Evaluation of novel SexedULTRA-4M technology for in vitro bovine embryo production

SexedULTRA-4M™ is made using an improved method of sex-sorting sperm in a less damaging environment for better retaining sperm integrity throughout the sorting process. The objective of this research was to compare conventional (CONV) and SexedULTRA-4M™ (ULTRA-4M) semen for bovine IVP using four Angus bulls. Matured slaughterhouse oocytes (n = 4000) were divided into the CONV group and the ULTRA-4M group (2000 COCs for each semen type). The IVF process was implemented with CONV and ULTRA-4M semen from the same bull. The cleavage rates, eight cell embryos and blastocysts on day 7 of culture were evaluated for each semen type and each bull. The statistical analysis was carried out with the ANOVA procedure SAS software. The results were 54.45% ± 1.03 and 58.10% ± 1.07; 35% ± 1.57 and 39.15% ± 1.62; 22.8% ± 1.09 and 27.15% ± 1.12 for CONV and ULTRA-4M, respectively, for cleavage rate, eight cell embryos and blastocysts on day 7 for the average of all bulls, comparing only the semen type. Concerning only the semen type, ULTRA-4M was significantly superior to CONV for cleavage rates (P = 0.01) and blastocysts on day 7 (P = 0.009). There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analyzed for Bull 1 and Bull 4, however, for Bull 2 ULTRA-4M was significantly superior to CONV for cleavage rates and blastocysts on day 7 (P< 0.05). In Bull 3, ULTRA-4M was significantly higher (P< 0.05) for blastocysts on day 7 compared to CONV. In conclusion, under the conditions of this research the ULTRA-4M and CONV semen produced similar bovine IVP results overall.     

Kisspeptin在女性生殖内分泌和辅助生殖技术中的研究进展

近年来研究显示,kisspeptin在大脑的性别分化、性激素正负反馈调节、青春期始动以及机体能量信号转导等生理过程中起到重要作用,表明kisspeptin可能是女性生殖功能成熟及调控的一个关键性信号因子。除下丘脑分泌的kisspeptin之外,生殖器官局部表达的kisspeptin在机体正常生殖过程中的作用也不断得到证实。研究表明,很多生殖内分泌疾病,如单纯性促性腺激素分泌不足的性腺机能减退症(isolated hypogonadotropic hypogonadism, IHH)、多囊卵巢综合征(polycystic ovary syndrome, PCOS)、卵巢早衰(premature ovarian failure, POF)、病理性高泌乳素血症等,都与kisspeptin的异常表达有关。通过给予外源性kisspeptin可解决辅助生殖技术应用中的一些问题。本文主要就kisspeptin在女性生殖内分泌尤其是在辅助生殖领域研究中所取得的进展进行论述。     

生物技术生产虾青素研究的最新进展

虾青素是一种具有极强的生物抗氧化性的酮式类胡萝卜素在医药,食品,化妆品等方面有着极广阔的应用前景。对目前生物技术生产虾青素的两种主要方法：即雨生红球藻(haematococcus pluvialis)培养法和红发夫酵母(xanthopltyllomyces dendrorhous,formedy Phaffia raodazyma)发酵法的最新研究进展,包括采用的微生物的生理代谢特性、虾青素合成途径、生产技术特点、工程菌种的构建等方面进行了系统的总结。针对天然虾青素生产技术研究中存在的问题和不足,提出了建议和解决的方法。     

Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites

For a long time people are using plants not only as crop cultures but also for obtaining of various chemicals. Currently plants remain one of the most important and essential sources of biologically active compounds in spite of progress in chemical or microbial synthesis. In our review we compare potentials and perspectives of modern genetic engineering approaches for pharmaceutical biotechnology and give examples of actual biotechnological systems used for production of several promising natural compounds: artemisinin, paclitaxel and scopolamine.     

Leukemia inhibitory factor: Recent advances and implications in biotechnology

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with several functions in health and disease ranging from inflammation to cancer. LIF is also a potential target and/or therapeutic agent for diseases such as multiple sclerosis, stroke and even psychological disorders, where the function of LIF as a neurotrophic factor has only recently been explored. In recent years, a limited number of LIF clinical trials have been completed, which partially explains the shortage of effective applications as a therapeutic agent. With the increasing interest from biotechnology companies producing recombinant LIF, this status quo will certainly change, and the potential impact of LIF in terms of disease diagnosis, treatment and management will be realized.     

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.     

The effects of superovulation and reproductive aging on the epigenome of the oocyte and embryo

A societal preference of delaying maternal age at first childbirth has increased reliance on assisted reproductive technologies/therapies (ART) to conceive a child. Oocytes that have undergone physiologic aging (≥35 years for humans) are now commonly used for ART, yet evidence is building that suboptimal reproductive environments associated with aging negatively affect oocyte competence and embryo development—although the mechanisms underlying these relationship are not yet well understood. Epigenetic programming of the oocyte occurs during its growth within a follicle, so the ovarian stimulation protocols that administer exogenous hormones, as part of the first step for all ART procedures, may prevent the gamete from establishing an appropriate epigenetic state. Therefore, understanding how oocyte. Therefore, understanding how hormone stimulation and oocyte physiologic age independently and synergistically physiologic age independently and synergistically affect the epigenetic programming of these gametes, and how this may affect their developmental competence, are crucial to improved ART outcomes. Here, we review studies that measured the developmental outcomes affected by superovulation and aging, focusing on how the epigenome (i.e., global and imprinted DNA methylation, histone modifications, and epigenetic modifiers) of gametes and embryos acquired from females undergoing physiologic aging and exogenous ovarian stimulation is affected.     

Probing morphological,genetic and metabolomic changes of in vitro embryo development in a microfluidic device

Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10–12 embryos. Development rates, mitochondrial polarization and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation.     

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.     

Recent advances for the production and recovery methods of lysozyme

Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.     

The past, present, and future of embryo selection in in vitro fertilization: Frontiers in Reproduction Conference

Assisted reproductive technology (ART) has been recognized for its success in treating infertility, a condition that affects 15 percent of couples in the United States. The most popular option is in vitro fertilization (IVF), which relies on embryo culture, selection, and transfer for implantation, with the ultimate aim of pregnancy. Previous embryo selection methods relied on morphological factors to select for greatest viability. At Yale's Frontiers in Reproduction Conference on April 29, 2011, at the New Haven Lawn Club, Dr. Denny Sakkas of Yale's Department of Obstetrics, Gynecology, and Reproductive Sciences presented a paradigm shift: using morphological factors along with metabolic, protein, and genetic markers in culture media to enhance embryo selection and IVF success rates.     

Improvement in bovine embryo production in vitro by glutathione-containing culture media

Bovine oocytes were matured, fertilized, and cultured (TCM 199 with serum and co-culture) in vitro (IVMFC) with addition, during different phases of the procedure, of antioxidants: superoxide dismutase (SOD) and reduced glutathione (GSH). The addition of SOD (1,500 or 3,000 IU/ml) did not improve proportions of oocytes undergoing cleavage or the development of embryos to morula and blastocyst stages. The cleavage rates were significantly lower than in the control group (CTR 57.5%) when SOD was present during the insemination interval (IVF) or throughout the entire procedure (IVMFC). Thus when the lower concentration was present for IVF and IVMFC, 35.1% and 36.4% of inseminated oocytes cleaved (P < 0.01 compared to CTR) and cleavage results with the higher concentration during IVF and IVMFC were 38.5% and 29.2% (P < 0.025 and P < 0.001 compared to CTR, respectively). Significant improvements in proportions of oocytes undergoing cleavage (84.5% vs. 57.0%, P < 0.001) and morula/blastocyst development (33.3% vs. 13.9%, P < 0.005) were achieved when GSH (1 mM) was added to the culture medium. In a defined medium for culture (mSOF and BSA) the presence of SOD (3,000 IU/ml) was ineffective, but in a defined medium supplemented with GSH (1 mM) at day 6 postinsemination (i.e., when 90% of developing embryos were in 8–16 cell stages), development to the morula and blastocyst stages was supported for 35.5% of cultured oocytes (P < 0.005 compared to 19.2% for CTR). These data suggest that bovine embryos are sensitive to oxidative stress and that medium supplementation with the radical scavenger glutathione can improve embryo development in vitro. © 1996 Wiley-Liss, Inc.     

In vitro embryo production (IVEP) in camelids: Present status and future perspectives

Camels are a fundamental livestock resource with a significant role in the agricultural economy of dry regions of Asia and Africa. Similarly, llamas and alpacas are an indigenous resource considered as beasts of burden in South America because of their surefootedness and ability to adapt. Camel racing, a highly lucrative and well-organized sport, camel beauty contests, and high demand for camel milk lead to a steady interest in the multiplication of elite animals by in vitro embryo production (IVEP) in this species during the last few decades. Although offspring have been produced from in vitro produced embryos, the technique is still not that well developed compared with other domestic animal species such as cattle. IVEP involves many steps, including the collection of oocytes from either slaughterhouse ovaries or live animals through ultrasound-guided transvaginal aspiration; in vitro maturation of these collected oocytes; collection and preparation of semen for fertilization; culture and passaging of cells for nuclear transfer, chemical activation of the reconstructed embryos, and in vitro culture of embryos up to the blastocyst stage for transfer into synchronized recipients to carry them to term. This review discusses the present status of all these steps involved in the IVEP of camelids and their future perspectives.     

Assisted reproduction technologies and reproductive justice in the production of parenthood and origin: Uses and meanings of the co-produced gestation and the surrogacy in Brazil

This article examines the construction of parenthood, drawing on Brazilian cisgender, heterosexual, and homosexual couples' experiences in using assisted reproduction technologies (ART), particularly the surrogacy. For that purpose, we interviewed: 1) a lesbian woman who had her daughter through her partner's pregnancy, using ART with anonymous donor semen; 2) a gay man who, together with his partner, used a surrogacy service under contract via a specialised offshore agency; 3) a woman who was a surrogate, in Brazil, for her sister-in-law and brother who lived abroad and, from abroad, sent an embryo fertilised for surrogacy; 4) a woman who resorted to her sister-in-law in order to be a mother by surrogacy, with ovules from the woman herself fertilised with semen from her husband; and 5) the sister-in-law mentioned in 4), who acted as surrogate for her brother and his wife. These interviews made it possible to think about the discursive construction of the legitimacy of such parenthoods, as it is produced by access to, and manipulation and circulation of, reproductive technologies and persons. This biomedical management of bodies sets up a material and discursive circuit that, in turn, produces a complex web of personal, normative, legal, professional and market relationships, particularly with a view to construction of a parenthood anchored in a notion of biologically-constituted origin. In this respect, biological, affective and social bonds merge to produce a precise placement of who is the father and/or who is the mother, as well as who are the important others and how they are linked to the child in a broader web of parenthood.     

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.     

Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts.     

Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72?h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.     

Infectious agents in bovine embryo production: hazards and solutions

Infectious agents in systems for producing bovine embryos might reduce the number and quality of embryos generated, result in transmission of disease to recipients and offspring, or confound findings of research. Embryo-associated pathogens might also jeopardize human health when the goal of embryo production is creating transgenic animals intended to be a source of pharmaceuticals or organs. This paper addresses risks and resulting hazards of pathogen and microbial contaminant introduction into in vivo or in vitro embryo production systems. Additionally, methods for prevention and quality control are discussed.