Volume 16, Issue 11 (February 2023)
Qom Univ Med Sci J 2023, 16(11): 928-941 |
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Hosseinchi M, Najafi G. Protective Effects of Hydroalcoholic Extract of Achillea Millefolium on In-vitro Fertilization in Streptozotocin-Induced Diabetic Male Mice. Qom Univ Med Sci J 2023; 16 (11) :928-941
URL: http://journal.muq.ac.ir/article-1-3593-en.html
URL: http://journal.muq.ac.ir/article-1-3593-en.html
1- Gholamreza Najafi , dvm_06@yahoo.com
2- Department of Basic Science, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
2- Department of Basic Science, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Introduction
Diabetes is a chronic diseases characterized by chronic hyperglycemia which may have adverse effects on male reproductive function. Achillea Millefolium (AM), a medicinal plant used for over 3000 years, have been shown to have anti-inflammatory, antitumor, antimicrobial and antioxidant properties. The main compounds found in this plant include volatile oils, poly-phenol, flavonoids, sesquiterpene, betaine and tannins. This study aims to examine the effect of AM hydroalcoholic extract on sperm parameters and In-vitro fertilization (IVF) of diabetic mice.
Methods
In this study, 40 adult male Syrian mice (weighing 30±5 g) were kept in the Laboratory Animal House of Urmia University at a temperature of 21±2 ˚C and 12/12 light-dark cycle, fed with a standard pellet diet and had free access to water. The mice were randomly divided into five groups (n=8), including control group (receiving 0.20 mL physiological serum orally), diabetic group (receiving a single intraperitoneal injection of Streptozotocin at a dose of 45 mg/kg), diabetic+low AM (receiving Streptozotocin plus 37.5 mg/kg of AM hydroalcoholic extract by gavage for 30 days), diabetic+moderate AM , (receiving Streptozotocin plus 75 mg/kg of AM hydroalcoholic extract by gavage for 30 days), and diabetic+high AM (receiving Streptozotocin plus 150 mg/kg of AM hydroalcoholic extract by gavage for 30 days).
After anesthesia with a ketamine/xylazine solution, mice were euthanized by dislocation of their cervical vertebrae and their testis and epididymis were removed and placed in 1 mL of Human Tubal Fluid (HTF) medium. The tail of epididymis was cut into three parts and incubated at 37 ˚C for 30 min using a 5% CO2 incubator to allow sperms to swim out of the epididymal tubules. Sperm concentration was determined using a hemacytometer (Neubauer, HBG, Germany). After preparing a 1:20 dilution from the epididymal sperm, about 10 μL of this diluted specimen was transferred to the hemocytometer. The sediment sperms during this time were counted with a light microscope at ×400 and expressed as the number of sperm per mL of suspension (n×50000×d), where, n is the number of counted sperms and d is the reverse of dilution. Sperm suspension was stained with Eosin-Nigrosin staining technique. Slides were viewed by light microscope. Dead sperms appeared pink and live sperms were not stained.
Sperm smear was prepared, dried and fixed by the glutaraldehyde for 30 min. Then, the smears were stained by 5% aniline blue for 5-8 min and the percentage of mature sperms (with colorless head) and immature sperm (with blue head) were observed by using a light microscope. For assessment of sperm DNA integrity, dried smears were fixed in methanol. Then, the slides were stained with Acridine orange. Sperms without damaged DNA (with green color) and with damaged DNA (with orange color) were observed by a florescent microscope. Female mice were super ovulated with intraperitoneal injection of 10 IU of PMSG (Pregnant mare serum gonadotropin) hormone and 10 IU of HCG (Human chorionic gonadotropin) hormone. After 12 hr of HCG injection, oocytes of mice were collected by dissecting from ampulla of the oviduct and transferred to the fertilization droplet. Then, sperms were added to the fertilization medium (1×106/1 mL of HTF). Fertilization rate was determined about 6 hours after releasing the sperm by observing two pronuclei. In addition, the formation of two-cell embryos and blastocysts were evaluated. The data for various sperm and IVF parameters were expressed as Mean±SD and were compared by using one-way ANOVA followed by Tukey’s test for multiple comparisons. P<0.05 was statistically significant.
Results
The results revealed that sperm count and percentage of sperm viability decreased, and the percentage of mature sperm and sperm with damaged DNA increased significantly in the diabetic group compared to the control group (P<0.05). Sperm count and percentage of sperm viability increased significantly in the diabetic+high AM in comparison with the diabetic group. There was no significant difference in sperm maturity among the groups received low, moderate, and high doses of AM and the diabetic group (P≥0.05). Percentage of sperms with DNA damage in the groups received low, moderate, and high doses of AM decreased significantly in comparison with the diabetic group (P<0.05)
The diabetic group showed a significant decrease in fertilization rate, percentage of two-cell embryos, percentage of blastocysts, and percentage of hatched embryos (P<0.05) and a significant increase in the percentage of arrested embryos (P<0.05) in comparison with the control group. The increase of fertilization rate, percentage of two-cell embryos, percentage of blastocysts, and percentage of hatched embryos was significant only in the diabetic+high AM group in comparison with the diabetic group (P<0.05). In the diabetic groups received AM no significant decrease was observed in the percentage of arrested embryos (P≥0.05) in comparison with the diabetic group.
Discussion
This study demonstrated the protective effect of AM on reproductive system dysfunction caused by streptozotocin-induced diabetes. Streptozotocin-induced diabetes leads to increased sperm with DNA damage and immature sperm, and decreased sperm count and sperm viability, leading to low fertility parameters such as infertility rate and embryo development. The AM hydroalcoholic extract can strongly ameliorate these parameters.
Ethical Considerations
Compliance with ethical guidelines
This study was approved by the ethics committee of Faculty of Vetarinary Medicine, Urmia University (Code: AP/1286/3).
Funding
This study was funded by Islamic Azad University of Urmia Branch.
Authors contributions
All authors contributed equally in preparing all parts of the research.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors would like to thank Ali Karimi.
Diabetes is a chronic diseases characterized by chronic hyperglycemia which may have adverse effects on male reproductive function. Achillea Millefolium (AM), a medicinal plant used for over 3000 years, have been shown to have anti-inflammatory, antitumor, antimicrobial and antioxidant properties. The main compounds found in this plant include volatile oils, poly-phenol, flavonoids, sesquiterpene, betaine and tannins. This study aims to examine the effect of AM hydroalcoholic extract on sperm parameters and In-vitro fertilization (IVF) of diabetic mice.
Methods
In this study, 40 adult male Syrian mice (weighing 30±5 g) were kept in the Laboratory Animal House of Urmia University at a temperature of 21±2 ˚C and 12/12 light-dark cycle, fed with a standard pellet diet and had free access to water. The mice were randomly divided into five groups (n=8), including control group (receiving 0.20 mL physiological serum orally), diabetic group (receiving a single intraperitoneal injection of Streptozotocin at a dose of 45 mg/kg), diabetic+low AM (receiving Streptozotocin plus 37.5 mg/kg of AM hydroalcoholic extract by gavage for 30 days), diabetic+moderate AM , (receiving Streptozotocin plus 75 mg/kg of AM hydroalcoholic extract by gavage for 30 days), and diabetic+high AM (receiving Streptozotocin plus 150 mg/kg of AM hydroalcoholic extract by gavage for 30 days).
After anesthesia with a ketamine/xylazine solution, mice were euthanized by dislocation of their cervical vertebrae and their testis and epididymis were removed and placed in 1 mL of Human Tubal Fluid (HTF) medium. The tail of epididymis was cut into three parts and incubated at 37 ˚C for 30 min using a 5% CO2 incubator to allow sperms to swim out of the epididymal tubules. Sperm concentration was determined using a hemacytometer (Neubauer, HBG, Germany). After preparing a 1:20 dilution from the epididymal sperm, about 10 μL of this diluted specimen was transferred to the hemocytometer. The sediment sperms during this time were counted with a light microscope at ×400 and expressed as the number of sperm per mL of suspension (n×50000×d), where, n is the number of counted sperms and d is the reverse of dilution. Sperm suspension was stained with Eosin-Nigrosin staining technique. Slides were viewed by light microscope. Dead sperms appeared pink and live sperms were not stained.
Sperm smear was prepared, dried and fixed by the glutaraldehyde for 30 min. Then, the smears were stained by 5% aniline blue for 5-8 min and the percentage of mature sperms (with colorless head) and immature sperm (with blue head) were observed by using a light microscope. For assessment of sperm DNA integrity, dried smears were fixed in methanol. Then, the slides were stained with Acridine orange. Sperms without damaged DNA (with green color) and with damaged DNA (with orange color) were observed by a florescent microscope. Female mice were super ovulated with intraperitoneal injection of 10 IU of PMSG (Pregnant mare serum gonadotropin) hormone and 10 IU of HCG (Human chorionic gonadotropin) hormone. After 12 hr of HCG injection, oocytes of mice were collected by dissecting from ampulla of the oviduct and transferred to the fertilization droplet. Then, sperms were added to the fertilization medium (1×106/1 mL of HTF). Fertilization rate was determined about 6 hours after releasing the sperm by observing two pronuclei. In addition, the formation of two-cell embryos and blastocysts were evaluated. The data for various sperm and IVF parameters were expressed as Mean±SD and were compared by using one-way ANOVA followed by Tukey’s test for multiple comparisons. P<0.05 was statistically significant.
Results
The results revealed that sperm count and percentage of sperm viability decreased, and the percentage of mature sperm and sperm with damaged DNA increased significantly in the diabetic group compared to the control group (P<0.05). Sperm count and percentage of sperm viability increased significantly in the diabetic+high AM in comparison with the diabetic group. There was no significant difference in sperm maturity among the groups received low, moderate, and high doses of AM and the diabetic group (P≥0.05). Percentage of sperms with DNA damage in the groups received low, moderate, and high doses of AM decreased significantly in comparison with the diabetic group (P<0.05)
The diabetic group showed a significant decrease in fertilization rate, percentage of two-cell embryos, percentage of blastocysts, and percentage of hatched embryos (P<0.05) and a significant increase in the percentage of arrested embryos (P<0.05) in comparison with the control group. The increase of fertilization rate, percentage of two-cell embryos, percentage of blastocysts, and percentage of hatched embryos was significant only in the diabetic+high AM group in comparison with the diabetic group (P<0.05). In the diabetic groups received AM no significant decrease was observed in the percentage of arrested embryos (P≥0.05) in comparison with the diabetic group.
Discussion
This study demonstrated the protective effect of AM on reproductive system dysfunction caused by streptozotocin-induced diabetes. Streptozotocin-induced diabetes leads to increased sperm with DNA damage and immature sperm, and decreased sperm count and sperm viability, leading to low fertility parameters such as infertility rate and embryo development. The AM hydroalcoholic extract can strongly ameliorate these parameters.
Ethical Considerations
Compliance with ethical guidelines
This study was approved by the ethics committee of Faculty of Vetarinary Medicine, Urmia University (Code: AP/1286/3).
Funding
This study was funded by Islamic Azad University of Urmia Branch.
Authors contributions
All authors contributed equally in preparing all parts of the research.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors would like to thank Ali Karimi.
Type of Study: Original Article |
Subject:
بافت شناسی و جنین شناسی
Received: 2022/10/14 | Accepted: 2022/11/29 | Published: 2023/01/1
Received: 2022/10/14 | Accepted: 2022/11/29 | Published: 2023/01/1
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