Male pronuclear formation and embryo development following intracytoplasmic injection of ovine pretreated sperm

Background: Failure of Male Pronucleus [MPN] formation is a major concern in the success of Intracytoplasmic Sperm Injection [ICSI] in some species. Despite the conducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some compounds

Methods: In experiment 1, sperm were treated with either 2-mercaptoethanol [2ME], glutathione [GSH], or DTT [dithiothreitol] in combination with Heparin [Hep]. Following DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head decondensation was applied for the second and third experiments. In experiment 2, in vitro matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract [SE] co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm

Results: The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control

Conclusion: In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control

Evidence of oocyte polarity in bovine; implications for intracytoplasmic sperm injection and somatic cell nuclear transfer

Objective: We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine

Materials and Methods: In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to [HNS] and far-from [FS] spindle] or trisection [into MII-spindle [S], the spindle-side half [NS], and the distal half unassociated with the spindle [FS]]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction [RT-qPCR]. To map the possible preferential sperm entry point [SEP], the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization

Results: The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S [Tead4, Nanog, Ctnb and Sox2], NS [Oct4], or FS [Gata6]. The SEP in almost [90%] fertilized oocytes was located in MII-hemisphere

Conclusion: The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection [where a sperm is injected far from the MII-spindle] and somatic cell nuclear transfer [where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation]

Reconstruction of mammalian oocytes by germinal vesicle transfer: a systematic review

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future

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

Comparison of three methods for mitochondria isolation from the human liver cell line [HepG2]

Aim: The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application

Background: Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders

Patients and methods: Mitochondrial isolation from the human liver cell line [HepG2] was performed using two commercially available kits, including Qproteome [Qiagen] and MITOISO2 [Sigma-Aldrich], as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods

Results: Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods

Conclusion: Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria

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

Male pronuclear formation using dog sperm derived from ectopic testicular xenografts, testis, and epididymis

Testis tissue xenografting and the resultant sperm in a xenograft may provide a unique approach to rescue the genetic material of males that die prematurely and is a model for the study of human spermatogenesis and can represent an alternative approach for fertility preservation in cancer patients. This study was aimed to evaluate the xenogenic dog sperm in formation of male pronucleus following injection into the sheep oocytes. The in vitro matured slaughterhouse derived sheep oocytes were subjected to Intracytoplasmic Sperm Injection [ICSI] with epididymal, testicular, and xenogenic dog sperm. The ICSI was performed after scoring of the sperm midpiece using an IX71-Olympus inverted microscope with Nomarsky optics. Within 1 hr after injection, the injected oocytes in activated group were exposed to 5 microM ionomycin for 5 min. The data were analyzed by Chi-square and ANOVA using Sigma Stat, version 3.5, and p<0.05 was considered significant. The formation of female pronuCIeus after ICSI of xenogenic sperm was higher than epididymal and testicular sperm in non-activated oocytes. The corresponding rate in activated oocytes was higher or comparable with testicular and epididymal sperm. The rate of male pronucleus formation after ICSI of xenogenic sperm was comparable with injection of two other sperm sources. Oocyte activation had an inductive role in female and male pronuclear formation. Dog xenogenic sperm was capable to induce oocyte activation and proportion of male pronucleous formation was comparable to the testicular and epididymal sperm

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

in vitro fertilization of ovine oocytes in the presence of oviductal cells and its effect on the expression of zygote arrest 1 [Zar1] and subsequent embryonic development

The cells of mammalian female reproductive tract have been widely used for in vitro fertilization [IVF]. This study was designed to study the effects of oviductal epithelial cells [OECs] and their conditioned medium during IVF on subsequent embryonic development and the relative abundance of zygote arrest 1 [Zar1] transcript in ovine zygotes. The in vitro matured ovine oocytes were randomly fertilized in the following culture conditions: I] SOFaaBSA 20% sheep serum [control], II] SOFaa BSA 20% sheep serum [50 microl] in the presence of OECs, III] SOFaaBSA 20% sheep serum [100 microl] in the presence of OECs, and IV] OECs conditioned medium [CM]. Sigma Stat [Version 2.0] software and one-way ANOVA were considered for statistical analysis. A p<0.05 was considered statistically significant. The cleavage, blastocyst, and hatched blastocyst rates in OECs and CM groups were significantly lower than the control group [p<0.01]. In co-cultured groups, the application of two different volumes of IVF medium showed no difference in embryonic developmental indices. The Zar1 gene expression in zygotes produced in the presence of OECs was significantly higher than those produced in the control and CM groups [p<0.05]. Neither the presence of oviductal epithelial cells nor their conditioned medium could improve the developmental potential of ovine embryos during IVF. Moreover, no relationship was observed between the relative abundance of Zar1 transcript in zygotes produced in different conditions and the corresponding subsequent embryonic development

effect of macromolecule source and type of media during in vitro maturation of sheep oocytes on subsequent embryo development

Oocyte maturation and subsequent in vitro production [IVP] of embryos are affected by diverse groups of chemicals in maturation medium which are needed for successful mammalian oocyte maturation during which the dramatic cytoplasmic and nuclear reprogramming events take place. This study was designed to evaluate the effects of protein source [fetal bovine serum, FBS, and bovine serum albumin, BSA] as well as two different maturation media during in vitro maturation of ovine oocytes on subsequent embryo development. Cumulus oocyte complexes were recovered from ovaries obtained from slaughter house and cultured for 24 hr in either TCM-199 or SOFaa maturation medium supplemented with 10% [v/v] FBS or 0.8% [w/v] BSA. Data were analyzed by one-way ANOVA using Sigma Stat [Ver. 2]. A p-value smaller than 0.05 was considered statistically significant. The proportions of cleavage and total blastocyst [evaluated on days 3 and 6, respectively] were significantly higher in FBS than BSA supplemented groups, though no differences were observed between the two used different maturation media. The cryotolerance of blastocysts was negatively influenced by the presence of FBS rather than BSA during IVM. The quality of produced embryos, however, was affected neither by the source of macromolecules nor the maturation medium in terms of hatching rate, total blastocyst cells and inner cell mass/total cell ratio. The rate of oocyte development was improved by the presence of FBS, though the cryosurvival of resulting blastocysts was negatively influenced by the presence of the serum during in vitro production of sheep oocytes

[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

Penicillin - streptomycin in the culture medium during in vitro maturation [IVM] of bovine oocytes affects nuclear maturation and subsequent embryo development

Standard concentrations of antibiotics in culture media are thought to have no detectable toxic effects on the cultured cells. However, since antibiotics are biologically active substances, the possibility that they interfere to some extent with cellular processes occurring in the cultured cells can not always be totally excluded. This study, therefore, was conducted to assess whether the presence of penicillin-streptomycin [pen-strep] during in vitro maturation [IVM] of bovine cumulus oocytes complexes [COCs] affect nuclear and cytoplasmic maturation and subsequent embryo development. Bovine COCs were matured at 39oC in a humidified atmosphere with 5 percent CO[2] in air for 24 h in: 1- culture medium M199 supplemented with 10 percent FCS [Fetal calf serum], 0.05 lU/ml rhFSH [recombinant human FSH] and 100 units penicillin and 100 fig streptomycin/ ml. 2- culture medium Ml99 without FCS and rhFSH in the presence of pen-strep. Cultures without antibiotics served as control. Six series of experiments, each consisted of at least 3 replicates, were performed. In vitro maturation in the presence of pen-strep in culture medium supplemented with FCS and rhFSH significantly [P<0.05] increased the percentage of Mil oocytes, however, when the COCs were divided, on the basis of appearance of the cumulus investment, into bright and dark groups, this effect was less obvious in both types of COCs, 76 percent vs. 72 percent in bright COCs [P =0.149] or 83 percent vs. 80 percent in dark COCs [P=0.296] in treated and control groups respectively. The percentage of oocytes with type III of cortical granules [CGs] distribution was not affected in the presence of pen-strep. The COCs expansion after IVM was not affected by the presence of antibiotics in culture medium. The subsequent embryo development of IVM/IVF produced ova, which were exposed to pen-strep during IVM, was significantly [P<0.05] decreased with respect to blastocyst formation by day 9. In vitro maturation in the presence of pen-strep in culture medium without FCS and rhFSH had no significant [P=0.402] effect on nuclear maturation. The results indicate that the nuclear maturation of bovine oocytes was .positively influenced by the presence of pen-strep during IVM when the culture media was supplemented with FCS and rhFSH. Moreover, despite of no notable effect of pen-strep on CGs distribution the subsequent embryo development was negatively influenced by the presence of pen-strep