The Effects of Supplementation of the Freezing Extender with Silymarin on the Quality Parameters of Frozen-Thawed Arabian Stallion Sperm: A Preliminary Evaluation.

This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 µg/mL, 50 µg/mL, 75 µg/mL, and 100 µg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane integrity, lipid peroxidation, and DNA fragmentation. The findings indicated that 25-100 µg/mL of silymarin significantly improved viability and mitochondrial membrane potential while reducing the stallion sperm lipid peroxidation, DNA fragmentation, and apoptosis compared with the control group (p < 0.05). Silymarin concentrations of 75 µg/mL and 100 µg/mL significantly increased progressive motility and plasma membrane integrity (p < 0.05). Based on our findings, it can be inferred that silymarin exhibited a dose-dependent enhancement in the frozen-thawed Arabian stallion sperm quality. The most favorable outcomes were observed when 100 µg/mL silymarin was used.

Egg yolk plasma enriched with ß-carotene through the diet of laying hens and adding it to the extender improves the quality of frozen semen in Arabic stallions.

Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with ß-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, ß-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with ß-carotene. Subsequently, various variants of enriched extender (INRA-96 + 2.5% glycerol [G]) were gained by adding 2% EYP from four treatment groups. The sperm characteristics, including motility, viability, morphology, plasma membrane integrity (HOS test), lipid peroxidation (MDA) and DNA fragmentation, were evaluated after thawing. According to the results obtained in this study, the addition of EYP from T2 and T4 (500 and 2000 mg/kg of ß-carotene in hens' diet) to the extender (INRA-96 + 2.5% G) leads to an increase in total motility (50.50% and 49.49%, respectively), progressive motility (32.6% and 31.8%, respectively), viability (68.7% and 66.1%, respectively) and plasma membrane integrity (57.7% and 50.6%, respectively). Moreover, lipid peroxidation (1.3 and 1.4 nmol/mL, respectively) and DNA fragmentation (8.6% and 9.9%, respectively) were diminished using the mentioned treatments. However, sperm morphology was not affected by the treatments. In the current study, we concluded that the optimal concentration of ß-carotene in the laying hen's diet (500 mg/kg) could reveal the best results about sperm quality. So, EYP enriched with ß-carotene acts as a valuable natural and safe supplementary material that could be exploited for enhancing stallion sperm quality in cryopreservation conditions.

L-carnitine improves quality parameters and epigenetic patterns of buck's frozen-thawed semen.

Buck sperm cryopreservation is an effective method to distribute qualified sperm for reproductive purposes, but this procedure reduces sperm quality. The current study was conducted to investigate the effect of L-carnitine (LC) on the quality and epigenetic patterns of buck's post-thawed semen. Semen samples were collected from five male goats twice a week and diluted in extenders supplemented with 0 (LC-0), 1 (LC-1), 5 (LC-5) and 10 (LC-10) mM LC. Samples were cryopreserved according to standard protocol. After thawing, motility characteristics, lipid peroxidation, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, epigenetic modifications, viability, apoptotic-like changes and DNA fragmentation were assessed. Samples supplemented with 5 mM LC showed greater (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, DNA methylation, viability, and lower (P ≤ 0.05) apoptotic-like changes. Lipid peroxidation was lower (P ≤ 0.05) in LC-5 and LC-10 compared to the control group. Addition of LC to the cryopreservation extender had no effect (P > 0.05) on velocity parameters, abnormal morphology, histone modifications, or DNA fragmentation. In conclusion, supplementing the cryopreservation extender with 5 mM LC significantly preserves the quality of buck sperm after the cryopreservation process.

Isolation and Proliferation of Spermatogonial Cells from Ghezel Sheep.

BACKGROUND: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations. Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations. METHODS: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep. RESULTS: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7%, but after the freezing process the viability rates were 74 percent. CONCLUSION: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed.

Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

PURPOSE: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. METHODS: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. RESULTS: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. CONCLUSION: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells.

PURPOSE: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. METHODS: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. RESULTS: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. CONCLUSION: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells.

Follicle stimulating hormone increases spermatogonial stem cell colonization during in vitro co-culture.

The complex process of spermatogenesis is regulated by various factors. Studies on spermatogonial stem cells (SCCs) have provided very important tool to improve herd genetic and different field. 0.2 to 0.3 percent of total cells of seminiferous tubules is consist of spermatogonial stem cells. To investigate and biomanipulation of these cells, proliferation and viability rate of cells should be increased in vitro, at first. Follicle stimulating hormone (FSH) has been suggested to play a determinant role in the survival of germ cells in addition to increasing spermatogonial proliferation. In this study, the in vitro effects of FSH on spermatogonial cell colony formation were investigated. Sertoli and spermatogonial cells were isolated from 3-5 months old calves. The identity of the Sertoli cells and spermatogonial stem cells were confirmed through immunocytochemistry and colony morphology, respectively. Co-cultured Sertoli and spermatogonial cells were treated with FSH in different dose of 10, 20 and 40 IU mL(-1) FSH, before colony assay. Results indicated that, FSH increased in vitro colonization of spermatogonial cells in comparison with control group. In conclusion, using FSH provided proper bovine spermatogonial stem cell culture medium for in vitro study of these cells.

Differentiation of bovine spermatogonial stem cells into osteoblasts.

Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining.