The effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa was investigated. Semen samples were collected from five healthy mithun bulls through rectal massage method and cryopreserved in liquid nitrogen. The samples were diluted in Tris–egg yolk–glycerol extender, equilibrated for 4 h at 4 °C and loaded into 0.50‐ml straws. The straws were then frozen in liquid nitrogen vapour for 10 min and finally plunged into liquid nitrogen for storage. The required amount of glycerol was added into the diluted samples either in a single dose (3%, 4%, 5%, 6% or 7%; added at 37 °C immediately before equilibration) or in split doses (5%, 6% or 7%; the total amount was divided into four equal parts, and a part was added at 37 °C immediately before equilibration, and the remaining parts were added subsequently at 1, 2 and 3 h of equilibration at 4 °C). In the single‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and proportion of live spermatozoa with intact acrosome (LSIA, %) in 5% glycerol (40.6 ± 1.7 and 43.4 ± 1.8 respectively) and lesser (p < 0.05) total morphological abnormalities (%) in 5% (14.1 ± 0.8) and 6% (13.7 ± 1.0) glycerol were observed compared to the other glycerol concentrations. In the split‐dose addition method, following freeze‐thawing, greater (p < 0.05) motility (%) and LSIA proportion (%) were found in 5% (50.2 ± 1.9 and 53.3 ± 1.8 respectively) compared to 6% or 7% glycerol, but the total morphological abnormalities were not different among the glycerol concentrations. In addition, in all the glycerol concentrations, better (p < 0.05) post‐freeze‐thaw motility and LSIA proportions were observed when glycerol was added in split doses compared to a single dose. In conclusion, Tris–egg yolk extender with 5% glycerol added in split doses was found most suitable for cryopreserving mithun sperm. 相似文献
Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen. 相似文献
Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca2+free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport. 相似文献
This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos. 相似文献
1. The effect of supplementing water-soluble vitamin E analogues 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and butylated hydroxy toluene (BHT) was studied in two separate experiments.
2. In the first experiment, trolox was supplemented at 0.2 mM, 0.4 mM and 0.8 mM concentrations along with N-methylacetamide (MA; 12% final concentration) and semen was cryopreserved in 0.5 ml French straws. Different semen parameters and fertility were assessed from post-thaw samples.
3. Sperm motility, live sperm, and mitochondrial activity were significantly lower (P < 0.05) in cryopreserved semen. Lipid peroxidation (LPO) was significantly higher (P < 0.05) in cryopreserved semen that was reduced by trolox supplementation. The treatment containing trolox at 0.2 mM concentration produced significantly higher (P < 0.05) fertility compared to unsupplemented cryopreservation treatment.
4. In the second experiment, BHT was supplemented at 0.25 mM, 0.5 mM, and 1 mM concentrations along with MA during semen cryopreservation.
5. Sperm motility, live sperm and MTT dye reduction test were significantly lower (P < 0.05) in cryopreserved semen. These parameters declined with increasing BHT concentration. Abnormal sperm was significantly higher (P < 0.05) in the BHT supplemented treatments. The sperm chromatin dispersion (SCD) test was significantly higher (P < 0.05) in cryopreserved samples and was highest in samples supplemented with 0.5 mM and 1 mM BHT. The percentage fertility was significantly lower (P < 0.05) in cryopreserved semen and BHT supplementation did not improve fertility.
6. In conclusion, trolox supplementation at 0.2 mM concentration during semen cryopreservation improved fertility, whereas BHT supplementation resulted in a decline in post-thaw semen parameters. 相似文献
Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low‐density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25‐ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post‐warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p <0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification. 相似文献
This study was conducted to evaluate the effect of nicotinic acid on plasma membrane integrity and fatty acid composition in frozen–thawed boar sperm. Boar semen was cryopreserved using freezing extender containing nicotinic acid (NA), then plasma membrane integrity, osmotic equilibration, lipid peroxidation and fatty acid were analysed. The plasma membrane integrity of frozen–thawed sperm was significantly higher in the 10 mM NA than in the 0 and 20 mM NA treatment groups (p < 0.05). Additionally, the osmotic equilibration ability was not different in treatment groups, but lipid peroxidation was significantly decreased in the 10 mM NA treatment group (p < 0.05). The saturated fatty acids were significantly decreased in the 10 mM NA treatment group, and C18:1n‐9, C18:2n‐6, C20:4n‐6, C22:5n‐6 and C22:6n‐3, and total polyunsaturated fatty acids (PUFAs) were significantly increased in the 10 mM NA treatment groups (p < 0.05). In summary, 10 mM NA improved plasma membrane integrity, inhibited lipid peroxidation and increased PUFAs in frozen–thawed boar sperm. These results suggest that NA may be useful to protect the plasma membrane and inhibit the loss of PUFAs for sperm cryopreservation in pigs. 相似文献
The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms. 相似文献