effect of media supplementation with angiotensin on developmental competence of ovine embryos derived from vitrified-warmed oocytes

Background: This study was aimed to assess the effects of angiotensin II [Ang II] supplementationto the In Vitro Maturation [IVM] and In Vitro Culture [IVC] media ofvitrified-warmed ovine oocytes on their developmental competence and expression ofNa +/K +/ATPase in resulting embryos

Methods: The slaughterhouse-derived immature oocytes [n=1069] were randomly distributedinto four experimental groups: groups I and II] IVM/IVF and IVC of fresh andvitrified oocytes without angiotensin supplementation [Control-Fresh and Control-Vitgroups, respectively]; group III] IVM of vitrified oocytes in the presence of Ang II followedby IVF/IVC [Vit-IVM group]; and group IV] IVM/IVF of vitrified oocytes followedby IVC wherein the embryos were exposed to Ang II on day 4 of IVC [Vit-D4 group].The embryos were immunostained with primary antibodies against Na +/K +/ATPasealpha 1andbeta 1 subunits

Results: In Vit-IVM and Vit-D4 groups, the rates of expanded and total blastocysts onday 7 as well as the proportion of blastocysts on day 8 were increased. The expressionof Na +/K +/ATPasealpha 1 andbeta 1 subunits were positively influenced by the addition of AngII on day 4 [Vit-D4 group].Conclusion: The addition of Ang II to the IVM and IVC media could improve blastocystsformation in vitrified sheep oocytes. This improvement might be related to thegreater expression of Na +/K +/ATPasealpha 1 andbeta 1 subunits when Ang II was added duringIVC

Effect of Angiotensin on the quality of In Vitro Produced [IVP] sheep embryos and expression of Na[+] /K[+] /ATPase

Background: The presence of rennin-angiotensin components in mammalian ovaries and their involvement in ovarian physiology have been established. In the present study, effects of angiotensin II [Ang II] on sodium-potassium adenosine triphosphatase [Na[+] /K[+] /ATPase] expression and development of sheep embryos was evaluated

Methods: The abattoir-derived Cumulus Oocyte Complexes [COC] were randomly allocated into three experimental groups; group I] in vitro Maturation [IVM] of oocytes in the presence of Ang II followed by in vitro fertilization [IVF]/in vitro Culture [IVC] [IVM group], group II] IVM/IVF of oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC [D4 group], and group III] IVM/IVF and IVC of oocytes without any angiotensin [Control]. The blastocyst and hatching rates were recorded on days 6 to 8. Day 8 embryos were immunostained with primary and secondary antibodies against Na[+] /K[+] /ATPase alpha 1 and beta 1 subunits

Results: Addition of Ang II during IVM and IVC significantly increased the hatching rate of blastocysts on day 8 compared to the control. The trophectoderm and total blastocyst cells' numbers were significantly increased by addition of Ang II to the IVM and IVC media, though the expression of Na[+] /K[+] /ATPase alpha 1 and beta 1 subunits were positively influenced by the addition of Ang II on day 4 [D4 group]

Conclusion: In conclusion, it seems Ang II through positive effects on embryos, expressed as the greater hatching rate and blastocyst cell number, could increase the sheep embryo developmental rate. These improvements might be partly related to the greater expression of Na[+] /K[+] /ATPase alpha 1 and beta 1 subunits when Ang II was added during IVC

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells [SSCs]. They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture [3, 4, 5, and 6 hr] and discontinuous percoll density with different gradients [20, 28, 30, and 32%]. The difference of percentage of undifferentiated SSCs [PGP9.5 positive] in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat [ver. 3.5]. The highest PGP9.5 [94.6 +/- 0.4] and the lowest c-Kit positive [25.1 +/- 0.7] in Percoll method was significantly [p

Culture of ovine IVM/IVF zygotes in isolated mouse oviduct: effect of basal medium

The basal medium that supports Isolated Mouse Oviduct [IMO] is important for supporting embryo development and quality. The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos [p>0.05]. The hatching rate, total and trophectoderm cells number in IMO groups' blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the quality of oocytes was poor, IMO could better support ovine embryo development either with SOFaaBSA or SOFaaciBSA than SOFaaBSA alone and there was a significant difference in blastocyst formation at day 6 with SOFaaBSA alone. The culture of ovine IVM/IVF zygotes in IMO using two highly efficient ruminant embryo culture media not only could support development of ovine embryos similar to the level in non IMO culture system [SOFaaBSA alone] but also could improve the quality of resulting embryos. Additionally, IMO could better support the development of ovine embryos derived from poor quality oocytes compared to the SOFaaBSA alone

Regulations and ethical considerations in animal experiments: international laws and islamic perspectives

Growing usage of animals in the research projects has drawn more attention to their welfare and ethics surrounding this practice. Dissemination of information about the existing ethical consideration and alternatives in animal experiments has two important functions; first, it increases the researcher's awareness of the possible methods of using animals in the experiment, and second, to ensure that potential users are aware of the established alternatives. For example, legislations enacted in many countries during the 1980s state that laboratory animal applications should be reduced, refined and replaced wherever possible according to principles of the 3Rs. Thus, scientists around the world tried to apply the 3Rs in their biomedical researches regarding welfare of the laboratory animals. However, the Qur'an, the holy book of Muslims, and also Hadiths contain the obligatory ways to keep and treat animals since their revelations. According to Islamic viewpoint, animals represent Allah's ability and wisdom, and humans must pay attention to their health and living conditions. Several Islamic manuscripts state that animals have their own position in the creation hierarchy and humans are responsible for supplying minimal facilities and their welfare. This paper has tried to review ethical consideration in animal experiments and regarding Islamic resources in this case to encourage providing comprehensive ethical regulations in animal experiments which its establishment could be beneficial for animal ethics committees or research institutes

[Cloning and interfering factors]

The term "Cloning" has originated from "Klon", a Greek word with the meaning of a small twig that can multiply by itself and turn to a generative tree. Cloning is an asexual reproduction in which a copy or multiple copies of an organism are generated by transferring the nucleus [DNA] of a somatic cell into an enucleated metaphase-II oocyte. Despite the benefits and potentially broad applications of this technology, its low efficiency, especially in the production of viable offspring, has implicated its application with serious challenges. In this article, we will review papers related to its emerging principles, with an emphasis on epigenetic modifications, which appear to govern the efficiency of cloning. The literature review was carried out by searching through knowledge-based data bases such as ScienceDirect, PubMed and Scopus on the internet. No time limit was considered for literature review of the relevant articles up to the time of submission. Considering the large varieties of factors affecting cloning, improvements in cloning efficiency are dependent on the increment of theoretical knowledge and technical expertise of its procedures. This can be achieved by improving oocyte and cytoplasmic maturation, optimizing synchronization between the nucleus of the donor cell and cytoplasm of MII stage oocyte, minimizing the physical insults to the cytoskeleton of oocyte during enucleation and nuclear transfer, improving the cellular fusion and culture conditions of reconstructed oocytes and in particular and more importantly by employing effective methods to qualitatively alter the epigenetic status of the incoming nucleus to an embryonic or totipotent state, leading to the improvement of donor cell reprogramming. Considering the importance of inherited maternal transcripts and proteins in cytoplasm of fully matured oocytes in supporting the embryos up to the embryonic genomic activation [EGA] and the capability of MII stage cytoplasm in dedifferentiating mammalian somatic cells and coincident of EGA with depletion of maternally originated transcripts, reprogramming of the somatic cell nuclei must be completed by the time that the embryonic genome is activated. Since the patterns of epigenetic modification are dynamic and not static during development, the optimum procedure to properly induce nuclear reprogramming should follow the pattern of epigenetic modifications in normal embryo development. Besides the all progresses in reproductive cloning using highly efficient methods, any deviation from the normal pattern of mRNA expression due to epigenetic changes induced by chemical interventions in early preimplantation embryo may persist throughout fetal development. The effects of these aberrations may manifest later in development. Nonetheless, understanding the kinetics of normal molecular events related to epigenetic modifications and identification of the specific factors present in the ooplasm, which are necessary for epigenetic reprogramming, will provide a better understanding of the underlying mechanisms and would improve cloning efficiency and other related technologies