Effect of tris-based diluent containing beta-carotene-enriched egg yolk on liquid storage of ram sperm

Introduction: Sperm cooling is very important to preserve the genetic capacity of livestock and animals that have a high economic value and to preserve some species of rare animals or animals at risk of extinction, and the researchers of Royan Jihad University Research Institute have implemented this plan and its results were published in the journal Journal of Equine Veterinary Science have published. The cooling of sperm causes a disturbance in its qualitative traits. The use of egg yolk prevents metabolic changes and preserves the quality of sperm (Purdy 2006). In order to reduce the damage caused by sperm freezing, cryprotectants are used in sperm diluents (Purdy 2006). Which is one of the most common components of egg yolk sperm diluents that protect sperm against cold shock (Aisen et al. 2000). Which consider lipoproteins and lecithin in egg yolk to be the most important protective factors for sperm. Semen diluents dilute the seminal plasma, control the pH and osmolarity and provide the necessary nutrients for the sperm and protect it against cold shock during the cooling and storage of the sperm. The properties of adding a diluent to the semen are increasing the length of the life period, protecting the motility and fertility, and keeping the sperm for a long time. The aim of this study was to determine the effect of using beta-carotene-enriched egg yolk on the quality of liquid sperm.
Material and methods: For this purpose, a total of 15 ejaculations from 3 rams were collected by artificial vagina. At first, sperm were collected for 3 weeks for habituation. After the habituation period of the rams, sperm samples were collected and evaluated. In the first group, a diluent containing ordinary egg yolk was used to cool the sperm, and in the second group, a diluent containing beta-carotene-enriched egg yolk was used. Sperm quality parameters were assessed on days 1, 5, 10, 15 and 20 after cooling. The samples were examined in two stages: A) examining the characteristics of fresh sperm: the characteristics include sample volume (using a special sperm collection tube with an accuracy of 0.1 ml), semen pH (using a paper pH meter from Merk, Germany), movement Wavy (by light microscope with ×100 magnification), progressive, in situ and whole sperm movements (by diluting the fresh sample at a ratio of 1:100 with 2.9% sodium citrate and examining with a light microscope with ×400 magnification), Viability and percentage of abnormal sperms (by diluting the fresh sample at a ratio of 1 to 100 with 2.9% sodium citrate and expanding with eosin-nigrosin and examining with a light microscope at 400x magnification), sperm concentration or density (dilution of the fresh sample with citrate Sodium 2.9% at a ratio of 1 to 300 + one drop of formalin 2% and examination by hemocytometer slide) and sperm morphobiometry (a drop of diluted sperm solution with sodium citrate 2.9% at a ratio of 1 to 100 is expanded with eosin-nigrosin The data were examined under a light microscope with 400× magnification in 5 different areas) and measured. B) Examining the characteristics of diluted sperm: After the cooling stage, progressive motility, in situ motility, total motility, sperm viability, sperm morphology or abnormality, and morph biometry were performed as the methods mentioned above. In addition to these traits, host tests or the health and integrity of the sperm membrane, the unhealthy acrosome membrane, morph biometry and MDA were measured in the following order. HOST (sperm membrane health and integrity): To evaluate the health and integrity of the sperm membrane, a hypoasmetic solution was used in such a way that 10 microliters of the chilled sample was diluted with 100 microliters of HOST solution and kept at 37°C for 30 minutes. They were incubated, then using a sampler, a drop of the incubated solution was placed on a slide at 37°C, and a spread was prepared with a drop of eosin-nigrosin dye, and after drying the prepared spread with a gentle blow of a hair dryer, the spread was prepared with magnification. 400x was checked and percentage in light microscope. Unhealthy acrosome membrane: To identify sperms with unhealthy acrosome membrane, put 10 microliters of a solution diluted 1:100 with 2.9% sodium citrate on a 37°C slide and add 10 microliters of eosin-nigrosin on it. Then, a spread was prepared with another slide, and after drying the prepared spread with a gentle blow of a hair dryer, 1 drop of cedar oil was placed on it, and under a light microscope with 1250x magnification, the number of sperms with an unhealthy acrosome membrane was determined and percentages were determined. MDA: To measure the amount of malondialdehyde in the beginning to precipitate proteins, 1 ml of the sample of each group was mixed with 2 ml of trichloroacetic acid in a sterile tube on the final day of the experiment, and then to prevent lipid peroxidation during the experiment, 1 ml of BHT solution along with 1 ml of EDTA were added to the desired solution and then the tubes were centrifuged for 15 minutes at ×1200 rotation. 1 ml of thiobar biothiouric acid 0.67% was mixed and placed in 95°C water for 20 minutes, and after cooling, the light intensity of the samples was measured at a wavelength of 532 nm by a spectrophotometer. Data were statistically analyzed by proc mixed procedure in SAS software.
Results and discussion: The results showed that beta-carotene treatment had a significant positive effects on progressive motility, total motility, viability, abnormal sperm, HOST, acrosome membrane integrity (P<0.05) , and were decreased significant MDA and abnormal sperm (P<0.05). According to the results, beta-carotene-enriched egg yolk increases sperm quality parameters during storage. Because one of the most important protective factors for sperm is lipoprotein and lecithin in the egg yolk, and in addition, because the egg yolk is enriched with an antioxidant called beta-carotene, which is one of the types of pro-vitamins and a precursor for vitamin A, it prevents from the production and increase of free radicals and has a double positive effect on sperm quality, and in addition to protecting sperm from cold shock, it improves and preserves sperm fertility and also increases sperm quality characteristics (Shafiipour Fard et al. 2017). Antioxidants are expected to affect sperm production and quality by quenching excess reactive oxygen species (ROS) produced by rapidly dividing spermatogonia in the testis (Tomar et al. 2017). Accumulation of ROS causes oxidative stress and reduces the body's ability to detoxify or repair the resulting damage (Agarwal et al. 2004). Antioxidant beta-carotene neutralizes ROS and reduces oxidative stress. As more time passed, the quality of the samples decreased. For successful insemination in the cervix, 150 million live and motile sperm are required, and for successful insemination by laparoscopic method, 20 million live and motile sperm are required (Evans and Maxwell 1987) and according to the results obtained from this research, up to 20 days After freezing, the qualitative parameters of sperm have been checked, for successful insemination in the cervix, refrigerated samples must be used up to 10 days after refrigeration, and for successful insemination by laparoscopic method, refrigerated samples can be used up to 15 days after refrigeration.