A Modified Handmade Cloning Method for Dromedary Camels.

Camels play very important economic and sociocultural roles for communities residing in arid and semi-arid countries. The positive impacts of cloning on genetic gain in camel species are indisputable, considering the unique ability of cloning to produce a large number of offspring of a predefined sex and genotype using somatic cells obtained from elite animals, live or dead, and within any age category. However, the current low efficiency of camel cloning seriously limits its commercial applicability. We have systematically optimized technical and biological factors for dromedary camel cloning. In this chapter, we present the details of our current standard operating procedure for dromedary camel cloning, namely, "modified handmade cloning (mHMC)."

Vitrification of camel oocytes transiently impacts mitochondrial functions without affecting the developmental potential after intracytoplasmic sperm injection and parthenogenetic activation.

Oocyte vitrification preserves the female genetic resources of elite dromedary camels. In the current study, we aimed to explore the effects of vitrification of camel oocytes on mitochondrial activity, redox stress, and expression of genes related to mitochondrial function, apoptosis, pluripotency, and cytoskeleton. Moreover, we investigated developmental competence of vitrified oocytes after parthenogenetic activation. Oocytes vitrified with the Cryotop method were compared with the fresh oocytes. Our results showed that vitrification led to increased ROS production in oocytes as evidenced by an increase in the DCFDHA fluorescence intensity, and lower mitochondrial activity. At the molecular level, vitrification reduced mRNA expression of many genes, including those related to mitochondrial function (TFAM, MT-CO1, MFN1, ATP1A1, NRF1), pluripotency (SOX2 and POU5F1), and apoptosis (p53 and BAX). In contrast, expression of KLF4 and cytoskeleton-related genes (ACTB and KRT8) was not affected. However, we found no difference in the rates of oocyte survival, cleavage, and blastocyst development, and blastocyst hatching between fresh and vitrified oocytes after warming. Our results indicate that although vitrification of camel metaphase II (MII) oocytes adversely affected mitochondrial functions, the effect was transient without compromising the developmental potential of the oocytes after parthenogenetic activation.

Effects of abundances of OCT-4 mRNA transcript on goat pre-implantation embryonic development.

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8-16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed.

Survival, re-expansion, and pregnancy outcome following vitrification of dromedary camel cloned blastocysts: A possible role of vitrification in improving clone pregnancy rate by weeding out poor competent embryos.

There is a clinical demand for efficient cryopreservation of cloned camel embryos with considerable logistic and economic advantage. Vitrification of in vivo derived embryos has been reported in camels, but there is no study on vitrification of cloned embryos. Moreover, whether characteristic differences between cloned and in vivo derived embryos imply different vitrification requirement is unresolved. Here, we compared survival, re-expansion and pregnancy rates of cloned embryos vitrified using two commercial vitrification kits (Cryotec and Kitazato), developed basically for human embryos, and a vitrification protocol developed for in vivo camel embryos (CVP). Cloned embryos responded dynamically to vitrification-warming steps in commercial kits, with a flat shrinkage in the final vitrification solution and a quick re-expansion to the original volume immediately after transferring to the isotonic warming solution. Contrarily, full shrinkage was not observed in CVP method, and majority of embryos were still collapsed post-warming. The immediate re-expansion was highly associated and predictive of higher survival and total cell number, and also better redox state of embryos vitrified by Cryotec and Kitazato kits compared to CVP method. Importantly, while 30% blastomere loss, verified by differential dye exclusion test, was tolerated in vitrified embryos, >50% blastomeres loss in non-expanded blastocysts implied the minimal essential cell survival rate for blastocoelic cavity re-expansion in vitrified cloned camel blastocysts, irrespective of vitrification method. A protocol-based exposure of embryos to cryoprotectants indicated that cryoprotectant toxicity, per se, may not be involved in lower cryosurvival of embryos in CVP vs. Cryotec and Kitazato. The initial pregnancy rates were numerically higher in Cryotec and Kitazato frozen transfers compared to fresh transfer (56.3, 60 and 33.3%, respectively), and importantly, a higher percentage of established pregnancies in vitrified groups passed the critical 3 months period of early embryonic loss compared to sibling fresh clone pregnancies (50, 40, and 10%, respectively). Results confirmed the suitability of Cryotec and Kitazato kits for vitrification of cloned camel embryos and that vitrification may improve pregnancy outcome by weeding out poor competent embryos.

Development of a modified method of handmade cloning in dromedary camel.

In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.

Functional characterization of NANOG in goat pre-implantation embryonic development.

Nanog as a novel pluripotent cell-specific gene plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in inner cell mass (ICM) and embryonic stem cells (ESC) in mouse. The molecular features and transcription regulation of NANOG gene in domestic animals are not well defined. In this study, we performed knockdown of NANOG mRNA in goat embryos and examined its effect on early embryonic development. Presumptive zygotes were injected with a volume of 8-10 pl NANOG or scrambled (SCR) siRNA, and subsequently cleavage and blastocyst formation rate were assessed. Furthermore, gene expression analysis was carried out in 6-8 cell and blastocyst derived embryos from non-injected controls, SCR - and siRNA-injected presumptive zygotes. Cleavage and blastocyst rates in siRNA groups were insignificantly lower than the control and SCR groups. Embryos with reduced expression of NANOG showed decrease in number of trophectoderm (TE) and total cells in blastocysts. Analysis of expression of developmentally important genes (SOX2, OCT4 and NANOG), which work as a network, showed that NANOG knockdown results in significant increase in expression of SOX2 and OCT4 and among the possible target genes (CDX2, REX1 and GATA4) of this network, only GATA4 showed increased expression. Our results suggest that NANOG is likely to be required for proliferation of trophoblastic cells.

Methylation Status of H19/IGF2 Differentially Methylated Region in in vitro Human Blastocysts Donated by Healthy Couples

BACKGROUND: Imprinted genes are a unique subset of few genes that have been differentially methylated region (DMR) in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies have shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception micro-environments often is occurred by assisted reproduction techniques (ART). This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART. METHODS: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites (CpGs) located in this region. RESULTS: The overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85±4.87% and 43.75±5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined (49.52±1.86% and 50%, respectively). CONCLUSIONS: Considering the absence of in vivo- produced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks.

Expression Profile of Developmentally Important Genes in preand peri-Implantation Goat Embryos Produced In Vitro.

BACKGROUND: Little is understood about the regulation of gene expression during early goat embryo development. This study investigated the expression profile of 19 genes, known to be critical for early embryo development in mouse and human, at five different stages of goat in vitro embryo development (oocyte, 8-16 cell, morula, day-7 blastocyst, and day 14 blastocyst). MATERIALS AND METHODS: In this experimental study, stage-specific profiling using real time-quantitative polymerase chain reaction (RT-qPCR) revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on their respective mRNA profiles. RESULTS: The gradual pattern of reduction in the maternally stored transcripts without renewal thereafter (cluster-1: Lifr1, Bmpr1, Alk4, Id3, Ctnnb, Akt, Oct4, Rex1, Erk1, Smad1 and 5) implies that their protein products are essential during early cleavages when the goat embryo is silent and reliant to the maternal legacy of mRNA. The potential importance of transcription augment at day-3 (cluster-2: Fzd, c-Myc, Cdc25a, Sox2) or day- 14 (cluster-3: Fgfr4, Nanog) suggests that they are nascent embryonic mRNAs which intimately involved in the overriding of MET or regulation of blastocyst formation, respectively. The observation of two expression peaks at both day-3 and day-14 (cluster-4: Gata4, Cdx2) would imply their potential importance during these two critical stages of preand periimplantation development. CONCLUSION: Evolutionary comparison revealed that the selected subset of genes has been rewired in goat and human/goat similarity is greater than the mouse/goat or bovine/goat similarities. The developed profiles provide a resource for comprehensive understanding of goat preimplantation development and pluripotent stem cell engineering as well.

Vitrification at Day3 stage appears not to affect the methylation status of H19/IGF2 differentially methylated region of in vitro produced human blastocysts.

One of the most widely used assisted reproductive technology (ART) is vitrification. The aim of this study is to evaluate DNA methylation of H19/IGF2 differentially methylation region (DMR) in in vitro produced human blastocysts derived from non-vitrified and vitrified day3 embryos. Day3 embryos derived from ICSI cycles from fertile couples referring for family balancing program were either biopsied or vitrified/warmed and subsequently biopsied. Following biopsy, embryos were cultured to day 5. Day5 blastocysts with desired sex were transferred or vitrified for future use. Blastocysts with un-desired sex were donated for research. The assessment of the embryos was performed in two non-vitrified and vitrified groups. Methylation level of H19/IGF2 DMR was analysed by bisulfite conversion and sequencing at 18 CpG sites (CpGs) located in this region. Results showed that the overall methylated CpGs percentages of this region in the vitrified and non-vitrified groups were 35.3% ± 3.6 and 38.27 ± 4.1%, respectively. The difference between the two groups was not significant. Vitrification of day3 embryo appears to have no adverse effect on DNA methylation status of H19/IGF2 DMR of embryos cultured in vitro to blastocyst stage. These data may have implications for performing frozen embryo cycles transfer instead of fresh embryo transfer cycles, owing to the naturally synchronized uterus and subsequently improved endometrial receptivity in frozen embryo transfer instead of imbalanced hormonal milieu in fresh embryo transfer cycles.

Targeting the transforming growth factor-ß signaling during pre-implantation development in embryos of cattle, sheep and goats.

Recently, application of chemical inhibitors against differentiation signaling pathways has improved establishment of mESCs. In this study, we applied inhibitors of TGF-ß (SB431542) and BMP4 (Noggin) from cleavage to blastocyst stage in cattle, goat and sheep embryos. SB significantly decreases blastocyst rate and total cell number (TCN) in sheep blastocysts, whereas only TCN was significantly decreased in cattle blastocysts. In contrast to SB, Noggin significantly improved cattle blastocyst development but decreased TCN. However, Noggin treatment led to a significant increase in TCN in sheep blastocysts. Regarding pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment (REX1, CDX2, GATA4), SB led to a significant reduction in SOX2 expression in goat and cattle, while Noggin increased at least one or two of pluripotent markers in these species. Taken together, this data suggests that inhibition of TGF-ß by Noggin may be more favorable for derivation of stem cells in farm animals.

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals.

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

What does the cryopreserved oocyte look like? A fresh look at the characteristic oocyte features following cryopreservation.

In October 2012, the American Society for Reproductive Medicine (ASRM) and, in March 2012, the European Society of Human Reproduction and Embryology (ESHRE), lifted the categorization of oocyte cryopreservation as being "experimental" and endorsed its entrance into the mainstream of assisted reproductive techniques. This change in policy, with the considerable advantages that oocytes offer over embryos for cryopreservation, has increased applications of oocyte cryopreservation in assisted reproduction techniques. A deep understanding of oocyte cryobiology, however, is lagging behind the forces propelling the clinical application of oocyte cryopreservation. We have drawn attention to this shortcoming by initiating a debate on whether a vitrified-warmed oocyte has the same characteristics as its fresh sibling. The answer to this question may explain why the oocyte cryopreservation success rate is as yet far from satisfactory and why cryopreserved oocytes should be treated differently from their fresh siblings. A fresh look at the characteristic features of oocytes after cryopreservation is the main scope of this review as a stimulus to further improvement of oocyte cryopreservation.

Stage-Specific Profiling of Transforming Growth Factor-ß, Fibroblast Growth Factor and Wingless-int Signaling Pathways during Early Embryo Development in The Goat.

OBJECTIVE: This research intends to unravel the temporal expression profiles of genes in- volved in three developmentally important signaling pathways [transforming growth factor-ß (TGF-ß), fibroblast growth factor (FGF) and wingless/int (WNT)] during preand peri-implan- tation goat embryo development. MATERIALS AND METHODS: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the down- stream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 (D7) blastocysts and day-14 (D14) blas- tocysts] were separately prepared in triplicate for comparative real-time reverse tran- scriptase polymerase chain reaction (RT-PCR) using the selected gene sets. RESULTS: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-ß, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-ß, FGF and WNT components with renewal thereafter. CONCLUSION: The results suggested that TGF-ß, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of preand peri- implantation goat embryo development.

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

BACKGROUND: The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. RESULTS: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. CONCLUSIONS: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia.

Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats.

Cytoplasmic, rather than nuclear-DNA, insufficiencies as the major cause of poor competence of vitrified oocytes.

To unravel the differential contributions of nuclear-DNA and cytoplasm to the poor 'competence' of oocytes after cryopreservation, reciprocal exchange of metaphase II-spindle chromosomal complex (karyoplast) between vitrified and fresh oocytes was carried out in an ovine animal model. Karyoplast exchange per se was accomplished with high efficiency and in-vitro development of oocytes reconstituted with fresh-karyoplast and vitrified-cytoplast (FK/VC) showed no improvement over VK/VC and control-vitrification oocytes. Blastocyst development of oocytes that were reconstituted with vitrified-karyoplast and fresh-cytoplast (VK/FC) approached that of fresh-controls, however, and was significantly higher than FK/VC, VK/VC, and control-vitrification (all P ≤ 0.05). These results point toward 'cytoplasmic insufficiencies' as the main cause of poor 'competence' of matured oocytes after vitrification.

The effect of superoxide dismutase mimetic and catalase on the quality of postthawed goat semen.

Manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE) is a cell-permeable superoxide dismutase mimetic agent which can convert superoxide to hydrogen peroxide (H2O2). Supplementation of MnTE to a commercial semen extender can protect sperm from superoxide but not H2O2. Therefore, we proposed that addition of catalase (0.0, 200, or 400 IU/mL) in combination with MnTE (0.1 µM) may further improve the cryopreservation efficiency of goat semen in commercially optimized freezing media such as Andromed. Therefore, ejaculates were obtained from three adult bucks twice a week during the breeding season and diluted with Andromed supplemented with or without MnTE and catalase and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species contents were evaluated 2 hours after dilution (before freezing) and after freezing/thawing. The results revealed that all the treatments significantly (P ≤ 0.05) improved sperm motility, viability, and membrane integrity after freezing and reduced reactive oxygen species content compared with the control group, but maximum improvement was obtained in MnTE + 400 IU/mL catalase. In addition, supplementation with these antioxidants significantly (P ≤ 0.05) increases the cleavage rate after IVF. In conclusion, the results of present study suggest that addition of antioxidant MnTE or catalase to commercial optimized media, such as Andromed, improves total motility, membrane integrity, and viability of goat semen samples after thawing. But the degree of improvement for these parameters significantly (P ≤ 0.05) higher when MnTE and catalase were simultaneously added to the cryopreservation media.

Supplementation of sperm cryopreservation media with cell permeable superoxide dismutase mimetic agent (MnTE) improves goat blastocyst formation.

The aim of this study was to assess whether a cell permeable superoxide dismutase agent such as MnTE, can further improve the quality of frozen/thawed semen sample using a commercially optimized sperm cryopreservation media (Bioxcell). Bioxcell was supplemented with different concentration of MnTE. Sperm membrane integrity, motility, viability and acrosomal status were assessed after freezing. Optimized concentration of MnTE was defined and used to assess fertilization and developmental potential. 0.1 µM MnTE significantly improved membrane integrity while 0.01 µM MnTE significantly improved acrosomal integrity post thawing. Addition of 0.01 µM MnTE also improved blastocyst formation rate. Supplementation of commercially optimized cryopreservation media with MnTE further improves the quality of goat frozen semen sample and may have important consequence of future embryo development. This effect may be attributed to cell permeable behavior of this antioxidant which may protect sperm genome from ROS-induced DNA damage.

THY1 as a reliable marker for enrichment of undifferentiated spermatogonia in the goat.

Spermatogonial stem cells are unique cells of testes that can restore fertility upon transplantation into recipient testes. However, use of suitable markers for enrichment of these cells have important potential application. THY1, is an established conserved marker of spermatogonial stem cells in bovine, rodents, and primates, but there is no information available in goats. After three rounds of enzymatic digestion of prepubertal goat testicular tissues, undifferentiated spermatogonia positive for THY1 were isolated by magnetic-activated cell sorting and were used for immunocytochemistry, real-time polymerase chain reaction analysis for gene expression, protein expression, and transplantation into recipient mice. Immunocytochemical analyses showed that significantly higher percentage of THY1(+) cells were positive for PLZF and VASA when compared with unselected population. This result for PLZF was further confirmed at the protein level. Real-time polymerase chain reaction analysis revealed that expression of THY1, PLZF, VASA, BCL6B, and UCHL1 as SCCs characteristic genes in THY1(+) cells was significantly higher than in the initial population. Finally, transplantation of PKH26-labeled cells revealed that THY1(+) cells had higher capacity for colony formation when compared with unselected cells. In conclusion, the results provide indications that THY1 surface marker can be reliably used for enrichment of undifferentiated spermatogonial in the goats.

Can permeable super oxide dismutase mimetic agents improve the quality of frozen-thawed ram semen?

This study was carried out to assess the effects of MnTBAP, a cell permeable antioxidant, on motility, membrane integrity, capacitation status and in vitro fertilization ability of frozen-thawed ram semen. Fresh semen ejaculates were collected with artificial vagina from five rams, mixed and divided into five equal fractions, and diluted (1:20 v/v) with commercial extender, Bioxell®, containing 0 (control), 50, 100, 150 and 200 µM of MnTBAP. All diluted sperm suspensions were cooled to 5°C for 2h followed by transfer into 0.5 ml French straws before being stored in liquid nitrogen. The results showed that MnTBAP supplementation of extender improved ram semen quality in a dose-dependent manner. Accordingly, the extender supplemented with 150µM MnTBAP resulted in higher sperm motility and improved acrosomal membrane integrity compared to control. However, further supplementation (200µM) with MnTBAP not only did not improve the results but inversely affected motility and membrane integrity. The results of in vitro fertilization (IVF) indicated that the presence of MnTBAP in semen extender has a marginal beneficial effect on developmental competence of inseminated oocytes, though this improvement was not significant. In conclusion, this study demonstrated that semen extender supplemented with MnTBAP can reduce the oxidative stress provoked by freeze/thaw processes. Moreover, beneficial effect of 100 µM of MnTBAP on preservation of spermatozoa in a non-capacitated state post freezing, an important criterion for in vitro or in vivo fertilization, was observed. However, at 150 µM of MnTBAP, the harmful effects of cryopreservation on membrane integrity were decreased. Regarding to importance of non-capacitated spermatozoa during IVF or artificial insemination, the optimum MnTBAP concentration appears to be 100 µM for commercial ram semen extender tested here.