Flow cytometry study of post-thawed buck spermatozoa: Mito-TEMPO improves cryopreservation performance by controlling apoptosis rate, DNA fragmentation and ROS production.

Sperm cryopreservation is used to spread qualified semen for artificial insemination, but the freezing process reduces sperm quality. This study assessed the efficacy of Mito-TEMPO on post-thawed goat sperm quality. Semen samples divided to five equal groups and after dilution, received different doses of Mito-TEMPO (0, 1, 10, 100 and 1000 µM), and cryopreserved in liquid nitrogen. After thawing, flow cytometry analysis was performed to evaluate sperm mitochondria membrane potential, viability, apoptotic-like changes, DNA fragmentation and ROS concentration. According to the results, Mito-TEMPO (10 and 100 µM) improved (P ≤ 0.05) sperm viability and decreased (P ≤ 0.05) apoptotic-like changes and ROS concentration compared to the other groups. Mitochondria membrane potential was higher (P ≤ 0.05) in groups received 1, 10 and 100 µM Mito-TEMPO. The lowest (P ≤ 0.05) DNA fragmentation was observed in group received 10 µM Mito-TEMPO. In conclusion, mitochondria-targeted antioxidant Mito-TEMPO could be an efficient cryo-additive to enhance flowcytometric quality parameters of post-thawed buck semen.

Flow cytometry study of post-thawed bulk spermatozoa: Mito-TEMPO improves cryopreservation performance by controlling apoptosis rate, DNA fragmentation and ROS production.

Sperm cryopreservation is used to spread qualified semen for artificial insemination, but the freezing process reduces sperm quality. This study assessed the efficacy of Mito-TEMPO on post-thawed goat sperm quality. Semen samples divided to five equal groups and after dilution, received different doses of Mito-TEMPO (0, 1, 10, 100 and 1000 µM), and cryopreserved in liquid nitrogen. After thawing, flow cytometry analysis was performed to evaluate sperm mitochondria membrane potential, viability, apoptotic-like changes, DNA fragmentation and ROS concentration. According to the results, Mito-TEMPO (10 and 100 µM) improved (P ≤ 0.05) sperm viability and decreased (P ≤ 0.05) apoptotic-like changes and ROS concentration compared to the other groups. Mitochondria membrane potential was higher (P ≤ 0.05) in groups received 1, 10 and 100 µM Mito-TEMPO. The lowest (P ≤ 0.05) DNA fragmentation was observed in group received 10 µM Mito-TEMPO. In conclusion, mitochondria-targeted antioxidant Mito-TEMPO could be an efficient cryo-additive to enhance flowcytometric quality parameters of post-thawed bulk semen.

Supplementation of chilling storage medium with glutathione protects rooster sperm quality.

This study was aimed to evaluate the effect of addition of reduced glutathione (GSH) to the extender on the rooster's semen quality parameters and fertility potential. Semen samples were diluted with Lake extender contained 0, 0.5, 1, 2, 4 and 8 mM GSH. Then, were chilled to 5 °C and stored for a period of 48 h. Sperm motion characteristics, viability, membrane integrity, lipid peroxidation, mitochondrial activity and fertility potential were evaluated. At the initiation of the experiment (0 h), GSH did not affect sperm parameters, while 2-4 mM GSH improved (P ≤ 0.05) quality indicators during storage periods. Moreover, the samples treated with 2-4 mM GSH have had a lower lipid peroxidation compared to other groups (P ≤ 0.05). Artificial insemination using the semen samples, which had been stored in groups treated with 2-4 mM GSH for a period of 24 h, led to greater (P ≤ 0.05) fertilizing potential compared to the control group.

Cysteamine enhances quality and fertility potential of rooster semen in cooled storage.

This study investigated the effects of supplementing Lake extender with cysteamine (CYS) on rooster semen quality in cold storage and it's fertility performance. Semen samples were diluted with Lake extender supplemented with different concentrations of CYS (0, 1, 2, 4 and 8 mM) and were cooled and stored at 5 °C for a period of 46 h. Motility, membrane functionality, viability, lipid peroxidation, and mitochondria membrane potential were evaluated at 0, 23 and 46 h of storage. Fertility was assessed at 23 h of storage. Although at the beginning time (0 h), parameters were not affected, 1 mM of CYS improved (P ≤ 0.05) total motility, progressive motility and mitochondria membrane potential during 23 and 46 h storage. Moreover, 1 and 2 mM CYS improved (P ≤ 0.05) membrane functionality and viability compared to other groups. Lipid peroxidation was lower (P ≤ 0.05) in samples diluted with 1 and 2 mM CYS compared to the others. Artificial insemination with 23-hrs cooled-stored semen produced the higher (P ≤ 0.05) fertility rate in groups received 1 and 2 mM CYS compared to the control group. In conclusion, addition of 1 and 2 mM CYS to the extender could be helpful to protect rooster semen against structural and functional damages of cooling storage process.

The mitochondria-targeted antioxidant Mito-TEMPO conserves rooster's cooled semen quality and fertility potential.

The PUFAs content of rooster sperm cells makes them vulnerable to the thermal shocks during chilling storage, which reduces the fertility performance of cooled sperm. Extender supplementation with antioxidants is a reasonable method to conserve sperm fertility potential during cooling storage process. The aim of this study was to determine the effect of Mito-TEMPO addition to the Lake medium on rooster sperm quality and fertility potential during cooling process. Semen samples were diluted in the Lake medium and assigned into five equal aliquots and supplemented with 0, 0.5, 5, 50 and 500 µM Mito-TEMPO. Then, the samples were cooled at 5 °C and conserved up to 50 h. Total motility, progressive motility, morphology, viability, membrane integrity, lipid peroxidation and mitochondrial activity of samples were analyzed during 0, 25 and 50 h of cooling period. Artificial insemination was also conducted using 25 h-cooled semen. No significant difference was observed among different treatments during quality evaluations at 0 h storage. Extender supplementation with 5 and 50 µM Mito-TEMPO presented greater (P ≤ 0.05) total motility, progressive motility, viability, membrane integrity and lower lipid peroxidation compared to other groups during 25 and 50 h cooling storage. Mitochondrial activity was higher (P ≤ 0.05) in groups received 5, 50 and 500 µM Mito-TEMPO than others. Fertility rate of 25 h-cooled-stored samples was higher (P ≤ 0.05) in groups containing 5 and 50 µM Mito-TEMPO compared to control group. In conclusion, addition of 5 and 50 µM Mito-TEMPO as a mitochondria-targeted antioxidant to the storage medium could be a suitable method to conserve rooster semen quality against stressful conditions of cooling storage process.

Polymorphism within the intron region of the bovine leptin gene in Iranian Sarabi cattle (Iranian Bos taurus).

The main goal of breeders is breeding superior animals. Most traits of the economic importance in farm animals are determined by polygenic loci with environmental factors. Progress in animal breeding may be improved by combining traditional performance data with molecular genetic information on quantitative loci in selection index. Candidate genes are chosen for study on the basis of known relationships between biochemical and physiological processes. Leptin is 16 KD protein that is synthesized by adipose tissue and it is involved in the regulation of feed intake, energy balance, fertility and immune functions. In cattle, the leptin gene is located on chromosome 4. It consists of 3 exons and 2 introns of which only two exons are translated into protein. Sixty six animals were genotyped for this project. A PCR was carried out between 2 exon (intron 2). A strategy employing polymerase chain reaction was used to amplify a 422 bp from blood, semen, hair root and milk DNA. Digestion of polymerase chain reaction products with Sau3AI revealed two alleles: Allele A was 390, 32 fragments and allele B was 303, 88, 32 (only 303 fragment visible on the gel). Three patterns were observed and frequencies were 0.31, 0.43, and 0.14 for AA, AB and BB respectively. This polymorphism could be further evaluated for marker assisted selection and developed PCR methodology would expedite screening for large numbers of animals required for such studies.