تاثیر اِل -کارنیتین بر داده های حرکتی اسپرم اپیدیدیمی قوچ طی نگهداری در محیط کشت HTF با کاسا
محورهای موضوعی : مامایی و بیماریهای تولید مثل
1 - گروه علوم درمانگاهی دامپزشکی،واحد ارومیه،دانشگاه آزاد اسلامی، ارومیه،ایران
2 - تاثیر اِل -کارنیتین بر داده های حرکتی اسپرم اپیدیدیمی قوچ طی نگهداری در محیط کشت HTF با کاسا
کلید واژه: قوچ, اسپرم, اِل کارنیتین, HTF, کاسا,
چکیده مقاله :
اِل-کارنیتین در نقش اسید آمینه داخلی در پیش گیری از مرگ برنامه ریزی شده سلول بسیار موثر است. همچنین به عنوان آنتی اکسیدان، نقش حفاظتی از ساختار DNA را در برابر گونه های اکسیژن واکنشی بر عهده می گیرد. اِل کارنیتین فعالیت و میزان آنزیم های آنتی اکسیدان را نیز افزایش می دهد. در این تحقیق بلافاصله پس از ذبح اسلامی، 20 جفت بیضه قوچ بالغ از کشتارگاه صنعتی ارومیه جمع آوری شد. نمونه ها در کول باکس به آزمایشگاه دانشکده منتقل گردید. پس از تمییز و خشک کردن بیضه ها از خونابه، در نواحی بدون مویرگ روی دم اپیدیدیم برش ایجاد شد. اسپرم های محل برش به میکروتیوب های حاوی محیط کشت HTF با 10 درصد آلبومین سرم گاوی منتقل گردید. پس از تهیه رقت 30 الی 50 میلیون اسپرم در میلی لیتر محیط کشت، 5 سطح اِل کارنیتین (0- 2-4-6-8-10 میلی مول) به محیط کشت اضافه شد. سپس نمونه ها در دمای 5 درجه سلسیوس و تا 36 ساعت در یخچال نگهداری شد. در زمان های 1- 12- 24 و 36 ساعت، الگوی حرکتی اسپرم با سیستم کاسا ارزیابی گردید. نتایج آماری نشان داد اسید آمینه ِال کارنیتین در سطح 6-8-10 میلی مول و در زمان 24 و 36 ساعت، سبب افزایش واضح و معنی دار شاخص های حرکتی اسپرم اپیدیدیمی قوچ در مقایسه با شاهد گردید (p<0/05).
L-carnitine, an endogenous amino acid, is highly effective in preventing programmed cell death. As an antioxidant, it also protects DNA structure against reactive oxygen species and increases the activity and amount of antioxidant enzymes. In this study, 20 pairs of mature ram testicles were collected immediately after Islamic slaughter from the Urmia industrial slaughterhouse. The samples were transferred to the faculty laboratory in a cool box. After cleaning and drying the testes from blood, a cut was made in areas without capillaries on the tail of the epididymis. The sperm were transferred to microtubes containing HTF (Human Tubal Fluid) culture medium with 10% bovine serum albumin. After preparing a dilution of 30 to 50 million sperm per ml of HTF, five levels of L-carnitine (0, 2, 4, 6, 8, 10 mM) were added to the medium. The samples were then kept in a refrigerator at 5 degrees Celsius for up to 36 hours. At 1, 12, 24, and 36 hours, the sperm motility pattern was evaluated with CASA. Statistical results showed that L-carnitine at levels of 6, 8, and 10 mM and at 24 and 36 hours caused a clear and significant increase in the motility indices of ram epididymal sperm compared to the control group (p<0.05).
[1] Zhao J, Meng P, Jin M, Ma X, Ma H, Yang H. et al. Combined addition of L-carnitine and L-proline improves cryopreservation of dairy goat semen. Animal Reproduction Science. 2023; 257:107325. doi:10.1016/j.anireprosci.2023.107325
[2] Mohammadi V, Sharifi SD, Sharafi M, Mohammadi-Sangcheshmeh A, Shahverdi A, Alizadeh A. et al. Manipulation of fatty acid profiles in roosters’ testes, alteration in sexual hormones, improvements in testicular histology characteristics and elevation sperm quality factor by L-carnitine. Theriogenology, 2021. Theriogenology; 161: 8-15. doi.org/10.1016/j.theriogenology.2020.10.005
[3] Yang K, Wang N, Gou HT, Wang JR, Sun HH, Sun LZ. et al. Effect of L-carnitine on sperm quality during liquid storage of boar semen. Asian-Australasian Journal of Animal Sciences. 2020; 33(11): 1763-1769.
doi:10.5713/ajas.19.0455
[4] Pedreira AC, Malacarne AM, Dalmaso ACS, Carvalho KIFZ, Chagas TV, Gambetta MRI. et al. L-carnitine solution used on Rhamdia quelen thawed sperm activation boosts sperm movement, maintains larval quality, and permits to optimize the sperm use. Animal Reproduction Science, 2022. 245: 107054. doi:10.1016/j.anireprosci.2022.107054
[5] Arshadi N, Abdy K. The effects of Xylose monosaccharide on Water Buffalo (Bubalus bubalis) epididymal sperm kinetic parameters at 37 ˚ C. Journal of Basic and Clinical Veterinary Medicine. 2023; 4(1): 1-11. doi:10.30495/jbcvm.2023.1979108.1037
[6] Salinas MBS, Lertwichaikul T, Khunkaew C, Boonyayatra S, Sringarm K, Chuammitri P. et al. Freezability biomarkers in the epididymal spermatozoa of swamp buffalo. Cryobiology, 2022. 106: 39-47. doi:10.1016/j.cryobiol.2022.04.005
[7] Dziekońska A, Niedźwiecka E, Niklewska ME, Koziorowska-Gilun M, Kordan W. Viability longevity and quality of epididymal sperm stored in the liquid state of European red deer (Cervus elaphus elaphus). Animal Reproduction Science. 2020; 213: 106269. doi:10.1016/j.anireprosci.2019.106269
[8] Miró J, Morató R, Vilagran I, Taberner E, Bonet S, Yeste M. Preservation of epididymal stallion sperm in liquid and frozen states: effects of seminal plasma on sperm function and fertility. Journal of Equine Veterinary Science. 2020; 88: 102940. doi:10.1016/j.jevs.2020.102940
[9] Souza, CV, Brandão FZ, Santos JDR, Alfradique VAP, Dos Santos VMB, Morais MC. Effect of different concentrations of l-carnitine in extender for semen cryopreservation in sheep. Cryobiology. 2019; 89: 104-108. doi:10.1016/j.cryobiol.2019.05.009
[10] Ibănescu I, Leiding C, Ciornei SG, Roșca P, Sfartz I, Drugociu D. Differences in CASA output according to the chamber type when analyzing frozen-thawed bull sperm. Animal Reproduction Science. 2016; 166: 72-79.
doi:10.1016/j.anireprosci.2016.01.005
[11] Palacín I, Vicente-Fiel S, Santolaria P, Yániz JL. Standardization of CASA sperm motility assessment in the ram. Small Ruminant Research. 2013; 112(1-3): 128-135. doi:10.1016/j.smallrumres.2012.12.014
[12] Sarıözkan S, Ozdamar S, Türk G, Cantürk F, Yay A. In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage. Cryobiology. 2014; 68(3): 349-353. doi:10.1016/j.cryobiol.2014.04.006
[13] Agarwal A, Said TM. Carnitines and male infertility. Reproductive Biomedicine Online. 2004; 8(4): 376-84.
[14] Fattah A, Sharafi M, Masoudi R, Shahverdi A, Esmaeili V, Najafi A. l-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm. Cryobiology. 2017; 74:148-153. doi:10.1016/j.cryobiol.2016.10.009
[15] Abd El-baset SA, Abd El-Wahab SM, Mansour AMA, Mohamed EAH. Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men. Middle East Fertility Society Journal. 2010; 15(2): 95-105. doi:10.1016/j.mefs.2010.04.005
[16] Tatemoto H, Osokoshi N, Hirai M, Masuda Y, Konno T, Yamanaka K. Addition of l-carnitine to the freezing extender improves post-thaw sperm quality of Okinawan native Agu pig. Theriogenology. 2022; 188: 170-176. doi:10.1016/j.theriogenology.2021.12.030
[17] Zahmel J, Simonsen KS, Stagegaard J, Palma-Vera SE, Jewgenow K. Current state of in vitro embryo production in African Lion (Panthera leo). Animals. 2022; 12(11): 1424. doi:10.3390/ani12111424
Abstract
L-carnitine as an endogenous amino acid is very effective in preventing programmed cell death. Also, as an antioxidant, it protects the DNA structure against reactive oxygen species. L-carnitine also increases the activity and amount of antioxidant enzymes. In this research, immediately after Islamic slaughter, 20 pairs of mature ram testicles were collected from Urmia industrial slaughterhouse. The samples were transferred to the faculty laboratory in a cool box. After cleaning and drying the testes from blood, a cut was made in the areas without capillaries on the tail of the epididymis. The sperms were transferred to microtubes containing HTF (Human Tubal Fluid) culture medium with 10% bovine serum albumin. After preparing a dilution of 30 to 50 million sperm per ml of HTF, 5 levels of L-carnitine (0-2-4-6-8-10 mM) were added to the medium. Then the samples were kept in the refrigerator at 5 degrees Celsius for up to 36 hours. At 1, 12, 24, and 36 hours, sperm motility pattern was evaluated with CASA. Statistical results showed that the amino acid L-carnitine at the level of 6-8-10 mM and in 24 and 36 hours caused a clear and significant increase in the motility indices of ram epididymal sperm compared to the control (p<0.05).
Keywords: Ram, Sperm, L-carnitine, HTF, CASA
تاثیر اِل -کارنیتین بر داده های حرکتی اسپرم اپیدیدیمی قوچ طی نگهداری در محیط کشت HTF با کاسا
چکیده :
اِل-کارنیتین در نقش اسید آمینه داخلی در پیش گیری از مرگ برنامه ریزی شده سلول بسیار موثر است. همچنین به عنوان آنتی اکسیدان، نقش حفاظتی از ساختار DNA را در برابر گونه های اکسیژن واکنشی بر عهده می گیرد. اِل کارنیتین فعالیت و میزان آنزیم های آنتی اکسیدان را نیز افزایش می دهد. در این تحقیق بلافاصله پس از ذبح اسلامی، 20 جفت بیضه قوچ بالغ از کشتارگاه صنعتی ارومیه جمع آوری شد. نمونه ها در کول باکس به آزمایشگاه دانشکده منتقل گردید. پس از تمییز و خشک کردن بیضه ها از خونابه، در نواحی بدون مویرگ روی دم اپیدیدیم برش ایجاد شد. اسپرم های محل برش به میکروتیوب های حاوی محیط کشت HTF با 10 درصد آلبومین سرم گاوی منتقل گردید. پس از تهیه رقت 30 الی 50 میلیون اسپرم در میلی لیتر محیط کشت، 5 سطح اِل کارنیتین (0- 2-4-6-8-10 میلی مول) به محیط کشت اضافه شد. سپس نمونه ها در دمای 5 درجه سلسیوس و تا 36 ساعت در یخچال نگهداری شد. در زمان های 1- 12- 24 و 36 ساعت، الگوی حرکتی اسپرم با سیستم کاسا ارزیابی گردید. نتایج آماری نشان داد اسید آمینه ِال کارنیتین در سطح 6-8-10 میلی مول و در زمان 24 و 36 ساعت، سبب افزایش واضح و معنی دار شاخص های حرکتی اسپرم اپیدیدیمی قوچ در مقایسه با شاهد گردید (p<0.05).
کلمات کلیدی : قوچ، اسپرم، اِل کارنیتین، HTF، کاسا
1. Introduction:
The mammalian sperm membrane is rich in unsaturated fatty acid (PUFA). The composition of these fatty acids plays a significant role in sperm function. Any kind of manipulation of sperm in the environment outside the body such as lab conditions, due to the production of free radicals and reactive oxygen species (ROS) causes peroxidation of sperm membrane lipids and this oxidative damage reduces the quality and fertility of sperm cells[1]. As an endogenous amino acid, L-carnitine plays a role in mitochondrial function and oxidation of long-chain fatty acids. On the other hand, it is effective in preventing cell apoptosis. As an antioxidant compound, it is effective in preserving the DNA structure of cells against damage of free radicals and reactive oxygen species. L-carnitine can also increase the levels and activity of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase Therefore, it has a potential role in strengthening sperm resistance to oxidative damage [1]. Carnitine is a vitamin-like amino acid, it is actually a quaternary compound of ammonium and it has many functions in reproduction [2]. In the epididymal ducts, there is a high concentration of L-carnitine, which is necessary for energy balance and sperm maturation. L-carnitine increases sperm fertility indicators. L-carnitine plays a role in the metabolism of abdominal fat in geese and ducks and subcutaneous fat in pigs. Also, before the maturity of rats, detailed studies have been conducted regarding the effect of L-carnitine on the metabolism of Sertoli cells. On the other hand, there are also reports of the beneficial effect of L-carnitine after damage caused by gamma radiation[2].
Carnitine is a quaternary ammonium compound that is involved in energy metabolism in most mammals, plants and microorganisms and with the passage of fatty acids through the mitochondrial membrane, it plays a major role in the metabolism of fatty acids. In the inner and outer membrane of mitochondria, there are two transporters called palmitoyl transferase 1 and 2, which carry carnitine in its long chain form called acetyl carnitine and allows fatty acids to pass through the mitochondrial membrane to complete the beta-oxidation pathway [3].Carnitine is mainly produced in the liver and is present in high amounts in the sperm and epididymis of mammals. The epididymal epithelium and the sperms themselves obtain their energy from the epididymal fluids through carnitine. Increasing the concentration of carnitine in epididymal fluids increases sperm motility. In addition, carnitine acts as an antioxidant by transporting fatty acids into the mitochondria and producing ATP, reducing the fat available for peroxidation. On the other hand, carnitine plays a critical role in mitochondrial respiration by supporting the activity of pyruvate dehydrogenase enzyme and in various studies, the diluent containing L-carnitine has been effective in increasing the sperm quality of most species such as stallions, roosters, rabbits and bulls[3]. In the process of freezing and thawing sperm, a lot of damage is done to the structures of sperm cells, as a result, sperm motility and fertility are reduced. Also, sperm is exposed to the destructive and harmful effects of reactive oxygen species and nitrogen species, as a result, due to lipid peroxidation in the mitochondrial membrane, mitochondrial function is impaired. Free radicals or ROS cause sperm DNA fragmentation, so gene expression and embryo development do not develop. L-carnitine reduces the destructive effects of these oxidants on sperm cells by neutralizing free ferrous ions, radicals , nitrogen and competing with superoxide ions[4].
Materials and methods
Ethics statement
In this study all the samples were obtained immediately after the routine slaughter of animals according to the standards of Islamic Sharia in the industrial slaughterhouse of Urmia.
Regents
All media, chemicals and reagents used in this study were procured from Sigma-Aldrich chemical Co. Ltd (through Barnard Urmia Company (L-carnitine hydrochloride/c9500-25g/lot# 022P4058/Sigma-Aldrich. HTF (Human Tubal Fluid), EmbryoMax® HTF/MR070/Catalogue number :637428.
Sperm sampling
This study was conducted in the specialized veterinary clinic , for this purpose, 20 pairs of mature ram testicles were collected from the industrial slaughterhouse after routine slaughter. The samples were transferred to the laboratory inside the styrofoam box containing cold pack. After transferring the samples to the laboratory of the veterinary faculty of Islamic Azad University of Urmia, the testicles were first removed from the scrotum. After cutting the white covering of the testis, tunica albuginea, the tail of the epididymis was fixed between two fingers. In the testicle samples where the tail of the epididymis was prominent and relatively large, the tubules of the area were full of sperm and of course contained many blood capillaries. In order not to stain the blood with sperm during epididymal extraction, first the blood vessels were slowly extracted and the blood in the area was cleaned and dried with a tampon. Then, by cutting in the dry area without blood secretions of the tail of the epididymis, the sperm were collected and transferred to four ml microtubes containing two ml of HTF (Human Tubal Fluid) culture medium with 10% bovine serum albumin (BSA). BSA was added to the culture medium to protect the sperm membrane and prevent sperm heads from sticking together. Sperm samples from several testicles were transferred to microtubes. 950 µl of HTF with 10% BSA was transferred to each microtube, then 50 µl of the medium containing the sperm sample was a
dded so that the final dilution between 30 and 50 million sperm per ml of microtube was obtained. By using an autoclavable micropipette of 1 to 10 µl of Socorex brand (Acura manual 825, Swiss made), levels of 0-2-4-6-8 and 10 mM L-carnitine were added to one ml microtubes containing HTF and sperm cells. The microtubes were stored in a refrigerator at 5 degrees Celsius for up to 36 hours[5],[6],[7],[8],
Sperm evaluation
To assess sperm motility, at 1-12-24 and 36 hours, after storage of ram epididymal sperm at 5 degrees Celsius, 5 microliters of the sample from each micro tube was placed on a Makler chamber of CASA (HFTCASA Computer Aided Sperm Analysis, system Version 7.00, Hooshmand Fanavar Co. Limited) at 36 degrees Celsius. And after two minutes, the motility pattern of at least 1000 sperms was evaluated with CASA hardware and software [4],[9],[10].
Statistical analysis
In this research, after repeating the experiment three times, Statistical package for social sciences (SPSS version 26) software was used for statistical analysis. After the data were examined for the significance of the difference in variances, to perform multi-domain tests or after the experiment, when assuming the equality of the variances of the groups, Tukey's test and when assuming the inequality of the variances of the groups were used Tamaneh test was used at 95% confidence level (Statistical significance was set at a P value ≤ 0.05). The results were recorded as mean ± standard error of the mean in the tables 1-7.
3. Results
Table (1): Effect of different L-Carnitine levels on Mean±SEM rapid progressive motility (Class A, %) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 5.31±0.32b | 11.87±0.33b | 48.14±1.84 | 63.78±1.64 | L-Carnitine 0 |
| 1.17±0.19c | 16.58±3ab | 48.49±4.43 | 65.27±2.6 | L-Carnitine 2 |
| 3.02±0.46bc | 13.19±1.36b | 52.84±1.53 | 72.8±4.9 | L-Carnitine 4 |
| 12.25±1.37a | 20.91±2.55a | 48.11±3.39 | 69.11±1.4 | L-Carnitine 6 |
| 5.64±0.53b | 19.6±3.16a | 56.39±1.2 | 68.93±2.95 | L-Carnitine 8 |
| 6.8±1.75b | 16.01±2.3ab | 58.57±2 | 66.77±3.59 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05)
The results of the effect of l-carnitine on the epididymal sperm of ram with CASA showed that the average percentage of rapid-progressive motility in terms of numerical values and at 1 and 12 hours after storage in liquid form, was higher than the control at all levels of l-carnitine. Although the difference in means was not significant. But at 24 and 36 hours after sperm storage, the difference in means was often significant (p<0.05). At 24 hours, the difference between the levels of 6-8-10 mmol L-carnitine was significant with the control, and the average values of the L-carnitine group were higher than the control. At 36 hours, the average level of 6 mmol l-carnitine was significantly higher than the control (p<0.05),(Tab-1).
Table (2): Effect of different L-Carnitine levels on Mean±SEM motile sperm (Class A+B+C, %) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 17.98±0.86cd | 30.92±1C | 77.16±1.08 | 82.84±1.29 | L-Carnitine 0 |
| 14.79±1.12d | 42.95±4.31bc | 76.45±4.29 | 86.10±3.52 | L-Carnitine 2 |
| 23.04±4.89bcd | 41.14±2.16bc | 84.7±0.08 | 93.47±1.51 | L-Carnitine 4 |
| 36.55±0.32a | 53.17±6.55ab | 79.24±4.52 | 92.61±0.71 | L-Carnitine 6 |
| 27.88±1.92abc | 59.59±5.59a | 85.82±1.54 | 88.77±2.51 | L-Carnitine 8 |
| 31.32±7.5ab | 55.30±4.88ab | 85.3±1.71 | 91.8±2.29 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05)
The results of the effect of L-carnitine on the variable of motile sperms with CASA showed that the mean percentage of this data was higher than the control at 1 and 12 hours after storage in most of the treatment levels, but the difference between the means was not significant. However, at 24 hours, all treatment levels were higher than the control and the difference with the control was significant at the levels of 6-8 and10 mmol (p<0.05). At 36 hours, the difference between the treatment and the control was significant only at the level of 6 and 10 mmol, and the mean of L-carnitine was higher than the control (p<0.05), (Tab-2).
Table (3): Effect of different L-Carnitine levels on Mean±SEM curvilinear velocity VCL (µm s-1) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 8.91±0.51cd | 17.48±0.52d | 73.15±2.7 | 94.9±2.38 | L-Carnitine 0 |
| 6.39±0.48d | 27.68±5.54bc | 67.82±5.37 | 97.33±6.97 | L-Carnitine 2 |
| 10.42±2.31bcd | 25.04±1.3cd | 77.64±2.24 | 109.44±5 | L-Carnitine 4 |
| 21.70±0.71a | 35.72±4.61ab | 69.90±6.28 | 99.30±0.56 | L-Carnitine 6 |
| 14.14±1.01bc | 37.54±4.1a | 82.72±3.6 | 94.90±3.61 | L-Carnitine 8 |
| 15.85±3.13ab | 31.63±2.12abc | 80.88±2 | 105.17±7 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05)
In connection with the effect of L-carnitine on the curve line velocity of ram epididymal sperm, the average numerical values of this data, in terms of micrometers per second, at 1 hour, in all levels of L-carnitine were higher than the control, but the difference between the means was not significant. At 12 hours, all L-carnitine levels, except for 2 mM, had a numerical average higher than the control, but again the differences were not significant. At 24 hours, all L-carnitine levels were higher than the control, and the differences were significant at 2-6-8 and 10 mM lEvels (p<0.05). At 36 hours, the means of all L-carnitine levels was higher than the control, and the difference between the means at the levels of 6 and 10 mM with the control was significant (p<0.05), (Tab-3).
Table (4): Effect of different L-Carnitine levels on Mean±SEM straight-line velocity VSL (µm s-1) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 3.92±0.27bc | 9.28±0.3c | 50.81±2.57 | 73.59±1.98 | L-Carnitine 0 |
| 1.61±0.16c | 14.57±4.2abc | 45.93±4.66 | 72±5.8 | L-Carnitine 2 |
| 3.04±0.54bc | 11.05±0.58bc | 53.11±2.19 | 83.44±7.16 | L-Carnitine 4 |
| 10.09±0.97a | 17.64±2.8a | 47.12±3.86 | 72.68±3.05 | L-Carnitine 6 |
| 5.24±0.54b | 16.85±2.08ab | 58.79±2.61 | 72.49±3.28 | L-Carnitine 8 |
| 5.69±1.21b | 12.82±1.2abc | 57.23±1.24 | 75.5±5.07 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05).
Regarding the straight-line velocity data, the value of this index at 24 hours was higher than the control at all levels of L-carnitine, and the difference between the averages at the level of 6 and 8 mmol with the control was significant. At 36 hours, the means of 6 mM L-carnitine was significantly higher than the control (p<0.05), (Tab-4).
Table (5): Effect of different L-Carnitine levels on Mean±SEM average path velocity VAP (µm s-1) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 5.65±0.34bc | 12.10±0.34c | 58.36±2.56 | 80.12±2.07 | L-Carnitine 0 |
| 3.24±0.3c | 19.4±4.71ab | 53.84±5.1 | 79.61±5.82 | L-Carnitine 2 |
| 5.54±1.11bc | 15.93±0.48bc | 61.54±2.3 | 91.77±6.77 | L-Carnitine 4 |
| 14.19±1.03a | 23.71±3.58a | 55.16±4.31 | 81.44±2.33 | L-Carnitine 6 |
| 8.01±0.76b | 23.26±2.69a | 66.35±2.48 | 80.10±3.37 | L-Carnitine 8 |
| 8.82±1.88b | 18.55±1.59abc | 65.41±1.64 | 84.36±5.35 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05).
The last speed index evaluated by CASA is the average path velocity of the sperm. The results showed that up to 24 hours, all L-carnitine levels were faster than the control, and the difference between the means and the control was significant at the levels of 2-6 and 8 mM L-carnitine (p<0.05). At 36 hours, the difference between the means was significant only at the level of 6 mmol L-carnitine with the control (p<0.05), (Tab-5).
Table (6): Effect of different L-Carnitine levels on Mean±SEM amplitude lateral head displacement (ALH,µm) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 0.49±0.03cd | 0.85±0.26d | 2.62±0.56 | 2.95±0.63 | L-Carnitine 0 |
| D0.37±0.27d | 1.28±0.16bc | 2.58±0.16 | 3.23±0.18 | L-Carnitine 2 |
| 0.63±0.15bcd | 1.25±0.7cd | 2.91±0.63 | 3.42±0.35 | L-Carnitine 4 |
| 1.07±0.003a | 1.07±0.22ab | 2.69±0.24 | 3.38±0.12 | L-Carnitine 6 |
| 0.78±0.05abc | 1.87±0.18a | 2.97±1 | 3.13±0.11 | L-Carnitine 8 |
| 0.89±0.19ab | 1.69±0.13ab | 2.94±0.47 | 3.53±0.22 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05).
Regarding the amplitude lateral head variable, the amount of this data at 24 hours at the levels of 2-6-8 and 10 mM L-carnitine was higher than the control (p<0.05). At the 36th hour, the value of this parameter at the levels of 6 and 10 mM L-carnitine was higher than the control and the difference between the means was significant (p<0.05), (Tab-6).
Table (7): Effect of different L-Carnitine levels on Mean±SEM epididymal sperm linearity (LIN, %) | |||||
Treatment Time (hrs) | |||||
| 36 | 24 | 12 | 1 |
|
| 6.78±0.4bc | 13.46±0.48c | 45.23±1.42 | 57.92±1.25 | L-Carnitine 0 |
| 3.47±0.23c | 18.28±3.22abc | 44.64±3.76 | 57.96±3.07 | L-Carnitine 2 |
| 5.73±1.17bc | 15.24±0.78bc | 49.66±1.1 | 65.14±4.29 | L-Carnitine 4 |
| 13.51±0.78a | 21.62±3.18ab | 45.59±2.68 | 61.48±1.95 | L-Carnitine 6 |
| 8.37±0.74abc | 22.4±2.85a | 53.07±1.3 | 61.71±2.59 | L-Carnitine 8 |
| 10.41±2.84ab | 19.39±1.5abc | 52.91±1 | 59.86±2.47 | L-Carnitine 10 |
In each column values with different superscripts are significantly different (p < 0.05).
The results of the effect of different levels of L-carnitine on the linearity index of the sperm motility path showed that no significant difference was observed between the averages until the first 12 hours. At 24 and 36 hours, this data was significantly higher than the control only at the level of 6 mM L-carnitine.
4. Discussion
Motility is one of the important indicators in the evaluation of sperm quality and it provides important information about the energy status of mammalian sperm. The evaluation of sperm motility is done either visually, which results in a lot of differences and variations, or the evaluation is done by a computer, that is, by CASA (Computer Aided Sperm Analysis), which provides an accurate evaluation of sperm motility and various sperm motility parameters [11]. In the current research work to investigate l-carnitine on the motility patterns of ram epididymal sperm, CASA was used. Anyway, in the present study, L-carnitine had a significant and useful effect on the movement kinetics recorded by CASA, and this effect was dose-dependent.
L-carnitine is a type of amino acid with a vitamin-like structure and the ability to dissolve in water, which naturally exists in microorganisms, plants and animals. Its amount is very diverse in different animals according to species, type of tissue and nutritional status. L-carnitine is an important and necessary amino acid and is involved as the main cofactor in the metabolism of fats, and by facilitating the transfer of fatty acids into the mitochondria, it plays an important role in facilitating energy metabolism. L-carnitine is present in high dilutions in mammalian epididymis and sperm. Epididymal tissue and sperm supply their energy from L-carnitine in epididymal fluid[12].
Sariozkan et al.[12] have reported that L-carnitine at 12 and 24 hours, and in dose of 0.5, 1 and 2 mM caused a significant increase in rabbit sperm motility during liquid storage. In our study, the effect of L-carnitine in increasing sperm motility indices at 24 and 36 hours and especially at the level of 6 and 8 mmol was significantly higher than the control. Epididymal carnitine is significantly consumed by epididymal sperm and actually increases mitochondrial oxygen consumption in rat, rabbit, and bull sperm. Therefore, it is believed that carnitine plays a role in maintaining energy metabolism and increasing sperm quality. In addition, the start of sperm motility increases in parallel with the increase in carnitine concentration in the epididymal ducts[12]. The findings of the researchers indicate a relationship between carnitine and sperm motility, the sperms that are removed from the epididymis are not motile because they have not yet been affected by carnitine, and the addition of L-carnitine increases their motility[13]. In the study of Agarwal et al.,[14] in a clinical trial, the concentration of sperm and the percentage of linearity and progressive motility of sperm in men who received L-carnitine were significantly higher than the control. Of course, this is a confirmation of the results of our research. L-carnitine protects the genetic structure of the cell by stabilizing the mitochondrial membrane, and because it reduces the amount of fat available, the amount of fat peroxidation also decreases. On the other hand, L-carnitine plays a role in the activation of antioxidant enzymes such as superoxide dismutase and sperm glutathione peroxidase. These enzymes play an important role in cleaning ROS in chilled sperm. Beneficial effects of L-carnitine on sperm motility of human, pig, stallion, quail, bull, rainbow, trout and rooster have been determined [15]. In our research, the stimulating effect of carnitine in increasing the motility indices of ram epididymal sperm was determined.
In medicine, L-carnitine is also involved in improving the quality and function of human sperm and causes an increase in the decarboxylation of sperm mitochondria, and by improving the movement structures of sperm, it plays a role in the treatment of infertility cases such as oligoasthenozoospermia (decreased sperm motility) [16]. L-carnitine reduces the harmful effects of cold shock during the cooling and freezing stages of pig sperm and, as a result, increases sperm motility kinetics after freezing and thawing [17]. In Heidari et al.'s study on the effect of l-carnitine on goat sperm freezing, the results showed that the addition of l-carnitine in the diluent increases the overall motility and progressive motility of goat sperm, and between doses of 1, 5 and 10 mM, the best result in the dose 5 mM was obtained[18].
Contrary to the research conducted in different species of mammals, which indicates the beneficial effects of L-carnitine on quality parameters and especially sperm motility, In Souza et al.'s [9] study on the effect of L-carnitine in two types of Tris-egg yolk extender and commercial IMV extender (optiXcell) on ram sperm motility parameters was done. They stated that although L-carnitine improves some sperm motility parameters such as VAP, LIN, VSL, WOB and STR, especially in the optiIxcell commercial extender, and although L-carnitine improves sperm motility in humans and rabbits, the effect of L-carnitine is probably different, depending on the dose and species of mammal, and more studies and further investigation are needed in this field. Contrary to Suzy's study[9] and in agreement with the researches that have been mentioned above, in our research, although the study was carried out in HTF culture medium and in liquid storage mode at 5 degrees Celsius, but in conclusion, L-carnitine in dilutions of 6-8 and 10 mmol and especially at 24 hours after storage is effective on the motility indexes of ram epididymal sperm in HTF culture medium, and its use is recommended to increase the quality of ram epididymal sperm. Of course, it is better to study the effect of L-carnitine on the motility parameters of ram epididymal sperm in other types of culture media and types of diluents with or without the presence of egg yolk or quail and ostrich egg yolk in the state of sperm storage in liquid form or after freeze and thawing.
Conflicts of interest
The authors have no conflict to report.
References
1. Zhao, J., et al., Combined addition of L-carnitine and L-proline improves cryopreservation of dairy goat semen. Animal Reproduction Science, 2023. 257: p. 107325.DOI: https://doi.org/10.1016/j.anireprosci.2023.107325.
2. Mohammadi, V., et al., Manipulation of fatty acid profiles in roosters’ testes, alteration in sexual hormones, improvements in testicular histology characteristics and elevation sperm quality factor by L-carnitine. Theriogenology, 2021. 161: p. 8-15.DOI: https://doi.org/10.1016/j.theriogenology.2020.10.005.
3. Yang, K., et al., Effect of L-carnitine on sperm quality during liquid storage of boar semen. Asian-Australas J Anim Sci, 2020. 33(11): p. 1763-1769.DOI: 10.5713/ajas.19.0455.
4. de Oliveira Pedreira, A.C., et al., L-carnitine solution used on Rhamdia quelen thawed sperm activation boosts sperm movement, maintains larval quality, and permits to optimize the sperm use. Animal Reproduction Science, 2022. 245: p. 107054.DOI: https://doi.org/10.1016/j.anireprosci.2022.107054.
5. Arshadi, N. and K. Abdy, The effects of Xylose monosaccharide on Water Buffalo (Bubalus bubalis) epididymal sperm kinetic parameters at 37 ˚ C. Journal of Basic and Clinical Veterinary Medicine, 2023. 4(1): p. 1-11.DOI: 10.30495/jbcvm.2023.1979108.1037.
6. Segundo Salinas, M.B., et al., Freezability biomarkers in the epididymal spermatozoa of swamp buffalo. Cryobiology, 2022. 106: p. 39-47.DOI: https://doi.org/10.1016/j.cryobiol.2022.04.005.
7. Dziekońska, A., et al., Viability longevity and quality of epididymal sperm stored in the liquid state of European red deer (Cervus elaphus elaphus). Animal Reproduction Science, 2020. 213: p. 106269.DOI: https://doi.org/10.1016/j.anireprosci.2019.106269.
8. Miró, J., et al., Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility. Journal of Equine Veterinary Science, 2020. 88: p. 102940.DOI: https://doi.org/10.1016/j.jevs.2020.102940.
9. Souza, C.V.d., et al., Effect of different concentrations of l-carnitine in extender for semen cryopreservation in sheep. Cryobiology, 2019. 89: p. 104-108.DOI: https://doi.org/10.1016/j.cryobiol.2019.05.009.
10. Ibănescu, I., et al., Differences in CASA output according to the chamber type when analyzing frozen-thawed bull sperm. Animal Reproduction Science, 2016. 166: p. 72-79.DOI: http://dx.doi.org/10.1016/j.anireprosci.2016.01.005.
11. Palacín, I., et al., Standardization of CASA sperm motility assessment in the ram. Small Ruminant Research, 2013. 112(1–3): p. 128-135.DOI: http://dx.doi.org/10.1016/j.smallrumres.2012.12.014.
12. Sarıözkan, S., et al., In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage. Cryobiology, 2014. 68(3): p. 349-353.DOI: http://dx.doi.org/10.1016/j.cryobiol.2014.04.006.
13. Agarwal, A. and T.M. Said, Carnitines and male infertility. Reprod Biomed Online, 2004. 8(4): p. 376-84.
14. Agarwal, A. and T.M. Said, Carnitines and male infertility. Reproductive biomedicine online, 2004. 8(4): p. 376-384.
15. Fattah, A., et al., l-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm. Cryobiology, 2017. 74: p. 148-153.DOI: http://dx.doi.org/10.1016/j.cryobiol.2016.10.009.
16. Abd El-baset, S.A., et al., Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men. Middle East Fertility Society Journal, 2010. 15(2): p. 95-105.DOI: 10.1016/j.mefs.2010.04.005.
17. Tatemoto, H., et al., Addition of l-carnitine to the freezing extender improves post-thaw sperm quality of Okinawan native Agu pig. Theriogenology, 2022. 188: p. 170-176.DOI: https://doi.org/10.1016/j.theriogenology.2021.12.030.
18. Zahmel, J., et al., Current State of In Vitro Embryo Production in African Lion (Panthera leo). Animals, 2022. 12(11): p. 1424.
