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

Comparison of proliferative and multilineage differentiation potential of sheep mesenchymal stem cells derived from bone marrow, liver, and adipose tissue

Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells [MSCs] has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time [PDT], growth curves, and Colony Forming Unit [CFU] of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat [ver. 2]. The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups [p<0.001]. Comparing different serum concentrations [5, 10, 15, and 20%], irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum [p<0.001]. Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm2 density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices

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

effect of biopsy during precompacted morula stage on post vitrification development blastocyst derived bovine embryos

Improvements on embryo micromanipulation techniques led to the use of embryo biopsy in commercial embryo transfer programs for genetic analysis of preimplantation bovine embryos. The aim of this study was to evaluate the quality of bovine blastocyst derived from embryos biopsied at different precompacted morulae stages by assessment of cryosurvivability of the resulting blastocysts. The in vitro produced bovine embryos were subjected to biopsy at days 2,3, and 4 post-isemination with different cell numbers [4 to 16-cells]. Embryo cell biopsy was carried out in a 100 micro l drop of H-SOF following pronase drilling by aspiration of one blastomrere. The biopsied embryos were then cutured in SOFFaaBSA co-cultured with oviduct cells-monolayer until blastocyst formation. The blastocysts were cryopreserved at room temperature after exposure of equilibration [glycerol 1.4 M for 5 min and then glycerol 1.4 M and ethylene glycol 3.6 M for 5 min] and vitrification solutions [3.4 M glycerol and 4.6 M ethylene glycol]. The blastocysts were loaded into the center of 0.25 ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. There was no significant difference in cryosurvivability of vitrified-warmed blastocysts derived from biopsied embryos at different pre-compacted morula stages. The quality of biopsy derived blastocysts was identical to that of non-biopsy derived ones in terms of post vitrification survival and hatching rates. In conclusion there was no preference between different times of embryo biopsy at precompacted morula stages in term of cryosurvivability of biopsy derived bovine blastocysts

Effects of timing on cell biopsy from pre-compacted morula stage bovine embryos on subsequent embryonic development

Embryo biopsy has potential applications in molecular research processes in domestic animals, besides its application in sex determination in embryo transfer programs. The objective of the present study was to assess the in vitro development of bovine embryos biopsied on different days of precompacted morula stage. Slaughterhouse-derived oocytes were matured in vitro, fertilized [Day-0] by frozen-thawed, Percol-separated spermatozoa and cultured on oviductal cell monolayer. The embryos were subjected to cell biopsy on Days 2, 3, and 4 postinsemination at 4-16-cell stages. The data were analyzed using ANOVA and Chi-squared tests [SigmaStat, version 2]. A p-value < 0.05 was considered significant. Biopsies carried out at 16-cell stage [Day-4] resulted in 94% of embryos developing to the blastocyst stage, which was significantly higher [p < 0.05] than the ones biopsied at 8-cell stage on Day-4 [64%], and those undergoing the procedure on Day-3 [49% and 46% at 4-cell and 8-cell stages, respectively] and Day-2 [39% and 33% at 4-cell and 8-cell stages, respectively]. No significant differences were observed between biopsied and non-biopsied embryos on a given day. The total cell number in biopsy-derived blastocysts ranged between 103 and 135. The difference in the number of total cells, dead cells and cell allocation to trophectoderm and inner cell mass between non-biopsied and biopsy-derived blastocysts was insignificant. Biopsy of bovine embryos at 4-16-cell stages had no adverse effects on in vitro developmental potentials and the 16-cell stage embryos, biopsied on Day-4 was the best stage for blastomere removal

effect of the duration of in vitro maturation [IVM] on parthenogenetic development of ovine Oocytes

The aim of this study was to compare the effect of time of parthenogenetic activation [22 hr versus 27 hr after In Vitro Maturation-IVM] on in vitro development of ovine oocytes using either single [lonomycin 5 microM for 5 minor Ethanol 7% for 7 m/n] or combined [ionomycin and ethanol with 6-DMAP 2 mM for 3 hr] activation treatments. The abattoir-derived in vitro matured activated oocytes were cultured in modified synthetic oviductal fluid and assessed for the cleavage, blastocyst, and hatching rates. The single-activated oocytes had a reduction in cleavage, blastocyst and hatching rates compared to the combined-activated oocytes [except for the cleavage at 27 hr]. In single-treated groups the rates of cleavage and blastocyst were increased as the maturation time was extended from 22 hrto 27 hr. The numbers of total cells and Inner Cell Mass [ICM], though insignificant, were greater in combined-treated groups compared to the single treatment. The number of ICM in Eth+6-DMAP group activated at 27 hr was lower than 22 hr. Nonetheless, irrespective of the activation protocol, development to the blastocyst stage, the numbers of total cell, ICM, and cell allocation [ICM/total cells] were significantly lower in parthenogenetic than fertilized embryos. In conclusion, though the cleavage and blastocyst rates in single-treated groups were positively influenced by the extension of duration of IVM [27 hr], there was a trend of decreased numbers of total cells and ICM in slightly aged oocytes. Moreover, developmental potential of ovine parthenotes, especially in young oocytes, was improved by the addition of 6-DMAP to the activation regimen