Assess the effects on the sperm production of Y10 on patients with sperm decline

the Ministry of Health of Vietnam and the guidance of OECD, WHO. 30 mice were divided into 3 groups, 10 each; the control group drank distilled water. Study group 1: Y10 224mg decoction / kg / day. Study group 2: Y10 672mg decoction / kg / day in 90 consecutive days. Their general condition, body weight, hematopoietic function, liver and kidney function, liver and kidney histopathology were observed and recorded before and after the treatment. * The study of toxicity on reproductive abiliity of Y10 The white mice of both sexes were randomly divided into 5 study groups, with 60 each including 20 males and 40 females. The control group consisted of 20 males and 40 females drinking distilled water. -Group 1: 20 males and 40 females taking Y10 384mg decoction/kg/ day. - Group 2: 20 males and 40 females taking Y10 1152mg decoction / kg / day. -Group 3: 20 males taking Y10 384mg decoction / kg / day and 40 females taking distilled water. - Group 4: 20 males taking Y10 1152mg decoction / kg / day and 40 females taking distilled water. The treatment duration was 60 days, at a certain time everyday (8 am). After 60 days, a male was kept with two females in a separate cage (generation P) and the reproduction process was monitored. - The conception and fetal development were monitored. * The mutagenic toxicity study on chromosomes of Y10 on white mice According to OECD guideline 475 (2002), 90 healthy white mice, divided into 3 groups, each group of 30 rats including 15 male rats and 15 female rats (male and female rats kept separately): + Group 1 (control group): distilled water. + Group 2: Y10 384mg dectoction / kg / day. + Group 3: Y10 1152mg decoction / kg / day. Mice were given the medication daily, once a day at 8 am, in 4 weeks (28 days). After the groups finished drinking the decoction, they were injected with colcemide into the abdominal cavity to stop cell division in the middle of cell division, when the chromosomes were most typical. 2 hours after the colcemide injection, the chromosome samples from the bone marrow were made by Ford method and the chromosomes from the testes by Evan method. 50 chromosome patterns of each mouse were analyzed at the Diakinesis-metaphase stage when the chromosomes were short, evenly sprung and were not overlapped. * Evaluation of the effects of sperm enhancement of Y10 on white rats that caused sperm decline with Natri valproate Adult male rats, randomly assigned to five study groups, 10 each. + Group 1: were not caused sperm decline, drank distilled water. + Group 2: caused sperm decline, drank distilled water. + Group 3: caused sperm decline, drank testosteron undecanoat 16 mg / kg / day. + Group 4 (batch 1): caused sperm decline, drank Y10 224 mg decoction / kg / day. + Group 5 (batch 2): caused sperm decline, drank Y10 448mg decoction / kg / day. The group of mice that were caused sperm decline by Natri valproate at the dose of 500mg / kg / day in 7 weeks. Mice in non-sperm-decline-caused groups were given distilled water at the same volume in 7 weeks. After 6 weeks, mice were killed to serve as samples for the evaluation of research indicators, including: testosteron, sperm density, sperm motility, sperm morphology, percentage of structural morphology sperm abnormalities, testicular histopathological changes, size of spermatogenesis diameter, genital organs (testes, epididymis, seminal vesicles, Cowper glands, glans, prostate gland, and levator ani muscles). The genital organs were determined out of every 100g the mice’s body weight. 2.4.2. Clinical research We conducted inclusive studies and open clinical trials, compared the differences in the indexes between before and after the treatment. The patients with sperm decline were examined according to modern and traditional medicine, they undertook all tests and if they were eligible, they would be selected for the study groups. * Criteria for selecting patients 30 patients (male soldiers) were selected based on the following criteria: Patients who voluntarily took part in the research, stopped using drugs that affect sperm count and quality for at least 75 days. - Criteria for selecting patients according to modern medicine: Age: 16 to 56; Sperm decline according to WHO standards onseminogram (2010). - Criteria for selecting patients according to traditional medicine: men had ‘deficiency of kidney and Jing’ - Criteria for excluding patients: The patients who did not agree to take part in the study, did not strictly followed guidances, or refused to continue the treatment. * Dosage: 4 tablets per day, twice, 2 hours after eating, in 2 months. * Methods of clinical examination and clinical monitoring The medical record for each patient is made according to a unified form based on the criteria of the World Association of Southern Studies, combined with với four methods for physical examination of traditional medicine, the patients were examined and their test results were recorded. * Laboratory tests - Biochemical blood before and after the treatment: urea, creatinine, AST, ALT. - Quantification of LH, FSH, and serum testosterones before the treatment; quantification of LH, FSH, and serum testosteron in selected patients after the treatment. - Seminogram before and after the treatment. The tests were done at the Institute of Medical Research - Military Medical Academy. * Evaluation criteria - Epidemiological characteristics: distribution of patients by age and types of infertility (infertility I or infertility II). - Some clinical signs due to unwanted effects of the medication: rashes, digestive disorders (loose stool, constipation , etc), dizziness, etc. - Clinical symptoms due to renal impairment before and after the treatment. - ALT, AST, urea, serum creatinine before and after the treatment. - Serum testosteron, LH, FSH concentrations before and after the treatment. - Semen before and after treatment (Table 2.3). - The percentage of patients whose wives were pregnant and gave birth after the treatment.  * Data processing The research data is processed by biomedical statistical method, using statistical software SPSS.17.0. The difference was statistically significant when p <0.05. Chapter 3. RESULTS 3.1. Research results on the acute and semi-chronic toxicity study 3.1.1 Acute toxicity results: Rats took up to 20.0g decoction / kg body weight (equivalent to 25.0 g of powder in a capsule / kg body weight) which was the maximum dose that mice could take orally to evaluate the acute toxicity of the reagent but no mouse died, no abnormal symptoms appeared after 72 hours and during 7 days after the experiment. 3.1.2. Result of the semi-chronic toxicity study During the experiment, the rats in all 3 groups were normal, agile, eat well, had silky hair and firm feces. Mice weight in all 3 groups increased. The frequency and amplitude of ECG did not change. The number of red blood cells, hemoglobin content, hematocrit concentration, leukocyte count, percentage of neutrophils and lymphocytes, number platelet count, enzyme activity of AST, ALT, total bilirubin urea concentration and serum creatinine concentration of white rats in all groups were not different (p> 0.05). * Changes in histopathology: macroscope morphology images of liver organs, spleens, and kidneys of rats in research groups 1 and 2 had even dark reddish brown color, smooth surface, no lumps or hemorrhages. When being pressed down, there were no differences compared to that of the control group. 3.1.3. Results of the toxicity study on fertility Table 3.1. The percentage of female mice conceived in the groups Groups P generation F1 generation % female mice conceived p % female mice conceived p Control group 60.94 % > 0.05 71.65 % > 0.05 Group 1 59.86 % 70.14 % Group 2 63.28 % 74.56 % Group 3 61.72 % 72.43 % Group 4 66.37% 77.91% Comments: There was no difference in the pregnancy rate between the Y10 and control groups in P and F1 generations (p> 0.05). Table 3.2: The number of corpus luteum / 1 mother mouse in the groups Groups P generation F1 generation corpus luteum / 1 mother mouse p corpus luteum / 1 mother mouse p Control group 12.36 ±2.08 > 0.05 13.64 ± 2.57 > 0.05 Group 1 12.45±3.14 14.22 ±2.86 Group 2 12.18±2.56 13.69±3,02 Group 3 13.09±2.81 14.06 ±2.65 Group 4 12.27±2.35 13.91 ±3.14 Comments: There was no difference in the average number of corpus luteum / 1 mother rat between the Y10 and control groups in P and F1 generations (p> 0.05). Table 3.3: Number of alive fetus / 1 mother mouse in the groups(%) Groups P generation F1 generation Number of alive fetus / 1 mother mouse p Number of alive fetus / 1 mother mouse p Control group 97.65 % > 0.05 98.02% > 0.05 Group 1 96.92% 97.65% Group 2 98.45% 98.69% Group 3 97.26 % 97.91% Group 4 98.19% 98.54% Comments: There was no difference in the number of pregnant fetuses / 1 mother rat between the Y10 and control groups in P and F1 generations (p> 0.05). Table 3.4: Number of early fetal deaths / 1 female mouse in the groups(%) Groups P generation F1 generation Number of early fetal deaths / 1 female mouse p Number of early fetal deaths / 1 female mouse p Control group 2.94 % > 0.05 3.62 % > 0.05 Group 1 3.16 % 3.09% Group 2 2.08 % 4.17% Group 3 2.75 % 2.98% Group 4 3.21 % 3.81% Comments: There is no difference in the number of early fetal death / 1 mother rat between the Y10 and control groups in P and F1 generations (p> 0.05). Table 3.5: Number of stillbirths / 1 mother mouse in each group (%) Groups P generation F1 generation Number of stillbirths / 1 mother mouse p Number of stillbirths / 1 mother mouse p Control group 1.62 % > 0.05 2.36% > 0.05 Group 1 1.81% 2.09% Group 2 2.03% 1.86% Group 3 2.16% 2.47% Group 4 1.25% 2.18% Comments: There was no difference in the number of stillbirth fetal fetuses / 1 female rat between the Y10 and control groups in P and F1 generations (p> 0.05). Table 3.6: Number of dissipated eggs / 1 mother mouse in the groups (%) Groups P generation F1 generation Number of dissipated eggs / 1 mother mouse p Number of dissipated eggs / 1 mother mouse p Control group 4.48% > 0.05 3.96% > 0.05 Group 1 4.62% 4.12% Group 2 4.26% 3.97% Group 3 4.32% 4.31% Group 4 3.98% 2.68% Comments: There was no difference in the number of dissipated eggs / 1 mother between the Y10 and control groups in P and F1 generations (p> 0.05). Table 3.7: Number of mice per a litter Groups F1 generation Number of mice per a litter p Control group 12.08 ±1.93 > 0.05 Group 1 12.14 ± 2.54 Group 2 12.19± 1.98 Group 3 12.23 ± 1.64 Group 4 12.45± 2.46 Comments: There was no difference in the number of mice / litter between the Y10 and control groups in P and F1 generations (p> 0.05). Because the proportion of male and female mice in each litter (F1 generation) in each group was equal, the number of mice per litter in groups was similar. We randomly selected the number of female mice and the number of male mice in the groups so that the number of pairs in the group was the same. Table 3.8: Number of dead mice per litter (%) Groups F1 generation Number of dead mice per litter p Control group 1.68 % > 0.05 Group 1 2.08 % Group 2 1.98% Group 3 1.62 % Group 4 2.15% Comments: Mice born to F1 generation mother mice: acted and moved normally. No deformities in all groups. Conclusion: Results of the genetic toxicity study (reproductive toxicity) showed that Y10 capsules did not significantly affect the normal development of fetus and mices born to P and F1 generations. 3.1.4. Research results on chromosomal toxicity Table 3.9. The effects of Y10 on the number of bone marrow cell chromosomes Indexes Groups p Group 1 Group 2 Group 3 Number of cells monitored 172 169 160 Number of Aneuploid 3 2 2 Rate of Aneuploid (%) 2.05 1.18 1.25 > 0.05 Number of polyploidy 6 5 3 Rate of polyploidy (%) 3.07 2.96 1.88 > 0.05 Comments: on chromosomal samples from bone marrow cells in groups taking preparations at both low and high doses continuously in 28 days, the rate of occurrence of chromosomal disorders was not different from that of the control group (p> 0.05). Table 3.10. Effect of Y10 on bone marrow cell chromosome structure Indexes Group 1 Group 2 Group 3 p Number of cells monitored 172 169 160 Number of chromosomal chromatid disorders 2 0 0 Rate of chromosomal chromatid disorders (%) 1.12 0.00 0.00 > 0.05 Number of chromosomal structural disorders 3 0 0 Rate of chromosomal structural disorders (%) 1.69 0.00 0.00 > 0.05 Rate of chromosome cluster disorders 0 0 0 Comments: The rate of chromosomal structural disorders in the chromosomal samples of bone marrow cells in the mice taking the decoction at low and high doses did not differ from that of the control group. (p> 0.05). Control group Low dose group High dose group Photo 3.5. White bone marrow cell chromosome s (X 1000) After the groups finished drinking the decoction, they were injected with colcemide into the abdominal cavity to stop cell division in the middle of cell division, when the chromosomes were most typical. . 2 hours after the colcemide injection, the chromosome samples from the bone marrow was made by Ford method and the chromosomes from the testes by Evan method. 50 chromosome patterns of each mouse were analyzed at the Diakinesis-metaphase stage when the chromosomes were short, evenly sprung and were not overlapped. Table 3.11. Effects of the decoction on testicular chromosomes Types of mutations Control group Group 2 Group 3 p Number of chromosome < 40% 6.48 ± 0.84 7.04 ± 0.80 7.30 ± 1.01 > 0.05 Number of chromosome = 40% 90.87 ± 1.32 90.98 ± 1.43 89.50 ± 0.65 > 0.05 Number of chromosome > 40% 1.23 ± 0.64 1.14 ± 0.54 1.08 ± 0.61 > 0.05 Number of normal chromosome % 1.25 ± 0.67 1.19 ± 0.59 1.10 ± 0.75 > 0.05 Number of gender chromosome % 7.82 ± 1.41 8.23 ± 1.52 8.90 ± 1.39 > 0.05 Comments: there was no difference in the frequency of chromosomal mutations of the testes between the study and control groups (p> 0.05). Conclusion: The Y10 did not cause chromosomal mutations in bone marrow and testes at the doses and times in the experiment. 3.2. Research on the effects of increasing sperm function of Y10 in the experiment 3.2.1. Effects of Y10 on mice’s serum testosterone Table 3.12. Ratio serum testosterone Groups Testosteron (ng/ml) % changes p Group 1 3.51 ± 2.10 - p1.3.4.5-2< 0.01 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 Group 2 1.55 ± 0.68 ↓ 55.95* % Group 3 3.10 ± 1.27 ↑ 99.81** % Group 4 2.77 ± 1.27 ↑ 78.71** % Group 5 2.94 ± 1.38 ↑ 89.35** % Comments: The serum testosteron concentrations in the groups 3, 4, 5 increased significantly compared to the group 2 (p 0, 05). There was no significant difference between the groups 3, 4, 5 (p> 0.05). 3.2.2. Effects of Y10 on sperm count and quality Table 3.13. Effects of Y10 on sperm density Groups sperm density (× 106/mL) % changes p Group 1 81.58 ± 23.99 - p1.3.4.5-2< 0.01 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 Group 2 39.14 ± 11.90 ↓ 52.03* % Group 3 69.11 ± 23.31 ↑ 76.57**% Group 4 79.09 ± 15.44 ↑ 102.09**% Group 5 82.45 ± 14.69 ↑ 110.67**% Comments: The sperm density of the group 2 decreased significantly compared to that of the group 1, the difference was statistically significant with p 0.05). Table 3.14. Sperm mobility (n = 10, ± SD) Groups Rate of Sperm mobility (%) Rapid progressive Slow progressive Non-progressive Non-mobile Group 1 38.20 ± 6.93 4.47 ± 1.54 6.34 ± 1.60 51.00 ± 9.02 Group 2 20.99 ± 8.84 9.13 ± 4.10 9.43 ± 3.39 60.32 ± 9.42 Group 3 34.58 ± 8.80 5.46 ± 1.48 6.38 ± 1.60 53.59 ± 9.15 Group 4 34.75 ± 8.05 4.60 ± 1.61 6.94 ± 2.17 53.72 ± 9.72 Group 5 35.50 ± 9.47 4.95 ± 1.54 6.47 ± 1.50 53.08 ±10.19 P p-2< 0.01 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 p-2< 0.05 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 p-2< 0.05 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 p-2< 0.05 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 Comments: Mice in the groups 3, 4 and 5 had significantly higher rates of rapid progressive sperms than the group 2 (p 0.05); meanwhile, the percentages of sperms that did not progress and progress slowly decreased significantly compared to the group 2 (p <0.05). Table 3.15. The proportion of the sperms having abnormal structural morphology Groups Proportion of the sperms having abnormal structural morphology % changes pso comparison between groups Group 1 8.15 ± 2.88 - p1.3.4.5-2< 0.01 p3.4.5-1> 0.05 p4.5-3> 0.05 p4-5> 0.05 Group 2 15.67 ± 4.77 ↑ 92.39* % Group 3 10.33 ± 2.61 ↓ 34.09** % Group 4 9.53 ± 2.89 ↓ 39.18** % Group 5 9.32 ± 3.06 ↓ 40.54** % * compared to the control group ** compared to the group 2 Comments: The percentage of sperms having abnormal structural morphology in the groups 3, 4, 5 decreased significantly compared to that of the group 2 (p 0.05). The figures for the groups 3, 4, 5 were higher than that of the 2 groups using Y10, but there was no statistically significant difference (p> 0.05). ). 3.2.3. Effects of Y10 on the weight of male genital organs Table 3.16. Weight of the genital organs (n ​​= 10) Lô nghiên cứu Weight of the genital organs (g/100g thể trọng) Testicle Epididymis seminal vesicles Prostate gland Cowper gland Glans Levator ani muscles Group 1 ± SD 0.889 ± 0.165 0.252 ± 0.031 0.221 ± 0.062 0.119 ± 0.030 0.032 ± 0.021 0.036 ± 0.019 0.326 ± 0.069 Group 2 ± SD 0.682 ± 0.174 0.213 ± 0.024 0.159 ± 0.028 0.098 ± 0.015 0.026 ± 0.018 0.035 ± 0.016 0.291 ± 0.038 p2-1 < 0.05 < 0.05 < 0.05 < 0.05 > 0.05 > 0.05 < 0.05 Group 3 ± SD 0.831± 0.206 0.247 ± 0.032 0.208 ± 0.030 0.113 ± 0.028 0.027 ± 0.016 0.036 ± 0.020 0.328 ± 0.058 p3-1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 p3-2 < 0.05 < 0.05 < 0.05 < 0.05 > 0.05 > 0.05 < 0.05 Group 4 ± SD 0.832 ± 0.201 0.250 ± 0.103 0.210 ± 0.041 0.114 ± 0.038 0.028 ± 0.012 0.035 ± 0.020 0.325 ± 0.126 p4-1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 p4-2 < 0.05 < 0.05 < 0.05 < 0.05 > 0.05 > 0.05 < 0.05 Group 5 ± SD 0.834 ± 0.194 0.252 ± 0.081 0.211 ± 0.062 0.115 ± 0.046 0.026 ± 0.014 0.036 ± 0.019 0.330 ± 0.107 p5-1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 p5-2 < 0.05 < 0.05 < 0.05 < 0.05 > 0.05 > 0.05 < 0.05 Comments: Testicle weight, epididymis, seminal vesicles, prostate gland, levator ani muscles of the groups decreased significantly compared to the group 1 (p 0.05). - The weight of penis and Cowper glands in the groups varied without statistical significance (p> 0.05). 3.2.4. Effects of Y10 on histopathology of mice’s testes A. Histopathology of mice’s testes (mouse 08, contol group). HE, x 400 B. Histopathology of mice’s testes (mouse 15, group 2). HE,x 400 C. Histopathology of mice’s testes (mouse 24, group 3). HE, x 400 D. Histopathology of mice’s testes (mouse 36, group 4). HE, x 400 E. Histopathology of mice’s testes (mouse 42, group 5). HE, x 400 Photo 3.8. Histopathological picture of mouse testicle histogram (HE x 400) Comments: In: The size and image of the sperm tubes of the two groups using Y10 (Groups 4 and 5) and the control group were similar to the group 1 and the tissue space was not much thicker than the physiological group. Table 3.17. Diameter of spermatophores of the study groups Groups Diameter of spermatophores (µm) p Group 1 (1) 128,56 ± 8,60 p1,3,4,5-2< 0,05 p3,4,5-1> 0,05 p4,5-3> 0,05 p4-5> 0,05 Group 2 (2) 116,81 ± 9,95 Group 3 (3) 125,95 ± 10,18 Group 4 (4) 125,86 ± 9,88 Group 5 (5) 126,83 ± 10,21 Comments: Y10 repaired testicular histopathological lesions, which significantly increased the diameter of the spermatic tubes compared to the non-medication-infected group. Y10 at two dosage levels (280 and 560 mg / kg / 24h) had the effect of restoring the diameter of the sperm tube to the equivalent of group 1 (p> 0.05). 3.3. Results of assessing the safety and increasing the ability of sperm stimulation of Y10 in patients with sperm decline 3.3.1. Result of serum testosterone, LH, and FSH concentrations Table 3.18. Serum concentration of testosterone, LH, FSH Indexes Before the treatment After the treatment Pbefore-after ± SD ± SD LH (IU/l) 6.02 ± 2.14 5.08 ± 2.06 < 0.05 FSH (IU/l) 8.16 ± 4.01 6.85 ± 3.69 < 0.05 Testosteron (nmol/l) 14.65 ± 6.27 16.89 ± 6.42 < 0.05 Comments: serum LH and FSH levels after the treatment decreased compared to before the treatment, the serum testosterone levels after the treatment increased compared to before the treatment. The LH and serum FSH of the patients with hormonal disorders increased while the serum testosterone reduced. After the treatment, LH and serum FSH decreased, the increase in the serum testosterone was a positive change, the concentration of sex hormones returned to the normal physiological limits. 3.3.2. Results of semen in patient Table 3.19. Ratio of semen samples according to sperm classification Number of semen samples Before the treatment After the treatment Pbefore-after n % n % Low sperm count 6 20.00 7 23.33 > 0.05 Weak sperms 9 30.00 8 26.67 > 0.05 Abnormal sperms 0 0 0 0 Low sperm count and weak 9 30.00 8 26.67 > 0.05 Weak and abnormal sperms 1 3.33 0 0 > 0.05 Low sperm count. weak and abnormal 2 6.67 1 3.33 < 0.05 Normal seminogram results 3 10.00 1 3.33 Total 0 0 5 16.67 Comments: The results from Table 3.31 show that the percentage of semen samples with low sperm count, weak sperm and deformity decreased after the treatment (p <0.05). The percentage of semen samples returned to normal and / or pregnant wives was 16.67%. Table 3.20 Volumetric volume, pH, and white blood cell count Indexes Before the treatment After the treatment Pbefore-after ± SD ± SD Volume (ml) 2.05 ± 1.08 2.34 ± 1.12 < 0.05 pH 7.42 ± 0.35 7.51 ± 0.27 > 0.05 Number of white blood cells (x106) 6.79 ± 1.26 6.34 ± 1.49 < 0.05 Comments: The semen volume after thettreatment increased, the number of white blood cells after the treatment decreased significantly (p <0.05). There was no statistically significant difference in semen pH after the treatment. Table 3.21. Comparison of semen indicators before and after the treatment Indexes Sperm density (T/ml) Number of sperms (T) Alive (%) Progressive (%) Non-progressive (%) Before the treatment 13.54 ± 10.62 31.05 ± 33.89 24.05 ± 12.94 8.26 ± 5.86 25.01 ± 11.24 After the treatment 22.96 ± 12.65 58.20 ± 49.01 31.94 ± 18.26 14.03 ± 6.98 34.12 ± 12.93 Pbefore-after < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Comments: The sperm density, alive sperm rate, progressive sperm rate increased after the treatment and the difference was statistically significant (p < 0.01). Table 3.22. Clinical treatment results Treatment results Number of patients % Very good 5 16.67%. Good 20 66.67%. Average 5 16.67% Bad 0 0%. Comments: Very good treatment results accounted for 16.67%. (The wives got pregnant or semen increased to normal) - Good (sperm count and quality increased compared to before the treatment) was 66.67%. - Average (either the sperm count or quality increased) was 16.67%. Bad was 0%. 3.3.5. The results of improving the symptoms according to traditional medicine Table 3.23. The changes of symptoms according to traditional medicine Symptoms Before the treatment After the treatment Pbefore-after n Tỉ lệ % n Tỉ lệ % Dizziness 22 73.33 4 13.33 < 0.01 Tinnitus 24 80.00 2 6.67 < 0.01 Backache 20 66.67 3 10.00 < 0.01 Fatigue 8 26.67 1 3.33 < 0.01 Deep fine or fine weak pulse 26 86.67 7 23.33 < 0.01 Low semen volume 19 63.33 7 23.33 < 0.05 Low sperm count 23 76.67 14 46.67 < 0.05 Comments: All the clinical signs due to the deficiency of kidney and Jing increased markedly after treatment (p <0.01 and p <0.05). 3.3.6. Clinical evaluation of the safety of Y10 There were no clinically unwanted results based on hematological and biochemical indexes test results before and after the treatment. CHAPTER 4: DISCUSSION 4.1. Toxicity of Y10 4.1.1. Acute toxicity: The LD50 of oral Y10 was not found