The beneficial effects of Moringa oleifera leaf on reproductive performance in mice

Abstract Moringa oleifera is a tropical plant with high nutritional and medicinal value. Recent studies have reported its remarkable effects in inflammatory, antioxidative, and anti‐diabetes modulations, but there was no significant report on its role in animal breeding. In this study, we investigated the effects of dietary Moringa oleifera leaf (MOL) on reproductive performances in mice. We studied the reproductive performance of mice for six consecutive gestations. Mice fed with 4% MOL diet showed improved litter size, litter birth weight, and litter survivals until weaning age compared to control mice fed with normal diet (p < 0.05). Mice fed with MOL diet did not change weight and organ coefficients. Serum malondialdehyde (MDA) concentrations in both male and female mice were significantly decreased by dietary MOL (p < 0.05), but glutathione peroxidase (GSH‐PX) and superoxide dismutase (SOD) were unchanged. For male, dietary MOL significantly reduced sperm abnormality rate (p < 0.05) and Bcl2‐associated X protein (Bax) expression in testis (p < 0.05), but did not affect serum testosterone and the expression levels of androgen receptor (AR), phosphoglycerate kinase 2 (Pgk2), protamine2 (Prm2), and B cell leukemia/lymphoma 2 (Bcl2) in testis. For female, dietary MOL did not change serum estradiol and the expressions of estrogen receptor beta (ERβ), Bcl2, Bax, and vascular endothelial growth factor receptor (VEGFR) in ovary. In summary, MOL increased litter size and antioxidant ability, reduced the rate of sperm abnormality and the expression of Bax. Therefore, MOL may serve as a functional feed addictive for improving animal reproductive performance.


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ZENG ). Moringa leaves also contain an abundance of bioactive compounds, principally polyphenols (phenolic acids and flavonoids) and four unique moringa isothiocyanates, with strong biological activities . Moringa oleifera leaves can be consumed either fresh or cooked, or be stored as dried powder for many months without refrigeration with little loss of nutritional value (Verma et al., 2009). For these reasons, Moringa oleifera leaf (MOL) has been used to treat a number of diseases including cardiovascular disease, insulin resistance, hepatic steatosis, and others (Almatrafi et al., 2017).
Moringa oleifera leaf has also shown protective activities in spermatogonial cells and mitigates the cell damage of mice injected with cyclophosphamide (Nayak, Honguntikar, et al., 2016). The hexane extract of MOL has been reported to enhance seminiferous tubule, epididymis, testis, and seminal vesicle functions in male mice (Cajuday & Pocsidio, 2010). In addition, Barakat, Khalil, and Al-Himaidi (2015) reported that MO combined with hormone supplementations improved the rate of maturation of sheep oocytes and could act as a promoter to induce mRNA expression and synthesis of essential proteins for the maturational processes.
Reproduction is an inevitable composition of life which plays an important role in the survival of human race. For efficient livestock production, advanced reproductive technology is crucial (Hayes, Lewin, & Goddard, 2013), and in mammals, food or nutrient is a key factor in regulating reproductive performance. Some natural plants are known as nutraceuticals, which including functional agents and could bring a positive effect for animal reproduction (Allan & Bilkei, 2005;Guroy, Sahin, Mantoglu, & Kayali, 2012).
However, there is little information on whether dietary MOL could improve reproductive performance in animals. Thus, in this study, we investigated the effects of dietary MOL powder on the reproductive parameters, serum hormones, serum antioxidant indicators, and expressions of essential genes in mice, thereby determining its role in animal reproduction. Not only could these results provide a series of significant data, but also enhance and enlighten the knowledge on development of MOL or its bioactive components in the field of animal reproduction.

| Data and sample collection
Litter size, litter birth weight, average birth weight, litter survival until weaning age (21 days), litter wean weight, and average wean weight were recorded for each pair of mated mice. After the seventh successful pregnancy of female mice, all male mice were sacrificed for the examination of sperm abnormality rate, blood samples, and testis tissues. For female mice, in order to exclude the effect of estrus cycle on the experiment, female mice were sacrificed to collect blood samples and ovary tissue on the 14th day after pregnancy. Observable pessus was regarded as a successful pregnancy (Liu et al., 2007;Ren et al., 2012). Pessus is a white mixture of male seminal vesicle secretion and female vaginal secretion after mating in mice. Generally, it is formed at the vaginal orifice of female mice within 2-4 hr after mating, and can remain at the vaginal orifice for 12-24 hr. Testis or ovary tissues were quick-frozen by liquid nitrogen. Blood samples were centrifuged at 1,000 × g for 20 min at 4°C for serum. The serum and tissue samples were stored at −80°C for further analysis.

| Sperm abnormality test
Mice sperm abnormality test was performed as described by Wyrobek and Bruce (1975). Mice were killed by cervical dislocation, and their cauda epididymides were removed. Two sperm suspensions were prepared, each from two cauda epididymides by mincing in 2 ml of phosphate buffered physiological saline, pipetting the resulting TA B L E 1 Chemical composition of the MOL (Dry matter basis)

| Serum analysis
The testosterone and estradiol in the serum were measured using enzyme-linked immunosorbent assay kits (ELISA, Nanjing Jiancheng Biotechnology Institute, China). Total SOD, MDA, and GSH-PX in the serum were determined using spectrophotometric kits (Nanjing Jiancheng Biotechnology Institute) according to the manufacturer's instructions.

| Gene expression analysis by quantitative RT-PCR
Total RNA was extracted from testis and ovary tissue using TRIzol reagent (

| Western blot analysis
The expressions of Bax protein in testis tissues were determined by Western blot analysis. Total protein was extracted from 100 mg testis tissue using 300 μl radio immunoprecipitation assay (RIPA)

| Statistical analysis
All data are expressed as means ± standard error of the mean (SEM).

| Effects of MOL on reproductive performance
We measured the reproductive performance of mice for six consecutive gestations, and found that average birth weight, litter wean weight, and average wean weight were not significantly different among the control and MOL groups (p > 0.05). However, mice fed with 4% MOL diet had higher litter size, litter birth weight, and litter survival than control mice (p < 0.05). Meanwhile, litter size of mice fed with 8% MOL diet (12.30 ± 0.70) is larger than control mice (10.92 ± 0.55; Table 3). All animals involved in the study looked healthy at the end of the study with no adverse effects observable.
We measured body weight and organ weight of male mice and pregnant female mice, and results showed that MOL had no significant effects on body weight or organ coefficients (p > 0.05; Tables 4 and   5). These results suggested that MOL can improve the fecundity of mice without harming their health.

| Effects of MOL on serum hormone and antioxidant indexes
Moringa oleifera leaf had no significant effects on the serum testosterone concentration in male mice and serum estradiol concentration in female mice (Tables 6 and 7 (Tables 6 and 7), which indicated that MOL could, in some extent, play an anti-oxidative role in mice.

| Effects of dietary MOL on sperm abnormality rate and expressions of genes in testis for male mice
The sperm abnormality rate of male mice was also determined to examine the effect of MOL on semen quality. The microscopic observation of sperm morphology is shown in Figure 1A-C. Interestingly, MOL-fed groups had lower sperm abnormality rate than control group ( Figure 1D). These observations demonstrated that MOL could improve semen quality of mice. We determined the expressions of genes related to spermatogenesis in testis tissue. The expression levels of androgen receptor (AR), phosphoglycerate kinase2 (Pgk2), protamine2 (Prm2), and B cell leukemia/lymphoma 2 (Bcl2) in testis remained unchanged after feeding dietary MOL ( Figure 1E-G).
Importantly, Bcl2-associated X protein (Bax) was reduced in the MOL groups male mice relative to the control group ( Figure 1I), which was further confirmed by Western blot (Figure 2A,B). These results indicated that MOL may boost mice reproduction by improving sperm quality and inhibiting testicular apoptosis.

| Effects of MOL on gene expression in ovary for mice
In female mice, mRNA levels of estrogen receptor beta (ERβ), Bcl2, Bax, and vascular endothelial growth factor receptor (VEGFR) in ovary tissue were not significantly different among groups ( Figure 3A-D). These results indicated that MOL did not change mRNA expressions of reproductive related genes in ovary. have regulative effects in fertility performance (Jaiswal, Rai, Kumar, Mehta, & Watal, 2009;Vongsak, Sithisarn, & Gritsanapan, 2014). In agreement with the above findings, the current study showed an improvement in reproduction performance of mice fed with MOL diet.
In the present study, the body weight and organ coefficient of the Malondialdehyde (nmol/ml) 8.00 ± 0.53 a 6.33 ± 0.50 b 6.14 ± 0.64 b 0.034 ab Different superscripts within a row represent significant differences (p < 0.05).  Malondialdehyde is considered a presumptive biomarker for lipid peroxidation in live organisms (Mateos, Lecumberri, Ramos, Goya, & Bravo, 2005). In our study, dietary MOL decreased serum MDA. Oparinde and Atiba (2014) also observed that serum MDA was significantly lower in rats fed with Moringa oleifera than those with normal diet. Additionally, Moringa oleifera leaf and fruit extracts were found to reduce MDA levels both in vitro and in vivo (Luqman, Srivastava, Kumar, KumarMaurya, & Chanda, 2012). Previous studies demonstrated that the MOL possessed potent antioxidant properties due to its high contents of phenolic compounds and isothiocyanate (Tumer, Rojas-Silva, Poulev, Raskin, & Waterman, 2015;Verma et al., 2009).

TA B L E 7 Serum estradiol and antioxidant indexes of female mice of fed MOL (n = 8)
An important discovery in our study is that MOL-fed groups had lower sperm abnormality rates than the control group. Sperm abnormalities have long been associated with male infertility and sterility in most species (Saacke, 2001 It is well known that mouse reproduction is regulated by a few crucial genes. AR and ERβ are essential for normal fertility efficiency of mice (Krege et al., 1998;Shiina et al., 2006). Pgk2 and Prm2 are crucial for maintaining the sperm motility, sperm chromatin integrity, and male fertility (Cho et al., 2003;Danshina et al., 2010 protein was an inhibitor of apoptosis, while Bax was an accelerator (Misao et al., 1996). The pre-set ratio of Bcl-2/Bax seemed to determine the survival or death of cells following an apoptotic stimulus (Albamonte et al., 2008). Our results suggested that MOL may suppress testis cell apoptosis by down-regulating Bax expression.
Meanwhile, it has been reported that Bcl-2/Bax was closely connected with antioxidative pathway (Yang, Wang, Xie, Sun, & Wang, 2010). Therefore, the antioxidative phytochemicals in MOL may change the expression of Bax. However, the exact mechanism has not been explored yet.

| CON CLUS ION
In summary, we have investigated the beneficial effects of MOL on animal reproduction. Our studies showed that dietary MOL im-

E TH I C A L S TATEM ENT
The authors declare that there is no conflict of interests. All experiments were conducted in accordance with "The Instructive Notions with Respect to Caring for Laboratory Animals" issued by