Search

Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3 h with the liposome-DNA complexes. The samples were collected after 1 h and after 3 h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1 µg of DNA and (5) incubation with liposomes and 10 µg of DNA. The CASA results for total motility and rapid motility were statistically significant (P < 0.01) between the control and the other treatments in the Percoll group as opposed to Swim-Up. Swim-Up was therefore chosen as the optimal selection method. In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail. [ABSTRACT FROM AUTHOR]

Multiple scattering limits the contrast in optical imaging of thick specimens. Here, we present gradient light interference microscopy (GLIM) to extract three-dimensional information from both thin and thick unlabeled specimens. GLIM exploits a special case of low-coherence interferometry to extract phase information from the specimen, which in turn can be used to measure cell mass, volume, surface area, and their evolutions in time. Because it combines multiple intensity images that correspond to controlled phase shifts between two interfering waves, gradient light interference microscopy is capable of suppressing the incoherent background due to multiple scattering. GLIM can potentially become a valuable tool for in vitro fertilization, where contrast agents and fluorophores may impact the viability of the embryo. Since GLIM is implemented as an add-on module to an existing inverted microscope, we anticipate that it will be adopted rapidly by the biological community. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of exogenous progesterone supplementation on superovulatory response in buffaloes that has undergone a multiple ovulation program. Fourteen Mediterranean buffaloes were divided into two groups and received a 4-day decreasing dosage of an equal mixture of 500 IU of FSH and LH starting on day 8 of the cycle. In group A ( n = 7) a progesterone-releasing intravaginal device was removed on day 8, whereas in group B ( n = 7) it was left till day 10, when $$ {\hbox{PG}}{{\hbox{F}}_{{2}\alpha }} $$ was administered. Eighty hours later, buffaloes were artificially inseminated and after 6 days they undergone uterine flushing. A higher ( P < 0.05) number of corpora lutea (8.3 vs. 5.7) and embryo/flushing/buffalo (2.3 vs. 1.3) were recorded in group B vs. group A if responsive buffaloes are considered ( n = 12) and the number of corpora lutea was highly correlated with the number of embryos ( r = 0.65; P < 0.05). In conclusion, progesterone supplementation during the first 2 days of the superovulation treatment seems to enhance the recovery rate in buffalo species. A high ovulation rate, associated with a high number of corpora lutea, can represent a parameter for estimating embryo recovery. [ABSTRACT FROM AUTHOR]

Introduction: In the past 20+ years, several studies of bovine embryo production showed how the ratio of male to female embryos changes if embryos are made in vivo or in vitro. It is known that in in vitro systems, the sex ratio is in favor of males when there are high levels of glucose, and favors females when the principal energetic substrate is one other than glucose, like citrate.Objectives: The aim of this study was to evaluate the embryo metabolism during three important periods of in vitro development: the early development (from day 1 until day 3), the middle of culture (day 3 until day 5), and later development (day 5 until day 7).Methods: To obtain this information we evaluated the spent medium from each time period by 1H NMR.Results: Our results confirm that embryo metabolism is different between sexes. The new information obtained by identifies markers that we can use to predict the embryo sex.Conclusion: These results open a new, non-invasive method to evaluate sex of the embryos before the transfer. In the first period of embryo culture, valine concentration is good indicator (66.7% accurate), while in the last phase of culture, pyruvate depletion is the best marker (64% accurate) to evaluate the sex of the embryo. [ABSTRACT FROM AUTHOR]