A comparative study of preimplantation embryos development of young and aged mice treated with L-carnitine

One of the most critical factors affecting female fertility is aging. Physiologically, female fertility decreased with aging. It has been noticed that fertility declines slowly and steadily in women aged 30–35 years, but it accelerates beyond 35 due to a decline in ovarian reserve and the quality of oocytes 1 . An oocyte's ability to restart meiotic division, be fertilized, implant in the uterus, and eventually grow into an intact embryo and subsequent progeny is determined by its developmental potential 2 . Therefore, female infertility is caused by poor oocyte quality 3 . It has been found that one of the causes that resulted in poor oocyte quality is reactive oxygen species (ROS) generation. Through the respiratory chain, β -oxidation of fatty acids generates ROS such as peroxide, hydroxyl, and superoxide radicals 4 . Excessive ROS production causes inactivation of the enzyme, peroxidation of lipids, and covalent DNA and protein modification, all of which can affect the oocytes and embryos 5 . Many nutrients and pharmaceutical medications, including proteins, hormones, growth factors, and antioxidants, are recommended to enhance oocyte quality and reduce fertility problems 6 . L-carnitine Abstract Female aging is one of the main factors influencing reproductive fertility. The existing experimental study was planned to investigate the L-carnitine (LC) treatment effect on the body weight of adult young 8–10 weeks old and aged 26–28 weeks old female mice and the preimplantation embryonic development of their embryos. This study involved 40 mature young (n=20) and aged (n=20) old female mice. The animals were weighed and divided into 4 groups according to their age (n = 10). The control groups of young and aged mice groups were orally administered distilled water, while the young and aged mice that were treated orally with 10 mg/kg LC daily for 2-3 estrous cycles were considered as treated groups. Then, all female mice were mated with adult males. The weight of pregnant mice on 1-day post coitum was recorded and then euthanized to harvest early cleavage embryos. The embryo development was examined and evaluated their grading according to A, B, C and D. The results showed that the body weight of mice in both young LC and aged LC groups reduced significantly (P≤0.01). The grade A embryo in 1-day post coitum in young and aged LC groups improved significantly (P≤0.01). However, the development of embryos grade A in the young LC group was higher than that of the aged LC group. It was concluded from these findings that the oral supplementation of LC can reduce body weight and improve the preimplantation embryonic development proportions.


Introduction
One of the most critical factors affecting female fertility is aging.Physiologically, female fertility decreased with aging.It has been noticed that fertility declines slowly and steadily in women aged 30-35 years, but it accelerates beyond 35 due to a decline in ovarian reserve and the quality of oocytes 1 .An oocyte's ability to restart meiotic division, be fertilized, implant in the uterus, and eventually grow into an intact embryo and subsequent progeny is determined by its developmental potential 2 .Therefore, female infertility is caused by poor oocyte quality 3 .It has been found that one of the causes that Published Online First: November, 2023 https://dx.doi.org/10.21123/bsj.2023.8923P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal (LC) can carry activated fatty acids into the matrix of the mitochondria, promoting the utilization of fatty acid and energy, while also simultaneously raising ATP concentration 7 .Thus, LC is important in lipid metabolism because it regulates the transport of fatty acids to the matrix of mitochondria for β-oxidation 8 .Furthermore, LC functions as an antioxidant, reducing cell stress and suppressing apoptosis, a response to mitochondrial dysfunction 3,9 .Lcarnitine also regulates oxidative and metabolic states in the reproductive system of female and is essential for mammalian oocyte in vitro maturation (IVM) and embryo culture 10 .It is discovered that adding LC to the IVM medium improved β-oxidation as well as the rates of nuclear maturation, fertilization, and blastocyst development in mice 11 .Also, LC increases levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2) in the blood.Furthermore, it increases certain sperm function parameters in vitro, e.g., sperm motility 12 .Consequently, the aim of this research is to study the effects of LC on the body weight and quality of early cleavage embryos of young and aged mice.

Animals' management:
This study complies with current  13 .

Detection of female estrous cycle:
Under a light microscope, a vaginal smear was utilized to assess the stage of the estrous cycle in each female mouse.Every day between the hours of 8:00 a.m. and 1:00 p.m., the smear was conducted.The four stages of the estrous cycle in female mice were detected using the vaginal smear method as described by Rafiee and her colleagues 14 .The slides were examined under a light microscope to determine the estrous cycle phase.

Mating procedure and timing of pregnancy:
After the adaptation of female mice, LC oral supplementation and, detection of the estrous cycle in them, mating was carried out by putting two females within the estrus stage in the breeding cage of one male overnight 15,16 , following which the females were checked for indications of a vaginal copulatory plug.The presence of a vaginal copulatory plug the next morning established the starting of gestation, and the day was identified as day 'zero' of pregnancy 17 .

Retrieval and grading of embryos:
Following animal CO2 gas inhalation, each animal's uterine horns and oviducts were isolated from the body and removed using sterile surgical tools, and the specimens were placed directly in a Petri plate loaded with phosphate-buffered saline (PBS with pH 7.3).The horns of the uterus were dissected under a stereomicroscope (Zeiss, Germany).The "flushing and dissection" technique was used to retrieve embryos from uterine horns or oviducts 18,19 .An inverted microscope (TECHNO, Japan) was used to examine the morphology of all retrieved early-stage (i.e., 2-cell) embryos.The early embryos (1-day post coitum) were graded into four categories: A, B, C, and D according to Khalili and Anvari 20 .analysis.Frequency and proportions were used to represent categorical data.The proportions were compared by using the Chi-square test.In order to detect statistical differences between means, one-way ANOVA was employed.Prior to the investigation, P ≤ 0.01 was selected to represent significance.

Results and discussion
L-carnitine and body weight gaining: According to the findings of Table 1, the weight of young mice after LC treatment showed a significant (P≤0.01)reduction in comparison with their weight before the LC treatment, while there was no significant difference in the weight of female mice of the young control group during the experiment period.On the other hand, the weight of aged mice after LC administration decreased significantly (P≤0.01)compared to their weight prior to the administration of LC as illustrated in Table 2.The present investigation established that the oral administration of LC to both young and aged mice for 2-3 estrous cycles resulted in body weight reduction.Several published studies demonstrated that LC supplementation can reduce body weight in human and experimental animals 21,22 .This observation may result from the mechanism of LC that leads to a reduction in fat mass 22,23 .It has been found that the action of LC in the transport of longchain saturated fatty acids into the mitochondria and later ß-oxidation resulted in a decrease in body weight 24,25 .In addition, the relationship between LC diet and weight loss involves multiple factors.While LC diets can lead to a decrease in plasma leptin levels and an increase in fat metabolism, these effects may contribute to limiting the proliferation of adipocytes (fat cells).This can ultimately result in reduced fat mass and weight loss 26 .Different small superscripts represent significant differences at P ≤ 0.01 between weights in the columns (before and after LC administration).The mean and standard error mean are used to represent the data (SEM).LC: L-carnitine group.

L-carnitine improves preimplantation embryos development:
Based on the results illustrated in Table 3, a significant raise (P≤0.01) in the mean number of 2cell embryos (1-day post coitum) grade A from young mice received LC was assessed in comparison with untreated mice of the same age group.On the other hand, the number of 2-cell embryos grade A from aged mice treated with LC improved significantly (P≤0.01) in comparison with the aged control group but declined considerably (P≤0.01) when compared to young mice in the LC group.
In the young LC group, the mean number of 2-cell embryos grade B revealed a significant (P≤0.01)reduction compared to the young control group.The proportions of the same grade embryos in the control and LC groups of aged mice revealed a nonsignificant (P>0.01)difference.Furthermore, the On the other hand, the results proved in the same table showed that the mean number of 2-cell of grade C embryos in LC and control groups from young female mice had no significant (P>0.01)difference between them. in the aged LC group, there was a significant (P≤0.01)reduction when compared to the aged control group.Whereas there was no considerable change in the mean number of 2-cell of embryos grade C between the young and aged LC groups.At the same time, the proportion of grade D embryos in the young LC group decreased significantly (P≤0.01) when compared to the young control group, and there were no significant (P>0.01)differences between the young LC and aged LC groups, as observed in Table 3.
The grades of the early embryo (A, B, C and D) are shown in Fig. 1.The current study confirmed that the treatment of mice with LC improved the mean of embryonic development in the early embryos and the quality of embryos after 24 hrs. of fertilization in both young and aged mice and decreased the mean of embryonic development of the 2-cell embryo of grade D, that characterized by unequal size blastomeres and sever fragmentation.It has been demonstrated that LC increases the preimplantation and developmental competence of embryos in experimental animals, due to the improvement of cytoplasmic and nuclear maturation of oocytes that play an initial role in the consequent development of fertilized oocyte to embryo, and the reduction of ROS levels 27 , which may be due to its vital role in increasing glutathione (GSH) levels inside the cells that can prevent free radical damage to embryos 28 .
On the other hand, it has been reported that LC tends to induce nuclear maturity and increase the number of early embryos in camels 29 .While, LC when added to the diet of pigs, rates of oocyte maturation, cleavage of early embryos, and formation of early embryos significantly improved 30 .In mice, the inclusion of LC has been demonstrated to improve the rate of oocyte maturation and fertilization, and early embryonic development 11 .It seemed that LC also increases the cleavage of preimplantation embryo of mice, which is likely due to the intracellular lipid storage utilization.L-carnitine has been shown in several studies to have good effects on early embryonic development in canines 31 , cattle 32, 33 , camels 29 , and mice 34 .

Conclusion
In conclusion, the study found an important benefit in LC oral administration to decrease the body weight of treated female mice and enhance the quality and count of preimplantation embryonic development.A significant decrease in the grading of abnormal embryos (i.e., Grade D) was noticed after treatment with LC.These results can be utilized for other mammals to increase the pregnancy outcome in young and aged females.

Figure 1 .
Figure 1.Shows 2-cell embryos; grade A good quality embryo with equal size blastomeres (Young LC group), grade B embryo of slightly unequal size blastomeres (Aged LC group), grade C embryo of moderately unequal size blastomeres (Aged control group) and grade D embryo with unequal size blastomeres and sever fragmentation (Young control group), by inverted microscope.Magnification x400.

Table 1 . The results of young female mice weight after LC administration.
Groups of 8-10 weeks young mice (n=10)Wt.Before LC-treatment (g) Wt.After LC-treatment (g) P value The mean and standard error mean are used to represent the data (SEM).LC: L-carnitine group.