et al., 2019). Flavonols have been found to possess potential health benefits as antioxidants, anticarcinogens and antimutagens. The consumption of foods rich in polyphenols has been associated with a decreased risk of cancers, stroke and coronary heart disease based on the study done on experimental animals and cell culture (Haratifar et al., 2014). It has been reported that the minimal doses of catechins for cancer prevention in human is 200 mg or higher but with more frequency (3+ times/day). The potential adverse effects of consuming very large amounts have not been extensively studied. Our results indicated that +(-)catechin supplementation (200 and 2000 mg/kg BW) resulted in significant (P < 0.05) increase in body temperature, blood oxygen and WBCs in addition to significant hypoglycemia (200 mg/kg). +(-)catechin can have pleiotropic effects and modulate many cellular activities, including gene expression, cell signaling and enzymatic activity. There are several factors affecting changes in body temperature such as metabolic reactions and hormones. This is accomplished through the body’s ability to regulate heat production and absorption in addition to its heat losses. The significant increase (P < 0.05) of body temperature in G3 +(-)catechin group might be due to anti-obesity of +(-)catechin (Wolfram et al., 2006) and therefore increase heat production. Moreover, the significant increase (P < 0.05) of partial pressure of oxygen in G3 +(-)catechin group is related to the insignificant increase (P > 0.05) of red blood cells (million/mm3) in +(-)catechin groups (Table 1) compared to G1 control group. In addition, white blood cells (thousand/mm3) in G3 +(-)catechin group were higher than G1 control. It has been indicated that tea catechins have been shown to effectively enhance immune activity and prevent cancer (Kim et al., 2016), although the mechanism is unclear. Kim et al., (2016) found that green tea catechin metabolites exert immune-regulatory effects on CD4+ T cell and natural killer cell activities. Significant hypoglycemia in G2 +(-)catechin group has been found compared to G3 +(-)catechin and G1 control group. In addition, total protein levels (g/dl) were higher (P < 0.05) in G3 +(-)catechin group compared to G2 +(-)catechin and G1 control group. Because catechins have the ability to bind to the signaling molecules, they may activate transcription factors in cells. 
        
The potential roles of low and high (+)-catechin on female reproduction were evaluated in this study through oocyte quality (brilliant cresyl staining and oocyte diameter) and offspring number and weight, which were un/significantly improved (Table 2). Fan et al., (2015) concluded that catechin had a positive effect on the reproductive performance, antioxidant and health status of sows when 200 and 300 mg catechin per kg diet added into the diet during the early gestation. Flavanols i.e. (+)-catechin play protective and antioxidant effective role for plants and the living organisms that consume the plants. Caro et al., (2019) concluded that catechin exhibits both antioxidant (superoxide-scavenging) and pro-oxidant effects under CYP2E1-dependent oxidative stress. Lipids oxidative damage in oocytes may cause persistently poor oocyte quality (Luderer, 2014). Elevation levels of oxidative stress caused a decrease in the number of follicles and oocytes, and these effects ultimately resulted in sub-fertility (Camlin et al., 2016). In addition, other animal studies indicated that oxidative stress were negatively influenced early embryo development (Harvey et al., 2002; de Castro et al., 2016), block the development of in vitro two-cell stage embryos by modifying the key transcription factors, transform gene expression and eventually resulting in female infertility (Jana et al., 2010; González-Fernández et al., 2016).
 
        
In experiment II, because of the significant increase of (+)-catechin on values of body temperature, blood oxygen and WBCs in addition to the significant decrease of glucose (200 mg/kg), the low and high (+)-catechin groups were tested after general anesthesia (xylazine and diazepam) for values of body temperature, heart rate, blood oxygen and glucose (Tables 3-6). The results indicated that +(-)catechin supplementation did not rescue the transient negative side effects of using diazepam and xylazine. Although +(-) catechin enhance energy consumption (Dulloo et al., 2000; Osaki et al., 2001), it did not rescue hypothermia due to anesthesia. Nevertheless, significant hypothermia found in G2 and G3 +(-)catechin groups compared to G1 control at 3h after anesthesia compared to G1 control group. In addition, the other parameters (heart rate, blood oxygen and glucose) were not improved in G2 and G3 +(-)catechin groups after anesthesia compared to G1 control. Although flavonoids have also been shown to reduce elevated blood pressure (Duarte et al., 2001), stimulate endothelium-dependent vasodilatation (Karim et al., 2000) and decrease blood glucose level (Igarashi et al., 2007), it did not rescue the negative effects of anesthesia in such parameters.
 
 

Table 3: Effect of +(-)catechin on body temperature (°C) in general anesthetized mice.


 

Table 4: Effect of +(-)catechin on partial pressure of oxygen (PO2) in general anesthetized mice.


 

Table 5: Effect of +(-)catechin on pulse rate (beats/min) in general anesthetized mice.


 

Table 6: Effect of +(-)catechin on blood glucose levels (mg/dl) in general anesthetized mice.

CONCLUSION

Supplementation of +(-)catechin could benefit animals’ health and reproduction through the improvement of physiological parameters and oocyte quality. Further studies are still required to investigate the developmental competence of in vitro matured oocytes and the resulting embryos upon +(-)cactechin supplementation in mammals and followed by embryo transfer. 

ACKNOWLEDGEMENT

This work was supported by the Deanship of Scientific Research of King Faisal University, Saudi Arabia (Grant number 160077).

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