Effects of Myo-inositol on Physiological and Reproductive Traits through Blood Parameters, Oocyte Quality and Embryo Transfer in Mice

Treatments of dysfunctions or improvements of physiological and reproductive performances were achieved through feed supplements (Mohammed, 2018; Mohammed and Al-Hozab, 2020; Sheoran et al., 2018) and assisted reproductive techniques (Mohammed et al., 2010; Mohammed et al., 2012; Mohammed et al., 2019; Mohammed 2019). The continuous interests for the use of dietary supplements have grown to human and animals as well in order to improve their productive, reproductive and therapeutic performances (Gonzalez-Uarquin et al., 2020). There has been a growing interest in recent years of the benefits of myo-inositol (Simi et al., 2017; Sortino et al., 2017; Zheng et al., 2017). It has been suggested that myo-inositol has antioxidant, anti-inflammatory and immune-enhancing properties (Bizzarri et al., 2016). It addition, myo-inositol aids in weight loss and treats diabetes and its complications. Dietary myo-inositol is designed to promote a hormonal balance in women, ovarian functions and egg quality, a healthy menstrual cycle and in men support sperm health. Facchinetti et al. (2015) stated that pretreatment of myo-inositol is a very new method carried out to manage poor ovarian response in multiple small studies. Zheng et al. (2017) found that inositol supplementation improves pregnancy rate of infertile females undergoing ovulation induction for ICSI or IVF-ET. Sortino et al. (2017) indicated the therapeutic approach of inositol for polycystic ovary syndrome. Simi et al. (2017) found a positive role of inositol in in vitro fertilization accompanied with embryo transfer. It has been indicated that oral supplementation of 10g/kg BW myo-inositol in mice is well-tolerated and relatively safe. Therefore, the present study aimed to explore two levels of dietary myo-inositol supplementation (30 mmol/l vs. 60 mmol/l) primarily on changes of physiological and reproductive parameters through blood profiles, oocyte quality and embryo transfer parameters; secondly, to assess if dietary myo-inositol supplementation is effective in alleviation of hypothermia and hyperglycemia upon general anaesthesia.

Myo-inositol was purchased from Sigma (Sigma Aldrich) and the remaining inorganic and organic compounds used in this study were purchased from Caisson Lab (USA). All media were prepared fresh and sterilized through a 0.22 µm filter (Acrodisc).
 
Animals feeding and management
 
The experiment was carried out on College of Agriculture and Food Sciences, King Faisal University from October to December 2019. The experiment was carried out following the procedures approved by the Ethics Committee of King Faisal University. Seventy-five virgin female albino mice of 6-8 week old (21.69 ± 0.21 g body weight) were kept in controlled room set to maintain a temperature of 25.0 ± 3oC and a relative humidity of 50.0 ± 10.0% on a 12-h light/dark cycle. All groups were fed commercial pellet diet (Arasco, KSA) composed of 21.0% protein, 2.9% fat, 3.3% fiber, 1% mixture of vitamins and minerals and 2800 kcal/kg energy. The animals were randomly assigned to three equal groups (Control T1; 30 mmol/L myo-inositol T2 and 60 mmol/L myo-inositol T3). Myo-insitol was daily drenched for thirty days.
 
Effects of myo-inositol on changes of physiological and reproductive performances
 
Body temperatures were recorded 10.0 am using digital infrared non-contact thermometer (Cofoe). The measurement time was less than 2 seconds, measurement range error was 0.1 – 0.3 degree Celsius and the measurement distance was 1-3 cm (Mohammed et al., 2018). The pulse oximeter and heart rate monitor was used (CMS60D-VET Handheld Veterinary Pulse Oximeter) to measure partial pressure of oxygen (PO2) and heart rate.
 
Blood sample collection and analysis
 
Blood samples were collected from orbital sinus of mice after a week of starting. Hemocytometer was used to determine RBCs and WBCs 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 at 5000 rpm for 15 min to obtain blood plasma for determination of total protein (g/dl) using clinical refractometer (SCHUCO, Japan).
 
Collections of germinal vesicle oocytes and grading
 
The females were injected with 7.5 IU of PMSG (Folligon, Intervet) a week after starting the experiment. The injected females were killed 44-48 h after PMSG injection. Ovaries were removed and GV oocytes were released from ovarian follicle puncturing using 30-G sterile needles under a stereomicroscope. GV oocytes released into HEPES medium (TCM199) supplemented with 5% fetal bovine serum. Oocytes were collected immediately using glass pipette with diameter larger than the cumulus-enclosed oocytes and graded into denuded GV or cumulus-enclosed GV oocytes.
 
Brilliant cresyl blue (BCB) staining
 
The collected cumulus-enclosed GV oocytes were washed
three times and incubated for 90 minutes at 37oC in humidified air atmosphere in KSOM medium supplemented with 4% BSA and 26 μM BCB. The oocytes were observed under stereomicroscope after the incubation time and classified according to BCB staining into dark blue cytoplasm (BCB+) and colorless cytoplasm (BCB–).
 
Oocytes’ diameter
 
The collected cumulus-enclosed GV oocytes were stripped from cumulus cells using glass pipette. 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 (Beijing).
 
Reproductive performance
 
Reproductive performances were evaluated of each group through the numbers and weight of pups per female. Females with vaginal plug after natural insemination were considered pregnant and checked for parturition 4 times/day after day 18 of vaginal plug formation. The numbers and weight of pups per female were counted and weighted for each group.
 
Vasectomy and embryo transfer
 
Vasectomized males were prepared (Bermejo-Alvarez et al., 2014) three weeks before embryo transfer process. Embryos collected 96 h after hCG injection from fallopian tubes in Dulbecco’s modified medium supplemented with 10% FCS were transferred to the uterus of pseudo-pregnant female mice inseminated with vasectomized male. Briefly, the females were anesthetized and an incision at the right dorsal of the skin gave access to the right uterine horn on right side. Five embryos were loaded in glass transfer pipette and flushed into uterine horn and then the horn returned to the body cavity and the incision was closed (Deb et al., 2006).
 
Body temperature and blood glucose values of anesthetized mice
 
The animals of control group were injected with 0.2 ml physiological saline whereas the animals of myo-inositol groups were anesthetized with diazepam (13.3 mg/kg) and Xylazine (26.6 mg/kg) (Mohammed et al., 2018). Body temperature and blood glucose levels were monitored at 0, 20 min, 40 min, 1h, 2h, 3h and 4h of injection. The tip of tail was punctured and the drop of blood put on strips for measuring blood values.
 
Statistical analysis
 
Statistical analysis was done according to general linear model (GLM) of SAS program (2008). Differences between control (T1) and myo-inositol treated groups (T2 and T3) were evaluated in physiological and reproductive characters by one-way ANOVA. Duncan Multiple Range Test was used to test the effect of treatments.

In the current study, myo-inositol supplementation (30 vs. 60 mmol/L) to virgin albino female improved body functions in case of body temperature (oC), heart rate (beats/min), blood profiles (RBCs, PCV, WBCs, total protein and glucose values), oocyte quality (cumulus enclosed, oocyte diameter and brilliant cresyl blue staining) and pups live-born after normal reproduction and embryo transfer. Furthermore, myo-inositol supplementation was significantly potential in modulating hypothermia and hyperglycemia upon general anaesthesia using diazepam and xylazine dosage, which indicated in our previous studies (Mohammed, 2018; Mohammed et al., 2018). The beneficial effects of feeding myo-inositol to mammals on physiological and reproductive functions and treatments of malfunctions have been reported (Simi et al., 2017; Sortino et al., 2017; Zheng et al., 2017; Chen et al., 2020). Several studies (Krauss and Haucke 2007; Gonzalez-Uarquin et al., 2020) demonstrated that myo-inositol improved body functions, which is consistent with this study.
 
Effects of myo-inositol on physiological parameters
 
Physiological parameters of myo-inositol (T2 and T3) and control (T1) groups  are presented in Table 1. Body temperature (P < 0.05) and partial pressure of oxygen (P > 0.05)  increased in T3 myo-inositol group compared to T2 myo-inositol and T1 control groups whereas pulse rate/min was non-significantly decreased in T3 myo-inositol group compared to the other groups. Values of RBCs, PCV and WBCs were greater (P < 0.05) in T2 and T3 myo-inositol groups compared to T1 control one. While the concentrations of plasma total protein (g/dl) increased (P < 0.05) in T2 and T3 myo-inositol groups compared to T1 control group, the levels of blood glucose (mg/dl) were lower (P < 0.05).



It has been indicated that myo-inositol plays key physiological functions (Leung et al., 2011). Myo-inositol is incorporated into phosphoinositides and inositol phosphates, which have multiple cellular functions of membrane trafficking (Krauss andHaucke, 2007). Myo-inositol treatment results in the increase in insulin sensitivity and blood glucose level (Corrado et al., 2011). The important role of myo-inositol as inositol triphosphates (IP3), phosphatidylinositol phosphate lipids (PIP2/PIP3) and possibly inositol glycans in various cellular processes has been reported. Myo-inositol is essential for cell funtions including cell growth and survival (Condorelli et al., 2012).
 
Effects of myo-inositol on oocyte quality and litter size and weight
 
Quality of oocytes was evaluated through the presence of cumulus cells, brilliant cresyl blue staining and diameters (Table 2). The numbers of collected cumulus enclosed oocytes per animals were higher in T2 (P > 0.05) and T3 (P < 0.05) myo-inositol groups compared to T1 group. The numbers of stained cumulus enclosed GV oocytes with +BCB were higher (P < 0.05) in T2 and T3 myo-inositol groups compared to T1 control group. In addition, the numbers of medium oocytes (70 µm) and large oocytes (80 µm) were higher in T2 (P > 0.05) and T3 (P < 0.05) myo-inositol groups compared to T1 group. Such improvement in oocytes’ quality reflects the significant increase of litter size and weight in T2 (P > 0.05) and T3 (P < 0.05) myo-inositol groups compared to T1 group.



The results indicated improvement oocytes’ quality in case of cumulus enclosed, diameter and +BCB staining and reproductive performances due to myo-inositol supplementation. It has been indicated that myo-inositol enhances reproductive axis and hormonal functions (Krauss and Haucke, 2007), oocyte and egg quality (Brown et al., 2016), sperm motility and fertilization rate in vitro (Condorelliet al., 2012). It appears likely that exogenous inositol is important during embryo development (Cockroft, 1991).

The effects of myo-inositol on pregnancy and birth rates after embryo transfer were tested using ten recipient females and transferring 50 embryos (5 embryo/female) (Table 3). Pregnancy rates (%) were respectively 50, 50 and 60% of T1 control and myo-inositol T2 and T3 groups. The numbers of live-born per recipient were respectively 2.20 ± 0.27, 2.60 ± 0.38 and 2.83 ± 0.32 of T1 control group and T2 and T3 myo-inositol groups.


 
Effects of myo-inositol on body temperature and blood glucose upon general anaesthesia
 
alues of body temperature and blood glucose of myo-inositol and control groups  are presented in Tables (4 and 5) after general anaesthesia. Body temperature values before anaesthesia did not differ between T1 control, T2 and T3 myo-inositol groups. anaesthesiaValues of body temperature were higher (P < 0.05) in T2 and T3 myo-inositol groups compared to T1 group. The lowest body temperature was recorded 2h after anaesthesia administration in all groups. Values were higher (P < 0.05) in T2 and T3 myo-inositol groups than T1 group at 3h and 4h of anaesthesia administration. 





Values of blood glucose before anaesthesia of T2 and T3 myo-inositol groups were significantly lower than T1 group. This effect of myo-inositol on hypoglycemia extended with starting the effects of anaesthesia drug dose. This effect was more pronounced (P < 0.05) at 20 min, 40 min, 3h and 4h of anaesthesia drugs’ injection.

The significant effect of myo-inositol in treating hypothermia and hyperglycemia upon general anaesthesia might be due to its key physiological functions as phosphoinositides and inositol phosphates as previously mentioned. To the best of our knowledge, this is the first study indicating the beneficial effects of myo-inositol supplementation in general anesthetized mice. Further studies are still required in other species of general anesthetized animals to confirm such information. In conclusion, myo-inositol supplementation could improve physiological and reproductive performances of mice through blood parameters and oocyte quality.

The authors acknowledge the Deanship of Scientific Research at King Faisal University for financial support under Nasher track (Grant No. 186154).