Effects of in vitro zinc sulphate additive to the semen extender on water buffalo [Bubalusbubalis] spermatozoa before and after freezing

The objective of the study was to investigate the effects of in vitro zinc sulphate additive to semen extender on sperm parameters [progressive motility, viability, membrane integrity and DNA stability] after cryopreservation. In this Prospective longitudinal laboratory study, semen samples of 5 buffalo bulls of 3-5 years old were collected at 5 different occasions from Iran, Urmia during summer and autumn 2011, 25 samples were used in each treatment. Sperm progressive motility, viability and abnormal morphology were measured before and at 0.5 [T[0]], 1[T[1]] and 2[T[2]] hours after diluting semen[1:10 v/v] in Tris-citric acid based extender [without egg yolk and glycerol] at 37°C containing none [control group], 0.072, 0.144, 0.288, 0.576 and 1.152 mg/L zinc sulphate to investigate dose and time effects. Next, a Tris-citric acid-egg yolk-glycerol extender [20% egg yolk and 7% glycerol] containing the same amount of zinc sulphate was prepared, diluted semen [1:10 v/v] was cooled and kept into a refrigerated chamber [4°C] for 4 hours to equilibrate. Sperm progressive motility, viability, abnormal morphology, membrane integrity and DNA damage were estimated. The equilibrated semen was loaded in 0.5 ml French straws and frozen in liquid nitrogen. Later, the frozen semen was thawed and the same parameters as well as total antioxidant capacity [TAC] of the frozen-thawed semen were determined. The results showed that zinc sulphate additive at the rate of 0.288 mg/L gave a higher protection of sperm progressive motility [53.7 +/- 1.8% vs. 40.5 +/- 1.7%], viability [70.8 +/- 1.8% vs. 60.1 +/- 1.5%], membrane integrity [67.3 +/- 1.6% vs. 56.6 +/- 1.7%], DNA stability [10.1 +/- 0.47% vs. 11.8 +/- 0.33% damaged DNA] through the process of dilution, equilibration and freeze-thawing and caused a higher TAC level [81 +/- 3.3% vs. 63 +/- 3.2 micromol/L] after freeze-thawing compared to the control group. Adding 0.576 and 1.152 mg/L zinc sulphate, however, was deleterious to the sperm and significantly reduced the studied sperm parameters. Adding 0.288 mg/L zinc sulphate to the extender, compared to the control group, gives a better sperm preservation upon freezing processes which in turn, may results in higher semen fertility. But, addition of higher zinc sulphate concentrations [0.576 and 1.152 mg/L] are detrimental to buffalo spermatozoa

Effect of leptin on in vitro nuclear maturation and apoptosis of buffalo [bubalus bubalis] oocyte

Leptin, as a 16 kDa adipokine, is a pleiotropic cytokine-like hormone that primarily secreted from adipose tissue. It also involves in the regulation of energy homeostasis, neuroendocrine function, immunity, lipid and glucose homeostasis, fatty acid oxidation, angiogenesis, puberty and reproduction. The aim of this study was to investigate the effects of in vitro addition of leptin to in vitro maturation [IVM] medium on buffalo oocyte maturation and apoptosis. In this experimental study, Ovaries from apparently normal reproductive organs of slaughtered adult buffaloes [Bubalus bubalis] with unknown breeding history were collected from Urmia Abattoir, Urmia, Iran, and were transported immediately to the laboratory in a thermos flask containing sterile normal saline with added antibiotics. Oocytes were aspirated from 2-8 mm visible follicles of the ovaries using an 18-G needle attached to a 10 ml syringe. IVM medium included tissue culture medium-199 [TCM-199], 10% fetal bovine serum [FBS], 22 microg/ml sodium pyruvate, 0.5 IU/ml ovine follicle-stimulating hormone [oFSH], 0.5 IU/ml ovine luteinizing hormone [oLH], 1 microg/ml oestradiol, 50 microg/ml gentamycin, and leptin [0 [control], 10, 50, and 100 ng/ml]. The good quality buffalo oocytes [batches of 10 oocytes] were placed in a culture plate containing six 50 microl droplets of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5?C with 5% CO2 in air for 24 hours. The maturation of oocytes was evaluated under a stereomicroscope by detecting the first polar body extrusion of oocytes. FITC-Annexin V - propidium iodide [PI] staining method was used to detect oocyte apoptosis. From a total of 115 collected ovaries, 1100 oocytes were recovered among which 283 oocyte were suitable for IVM. In the groups of leptin treated with 0 [control], 10, 50 and 100 ng/ml, the percentage of oocytes maturation was 74.65, 83.81, 77.85, and 75.40%, while the percentage of oocytes apoptosis was 9.83, 9.54, 9.93, and 10.42%, respectively. Our results showed that addition of 10 ng/ml leptin to buffalo IVM medium increased oocyte maturation, significantly, as compared with that in control group. However, addition of leptin to IVM medium had no significant influence on buffalo oocyte apoptosis. Our findings suggested that addition of 10 ng/ml leptin to IVM medium of buffalo oocyte can improve oocyte nuclear maturation. Furthermore, we showed that there is no relation between in vitro addition of leptin to buffalo oocyte IVM medium and oocyte apoptosis