ABSTRACT
Production of high quality embryos in vitro needs an efficient in vitro fertilization (IVF). Seminal origin is one of the important factors that affects the success of in vitro embryo production. So our goal was to determine the effect of using fresh and frozen semen in fertilization on developmental competence and cryo-survival of buffalo embryos. Buffalo oocytes were matured and fertilized in vitro by fresh and frozen semen. After embryos evaluation, good quality morula and blastocysts were vitrified using 0.25 ml straws and the post-warmed viability was assessed by further culture for 24 h. There was no significant difference in cleavage rate between embryos derived from fresh and frozen semen, whereas the rate of embryo development to the morula (P<0.05) and blastocysts (P<0.01) stages was significantly decreased in embryos derived from frozen compared to fresh semen. After warming the vitrified embryos, there was no significant difference between embryos derived from fresh and frozen semen in the percentages of morphologically viable embryos. However, 24 h after culture, the rate of morphologically normal and survived embryos was increased (P<0.05) in embryos derived from fresh compared to the frozen semen. In conclusion, in buffalo, the use of fresh semen could improve the rate of embryo development and their crytolerance compared to the frozen semen.
ABSTRACT
The aims of the present study were to assess the effects of cysteamine as an anti-oxidant on the rate of in vitro maturation (IVM) of buffalo oocytes (experiment 1), and their viability and nuclear status following vitrification (experiment 2). Immature oocytes with compact cumulus cells obtained from the ovaries of slaughtered animals were harvested and then cultured in the maturation medium with no cysteamine (control) or 50 µM cysteamine (treated). Oocytes were vitrified in vitrification solution 1 (VS1): 1.5 M ethylene glycol (EG) + 1.5 M dimethyl sulfoxide (DMSO) for 45 s (step one). After this initial exposure, oocytes were transferred to VS2: 3 M EG + 3 M DMSO in a holding medium for 25 s (step two). After warming, oocytes were evaluated morphologically and then cultured for a further 2 h in the cysteamine-supplemented or control maturation media. The oocytes were evaluated morphologically, stained with trypan blue for viability evaluation. The maturation rate of oocytes was higher (P<0.05) for IVM media with cysteamine compared with controls. There was no significant difference in morphology, survivability and maturation rate between the two vitrification groups (cysteamine-treated and untreated groups) but the morphology, survivability and percentages of metaphase-II oocytes in both groups of vitrified oocytes were lower compared with their respective controls. In conclusion, the addition of cysteamine to the maturation medium improved nuclear maturation of buffalo oocytes but had no positive effect on their cryoresistance during vitrification.
ABSTRACT
The present study aimed to compare the in vitro fertilizing capacity of frozen-thawed ejaculated and epididymal spermatozoa in order to standardize the semen preparation protocol for camel in vitro fertilization (IVF). Semen samples were collected from 7 Dromedary camels by means of artificial vagina (AV). Ten cauda epididymes were obtained from slaughtered adult camels, isolated, incised and rinsed for obtaining the sperm rich fluid. Ejaculated and epididymal spermatozoa were processed for cryopreservation. Fresh and frozen-thawed ejaculated and epididymal spermatozoa were evaluated for motility, livability, membrane and acrosomal integrities. Frozen-thawed ejaculated and epididymal spermatozoa were used to fertilize camel mature oocytes in vitro. The results showed that, the progressive motility of freshly collected epididymal spermatozoa was significantly (P<0.05) higher than ejaculated spermatozoa (49.25 ± 1.75 vs. 38.50 ± 1.50%, respectively). The viability index of epididymal spermatozoa was significantly (P<0.05) higher than that of ejaculated spermatozoa (96.63 ± 2.45 vs. 84.00 ± 4.08, respectively). The post-thaw acrosome and membrane integrities of epididymal spermatozoa were significantly (P<0.05) higher than those of ejaculated spermatozoa. Morula and blastocyst rates of camel oocytes in vitro fertilized by frozen-thawed epididymal spermatozoa (59.4 ± 0.8, 19.12 ± 0.7 and 10.29 ± 0.7%, respectively) were significantly (P<0.05) higher than those fertilized by frozen-thawed ejaculated spermatozoa (48.27 ± 3.1, 11.63 ± 1.1 and 5.43 ± 0.8%, respectively). In conclusion, the Dromedary camel frozen epididymal spermatozoa have the capacity to endure cryopreservation, fertilize oocytes and produce embryos in vitro better than ejaculated sperm.
ABSTRACT
This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination.
ABSTRACT
Cryopreservation and sexing of embryos are integrated into commercial embryo transfer technologies. To improve the effectiveness of vitrification of in vitro produced buffalo embryos, two experiments were conducted. The first evaluated the effect of exposure time (2 and 3 min) and developmental stage (morula and blastocysts) on the viability and development of vitrified buffalo embryos. Morphologically normal embryos and survival rates (re-expansion) significantly increased when vitrified morulae were exposed for 2 min compared to 3 min (P<0.001). On the other hand, morphologically normal and survival rates of blastocysts significantly increased when exposed for 3 min compared to 2 min (P<0.001). However, there were no significant differences between the two developmental stages (morulae and blastocystes) in the percentages of morphologically normal embryos and re-expansion rates after a 24 h culture. The second experiment aimed to evaluate the effect of viability on the sex ratio of buffalo embryos after vitrification and whether male and female embryos survived vitrification differently. A total number of 61 blastocysts were vitrified for 3 min with the same cryoprotectant as experiment 1. Higher percentages of males were recorded for live as compared to dead embryos; however, this difference was not significant. In conclusion, the post-thaw survival and development of in vitro produced morulae and blastocysts were found to be affected by exposure time rather than developmental stage. Survivability had no significant effect on the sex ratio of vitrified blastocysts; nevertheless, the number of surviving males was higher than dead male embryos.
