Fertility and flow cytometric evaluations of frozen-thawed rooster semen in cryopreservation medium containing low-density lipoprotein.

Frozen-thawed rooster semen is not reliable for use in artificial insemination in commercial stocks. Low-density lipoprotein (LDL) has been assessed for effectiveness as a cryoprotectant in the extender to improve the quality of frozen-thawed rooster semen. Although LDL has been evaluated in a few studies in other species for semen cryopreservation, so far no study has been conducted to examine this cryoprotectant for cryopreservation of fowl semen. Thus, this study aims to analyze the effects of different concentrations of LDL (0%, 2%, 4%, 6%, and 8%) in a Beltsville extender for cryopreservation of rooster spermatozoa. In experiment 1, motion parameters, membrane integrity, acrosome integrity, apoptosis status, and mitochondria activity were assessed after freeze-thawing. The highest quality frozen-thawed semen was selected to be used for evaluation of the fertility rate in experiment 2. Semen was collected from six roosters, twice weekly, then extended in a Beltsville extender that contained different concentrations of LDL as follows: 0% (control), 1% (Beltsville plus 1% LDL [BLDL1]), 2% (BLDL2), 4% (BLDL4), 6% (BLDL6), and 8% (BLDL8). Supplementation of the Beltsville extender with 4% LDL produced the most significant percentage of motility (43.1 ± 1.3), membrane integrity (59.4 ± 2.1),mitochondria activity (49.1 ± 1.19), and viable spermatozoa (45 ± 2.28) compared with the control treatment with the results of 22.7 ± 1.3 (motility), 38.4 ± 2.1 (membrane integrity), 40.25 ± 1.19 (mitochondrial activity), and 37.8 ± 2.28 (viability). In experiment 2, a significantly higher percentage of fertility rate was observed for frozen-thawed semen in the extender supplemented with 4% LDL (49.5 ± 1.6) compared with the control (29.2 ± 2.9). Progressive motility and acrosome integrity were not affected by LDL levels in the extenders. The results revealed that supplementation of the Beltsville extender with 4% LDL resulted in higher quality of frozen-thawed rooster sperm.

High level of intracellular sperm oxidative stress negatively influences embryo pronuclear formation after intracytoplasmic sperm injection treatment.

This study evaluates the relationship between sperm intracellular reactive oxygen species (ROS; H2 O2 , O2 ), DNA fragmentation (DF), low mitochondria membrane potential (MMP) of sperm and normal pronuclear formation among intracytoplasmic sperm injection (ICSI) patients. Semen samples were obtained from 62 infertile male who were candidates for ICSI treatment. After sperm processing, metaphase II (MII) oocytes were injected, and the mean percentages of intracellular ROS, MMP and DF were evaluated using flow cytometry. The mean percentages of pronuclear formation and zygote score (Z) were also recorded, and Pearson, Spearman's rank correlation coefficient and Kruskal-Wallis tests were applied to analyse the data. The amounts of sperm intracellular H2 O2 and O2-˙ had significant positive correlation with low MMP (P < 0.01). The intracellular ROS had a negative correlation with pronuclear formation (P < 0.05), and its effect was higher than 66.66%. In addition, the mean percentages of neither H2 O2 nor O2-˙ affected the quality of pronuclear embryos (Z-score). This study shows that although high levels of both sperm intracellular H2 O2 and O2-˙ in ICSI patients have deleterious effect on sperm MMP, only H2 O2 may interfere in pronuclear formation.

Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull.

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post-thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen-thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen-thawed spermatozoa.

Synchronizing cell cycle of goat fibroblasts by serum starvation causes apoptosis.

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5-year-old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.