Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

Background： Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i） how much linear DNA to inject and ii） when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein （EGFP） plasmid into buffalo zygotes. Results： First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/pL DNA was significantly higher than from those injected with 5 pg/mL. However, 50 ng/pL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/pL DNA. Second, zygotes injected early （7-8 h post-insemination [hpi]） developed better than those injected at mid （12-13 hpi） or late （18-19 hpi） time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections （16.4 % vs 7.7 %）. With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions： Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.

Preliminary Study on Transgenesis by Injecting Exogenous DNA into Zygote Cytoplasm of Buffalo

[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation. One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization （IVF）; the other group of oocytes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection （generally called ICSI-Mediated Gene Transfer, ICSI-Tr）. Expression of exogenous DNA was observed and recorded during the process of embryonic development. [Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF injection group showed no significant difference compared with that in ICSI-Tr group （P0.05）. In addition, the cleavage rate and early embryonic gene expression efficiency in IVF injection group were significantly higher with injection at 7-10 h post IVF than that at 18-20 h post IVF （P0.05）. [Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes, and the optimal injection time is 7-10 h post IVF.

Rejuvenation of Ovary and Thin Endometrium by Autologous PRP Injection in POR and Recurrent Implantation Failure

In India, the problem of infertility is growing and in the last 5 years, it has gone up to 20% - 30%. This ongoing prospective clinical study brings forth a novel, innovative, effective, simple, affordable, easily performed outpatient procedure (OP) and a promising therapeutic method in rejuvenating the Ageing Ovaries and Thin Endometrium, with autologous Platelet Rich Plasma (PRP). This clinical study proves to give a better result in rejuvenating Ovary and treating the Thin Endometrium. This pilot study included five women (28 - 44 years) with Poor Ovarian Response (POR), Premature Ovarian Insufficiency (POI) and Perimenopause and Thirty-nine women (22 - 43 years.) with recurrent implantation failure due to Thin Endometrium were subjected to autologous PRP instillation under Ultrasound Guidance, and Hysteroscopic guided PRP. After PRP, a significant output was obtained, with improved Anti Mullerian Hormone (AMH) and Antral Follicle Count (AFC) and out of five women three women conceived by Intra Cytoplasmic Sperm Injection (ICSI). PRP injected in women with Poor Ovarian Response found successful ovarian rejuvenation within 1 - 3 months and had a 60% of pregnancy rate, PRP into the endometrium had 53.8% successful pregnancies. We have not encountered any complications.

Embryo-mediated genome editing for accelerated genetic improvement of livestock

Selecting beneficial DNA variants is the main goal of animal breeding.However,this process is inherently inefficient because each animal only carries a fraction of all desirable variants.Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains.To realize rapid genetic gain,precise edits need to be introduced into genomicallyselected embryos,which minimizes the genetic lag.However,embryo-mediated precision editing by homology-directed repair(HDR)mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos.This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos.It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism,enable multiplex editing and multiply rare elite genotypes.Such a breeding strategy would enable a more targeted,accelerated approach for livestock improvement that allows stacking of beneficial variants,even including novel traits from outside the breeding population,in the most recent elite genetic background,essentially within a single generation.