Effects Of Different Culture Systems Research Articles

Co-cultured of red hybrid tilapia and Gracilaria changii (Gracilariaceae: Rhodophyta): Effects on water quality, growth performance and algal density

ABSTRACT We evaluated the potential for co-cultured of red hybrid tilapia with Gracilaria changii in two different densities in zero water exchange conditions. Five treatments were performed, consisting of three monoculture systems as controls: algae at two different densities (M1 and M2 with 1 g L−1 density and 3 g L−1 density, respectively) and fish without algae (Mfish). For comparison purposes, the other two treatments were co-cultured fish with algae at densities of 1 g L−1 (P1) and 3 g L−1 (P2). The effects of different culture systems and densities on the water quality, growth performance, nutrient content and C/N ratio of the algae were determined. During the experimental period, the dissolved oxygen (DO) and pH values in the algal tanks (monoculture and co-culture) were significantly higher than those in the Mfish tank. The ammonia and nitrate concentrations in P1 and P2 were significantly lower compared to initial measurements. The water parameters indicated that the algae improved the water quality of the culture system. Compared to the fish monoculture (Mfish), the fish grew better with algae (P1 and P2) maybe due to the more favourable water conditions in the tanks. On day 20, the algae growth rates in the co-culture tanks (P1 and P2) were significantly higher than those in the monoculture tanks. Additionally, P1 and P2 improved the nutrient composition and C/N ratio of the algae. This study found that although the co-culture system resulted in better water quality and nutrient content in the alga, the algal density had no significant effect on fish or algal growth.

Preparation and properties of biofilm-states Bifidobacterium adolensentis Gr19 under dynamic culture system and its application on probiotic ice cream manufacture

Biofilms are microbial aggregates that attach to the surfaces of specific substances, producing extracellular polymeric substances (EPS) to increase their resistance. However, the biofilm-state strains produced by the current static culture system are not industrially applicable. Therefore, this study aimed to establish a dynamic culture system to investigate the biofilm-forming ability of Bifidobacterium adolensentis (B. adolensentis) Gr19 on pea hulls, wheat bran, flaxseed bran, buckwheat bran, and quinoa bran (2% w/v), and analyze the effect of different culture systems on the probiotic properties of bacterial strains. Finally, the probiotic activity, physicochemical properties, stability, and sensory acceptability of ice cream supplemented with dynamically cultured biofilm-state strains were evaluated. B. adolensentis Gr19 formed the most biofilms on flaxseed bran in the dynamic culture system. It showed better cell adhesion ability and resistance to bile salts, gastrointestinal fluids, and low temperatures than planktonic-state strains. Adding dynamically cultured biofilm-state strains to ice cream significantly improved product hardness, reduced overruns and melting rates, and stabilized probiotic viability, yielding 6.1 log CFU/g viable bacteria after 18 weeks of storage. Although the sensory score of ice cream decreased due to a sandy texture, no significant changes were evident in consumer purchase patterns. The results indicated that by-products could be used to establish a dynamic culture system for biofilm strains. This might offer a theoretical basis for creating highly resistant probiotics for the food industry and reduce the possibility of contamination in the agro-industry.

Evaluation of the effect of different culture systems on photomixotrophic capacity during in vitro multiplication of pitahaya (Hylocereus undatus)

Pitahaya (Hylocereus undatus) micropropagation is an alternative for obtaining invigorated plants with high phytosanitary quality. The objective of this research was to evaluate different culture systems on photomixotrophic capacity during in vitro multiplication and their effect on survival in the acclimatization stage of H. undatus. Different temporary immersion systems (TIS) were used: temporary immersion bioreactors (TIB), Ebb-and-Flow bioreactor, Recipient for Automated Temporary Immersion (RITA), and semi-solid (SS) culture medium. At 60 d of culture, the percentage of explant response, developmental variables, chlorophyll and carotene content, stomatal index (SI), percentage of closed stomata (CS), PEP (Phosphoenolpyruvate) and Rubisco (Ribulose-1,5-bisphosphate carboxylase oxygenase) content and the percentage of survival in the greenhouse acclimatization stage were evaluated. The culture systems with the highest percentage of response were the SS culture medium (96%), Ebb-and-Flow (93%) and RITA (90%). The Ebb-and-Flow and RITA bioreactors showed the highest number of shoots, with 9.5 and 9.0, respectively, the greatest length, with 2.62 and 2.71 cm, the highest chlorophyll, with 0.30 and 0.28 mg g–1 FW, and carotenoid content, with 2.25 and 2.20 mg g–1 FW. The SS medium culture system showed the highest SI with (57%), while the TIB had the highest CS, with (85%). The Ebb-and-Flow and RITA culture systems showed the highest PEP content, with 15 and 18 nmol g–1 FW, respectively; whereas, for Rubisco content, no differences were found in the different culture systems. The Ebb-and-Flow and RITA culture systems presented the highest survival rate, with 99.6% during acclimatization. The use of Ebb-and-Flow and RITA bioreactors are a biotechnological alternative for in vitro propagation of H. undatus and guarantee high survival rates during acclimatization without going through a rooting stage.

Effects of integrated multi-trophic aquaculture on microbial communities, antibiotic resistance genes, and cultured species: A case study of four mariculture systems

Aquaculture is an important protein source in many countries, including China. Integrated multitrophic aquaculture (IMTA) is widely used to reduce the environmental pressure of aquaculture and to increase profits. However, effects of IMTA on host immune and metabolic responses, the dynamics of bacterial communities, and antibiotic resistance genes (ARGs) are relatively understudied. In this study, the IMTA mariculture systems with different combinations of the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), the whiteleg shrimp (Litopenaeus vannamei), and the alga (Gracilaria bailinae) were constructed to evaluate the effects of different culture systems on the aquatic environment and cultured species during the initial stage of aquaculture. Results showed that G. bailinae could effectively remove inorganic nutrients accumulated in water, which in turn significantly promoted growth and enhanced non-specific immunity and glycolipid metabolism in the hybrid grouper. Although ARGs were present throughout the culture process, the relative abundance of ARGs in three of the four culture systems decreased with culture time. This was especially the case in the monoculture and the fish-algal IMTA systems, indicating that these aquaculture systems had a scavenging effect on ARGs in the environment. Correlation and network analyses indicated that ARGs were not significantly correlated with environmental factors but were closely related to bacterial communities. Approximately 25 bacterial groups of Bacteroidetes and Proteobacteria were significantly correlated with ARGs, including aminoglycoside, beta-lactamase, chloramphenicol, and multidrug resistance genes. Moreover, tnpA-07, a transposase gene, indirectly affected ARGs through its interaction with Acteroidetes and Proteobacteria. This study provides insights into further optimizing culture methods and controlling the spread and ecological risk of ARGs in IMTA systems.

Effects of different culture systems on the culture of prepuberal buffalo (Bubalus bubalis) spermatogonial stem cell-like cells in vitro.

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3+ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.

Effects of different culture systems on growth, immune status, and other physiological parameters of tench (Tinca tinca)

Tench production is limited in traditional and low intensity cultures. However, its demand is increasing and makes necessary the evaluation of physiological parameters that could affect tench welfare under intensive conditions. This study analyses the growth performance and related indices, hematological and immunological parameters as well as hormone profile and electrolytes in plasma, in fish under three different culture systems: extensive (E), with initial density of 0.07kg/m3, no control of environmental parameters and natural feeding; semi-intensive (SI), with initial density of 0.42kg/m3, partial control of temperature and supplementation of artificial food; and intensive (I), with initial density of 2.5kg/m3, total control of photo- and thermo-period and exclusive artificial diet. Results showed that variables such as weight, length, density increase and specific growth rate (SGR) were higher in I. Higher hepatosomatic index were found in I and SI. In addition, E and SI cultures, which kept natural conditions, achieved greater gonadal increase in detriment of somatic growth. On the other hand, the analysis of hormones and electrolytes, hematological and immunological parameters did not reveal a disturbed immune function in I compared to SI and E. Thus, the general activation of the nonspecific immune system and the mobilization of energy reserves associated to gonadal maturation in fishes from S-I and E systems could be related to an adaptive, non-pathological, response to the natural environmental changes, resulting in reduction of productivity. With all in mind, it can be concluded that adequate artificial feeding and control of the environmental parameters can improve tench productivity by intensive systems without major perturbations of its welfare.

[Effects of Different Culture Systems on the Hematopoietic Differentiation Ability of iPS Cells].

To investigate the in vitro effects of different culture systems on hematopoietic differentiation ability of induced pluripotent stem (iPS) cells. Two culture systems including E8 and mTESR(freeder-free medium), and the classical ES culture medium were chosen for culture of iPS cells. The iPS cells maintaining in above mentioning culcure systems were co-cultured with OP9 cells(murine bone marrow stromal cells) in vitro to be induced to differentiate into hematopoietic stem/progenitor cells. Flow cytometry and real-time quantitative PCR were used to detect the expression of specific hematopoietic markers and the effects of different culture systems on the differentiation of iPS in vitro. iPS cultured in the 3 selected medium could be differentiated into hematopoietic stem cells. Efficiency of hematopoietic differentiation was up to 28.4% in classical ES culture system, which was significantly higher than that in E8 and mTESR system. Under the co-culture with OP9, iPS can differentiate into hematopoietic stem/progenitor cells, which shows higher efficiency when iPS maintained in the ES medium.

Effect of different culture systems on the production of foot and mouth disease trivalent vaccine.

Aim:This study aims to determine the effect of the stationary rawx, roller, and the suspension cell culture systems on the total virus yield infectivity and antigenicity.Materials and Methods:Three serotypes of foot and mouth disease virus (FMDV) (serotype A, O and SAT-2) were inoculated separately into baby hamster kidney-21 cell line in rawx, roller, and suspension cultivation systems using multiplicity of infection (1:100). Samples were taken from the total virus yield from each system at 15, 18, 21, and 24 h post-inoculation. Testing the total virus yield infectivity through virus titration and antigenicity through estimation of complement fixing titer and 146S content and evaluation of the potency of the vaccine prepared from the different cultivation systems were done.Results:The results showed that the FMDV titer of serotype A, O, and SAT-2 obtained from the roller cultivation system showed the highest level followed by suspension cultivation system then the rawx cultivation system. The FMDV titer showed its highest level at 21 h post-inoculation in all the cultivation systems and then decline at 24 h post-inoculation. The antigenicity reached its highest value content at 18 h post-inoculation either by complement fixation test or by quantifying the 146S intact virion. Montanide ISA 206 oil inactivated trivalent vaccines were prepared from the tested serotypes (A Iran O5. O Panasia and SAT-2/EGY/2012) harvested at 18 h post-inoculation from the 3 culture systems. The results of tracing the antibody response showed that the mean antibody response from the roller cultivation system start its protective antibody titer earlier at 2 weeks post-vaccination (WPV) than the vaccine prepared from the other two cultivation system and the immune protection period lasts longer for 36 WPV for the roller cultivation system vaccine than the other two cultivation systems.Conclusion:The best cultivation system used for the production of FMD vaccine regarding its highest infectivity and antigenicity is the roller system.

Tissue-specific Cd and Pb accumulation in Peruvian scallop (Argopecten purpuratus) transplanted to a suspended and bottom culture at Sechura Bay, Peru

In order to understand the effect of different culture systems on Cd and Pb accumulation, suspended long-line and bottom cultures of Argopecten purpuratus were conducted during January until April 2010 (120days). The Cd tissue levels were the highest at the middle of the experiment (30-d till 70-d) for suspended-cultured individuals, while bottom-cultured individuals showed an increasing trend. Gonad Pb levels were also higher during the same period for all cultures, while adductor muscle exhibited no considerable variations. Cd and Pb tissue concentrations were mainly greater in deeper cultures. There were no significant differences in Cd and Pb accumulation between individual sizes. The Cd and Pb levels in edible tissue (gonad+adductor muscle) did not exceed the EU and FDA maximum levels. Based on the target hazard quotient (THQ) and the provisional tolerance weekly intake (PTWI), no risk (THQ<1 and %PTWI<30) was found for human consumption.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10μM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

Low Oxygen Tension and Relative Defined Culture Medium with 3, 4‐Dihydroxyflavone are Beneficial for Yak–Bovine Interspecies Somatic Cell Nuclear Transfer Embryo

With an aim to improve the efficiency of yak-bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak-bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with qRT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (P<0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation (P<0.05). However, under the same oxygen tension, the mRNA abundance of IFNT of RDCM groups was higher than that of the mSOF groups. In addition, high oxygen tension during in vitro culture (IVC) with RDCM significantly increases the mRNA expression of oxidative stress-related genes (GPX-1, SOD-1, CAT and GSS) (P<0.05). 3, 4-Dihydroxyflavone (DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM (P<0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak-bovine iSCNT embryo during IVC.

Effect of Different Culture Systems and 3, 5, 3'-Triiodothyronine/Follicle-Stimulating Hormone on Preantral Follicle Development in Mice

The mechanical method to isolate preantral follicle has been reported for many years. However, the culture systems in vitro are still unstable. The aim of this study was to analyze the effect of the culture system of mice preantral follicles on the follicular development in vitro. The results showed that the 96-well plate system was the most effective method for mice follicle development in vitro (volume change: 51.71%; survival rate: 89%, at day 4). Follicle-stimulating hormone (FSH) and Thyroid hormone (TH) are important for normal follicular development and dysregulation of hormones are related with impaired follicular development. To determine the effect of hormone on preantral follicular development, we cultured follicle with hormones in the 96-well plate culture system and found that FSH significantly increased preantral follicular growth on day 4. The FSH-induced growth action was markedly enhanced by T3 although T3 was ineffective alone. We also demonstrated by QRT-PCR that T3 significantly enhanced FSH-induced up-regulation of Xiap mRNA level. Meanwhile, Bad, cell death inducer, was markedly down-regulated by the combination of hormones. Moreover, QRT-PCR results were also consistent with protein regulation which detected by Western Blotting analysis. Taken together, the findings of the present study demonstrate that 96-well plate system is an effective method for preantral follicle development in vitro. Moreover, these results provide insights on the role of thyroid hormone in increasing FSH-induced preantral follicular development, which mediated by up-regulating Xiap and down-regulating Bad.

Effect of different culture systems on mRNA expression in developing rabbit embryos

The rate of zygotes in vitro developed to hatched blastocyst stage was evaluated between two different commercial media (TCM-199 and Ham's F10) and two different culture systems (renewal and non-renewal single medium) to determine the effects of culture conditions on rabbit embryo preimplantation development. The relative transcript abundances of OCT4, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor 3 (erbB3) of resultant blastocysts were also analysed and compared with in vivo developed blastocysts. Results showed an important divergence in mRNA expression between embryos developed under in vivo and in vitro conditions despite there being no significant difference in hatching blastocyst rates between different culture systems and different media. For OCT4, transcript abundance of in vitro culture embryos differs from their in vivo chronological counterparts, but, when the medium is renewed, mRNA expression seemed similar to in vivo developed 4-day-old embryos. In addition, VEGF and erbB3 expression showed marked variation between different in vitro conditions. Therefore, the study of specific transcript abundance in rabbit blastocyst provides a more detailed description of which alterations in gene expression occur due in vitro conditions, and further studies should be carried out to reduce current limitations of long-term culture of rabbit pre-implantation embryos.

Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.

188 EFFECTS OF CULTURE MEDIUM AND PROTEIN SUPPLEMENTATION ON mRNA EXPRESSION OF IN VITRO-PRODUCED PREIMPLANTATION BOVINE EMBRYOS

Numerous studies have reported aberrant gene expression levels in embryos attributed to suboptimal culture conditions. This study investigated the effects of different culture systems and protein sources on the development of IVP embryos as measured by cleavage and blastocyst rates, cell number, and relative abundance levels of Oct-4, Connexin 43, Nanog, and glucose transporter-1 (Glut-1) when compared with in vivo embryos. Experiment (Exp) 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium (KSOMaa) supplemented with amino acids. Exp 2 compared the same two culture systems with and without the addition of calf serum (CS). Oocytes were matured for 22 h, fertilized in vitro, and then cultured in the appropriate treatment medium. RNA was extracted from pools of blastocysts, reverse transcribed to cDNA, and primer-specific amplified via Q-PCR. Exp 1 analyzed 10 pools per treatment, and either 10 (in vivo) or 11 pools were analyzed per treatment in Exp 2. One-way ANOVA followed by multiple pair-wise comparisons using Tukey's test was used to detect differences between treatments and in vivo embryos (P &lt; 0.05). Results from both experiments indicated that, despite similar cleavage and blastocyst rates among treatments, significant differences were detected at the mRNA level and in cell numbers between treatments. In Exp 1, Oct-4 was found to have a mean abundance mRNA level significantly greater in KSOMaa-cultured blastocysts than in either SOFaa-cultured blastocysts or in vivo embryos. The same pattern of upregulation of Oct-4 in KSOMaa or KSOMaa with CS-cultured blastocysts was detected in Exp 2. In contrast to that reported by others, Connexin 43 was not expressed at detectable levels in the in vivo embryos analyzed in our studies. Connexin 43 was not detected in IVP blastocysts used in Exp 1. Conversely, Connexin 43 was detected in KSOMaa, SOFaa, and SOFaa with CS-cultured blastocysts in Exp 2. There was no significant difference in expression of the ICM-specific transcript Nanog in either experiment. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of Glut-1 when compared with other treatments and in vivo embryos. Overall, the transcript levels of the majority of the genes analyzed were altered by the in vitro culture condition. Differences continue to be observed between in vitro-cultured and in vivo embryos, and until these differences are minimized, aberrations in in vitro development will continue to arise. Further research will possibly modify the current culture conditions, allowing for the production of in vitro embryos of higher developmental potential similar to that observed in in vivo-derived embryos.

Developmental regulation of hyaluronan-binding protein (RHAMM/IHABP) expression in early bovine embryos.

Hyaluronan or hyaluronic acid (HA) is a normal component of mammalian follicular, oviduct, and uterine fluids. Granulosa and expanding cumulus cells secrete large amounts of HA, and when HA is added in maturation and culture media, it improves the developmental potential of oocytes and embryos. HA regulates gene expression, signaling, proliferation, motility, adhesion, and morphogenesis. Many of these biological activities of HA are mediated through binding to the receptor for HA-mediated motility/intracellular HA-binding protein (RHAMM/IHABP). We evaluated the presence and dynamics of RHAMM/IHABP mRNA and protein expression in different stages of in vitro-produced bovine embryos using quantitative reverse transcriptase-real time-polymerase chain reaction and immunohistochemistry. We also analyzed the effects of different culture systems on the relative abundance of RHAMM/IHABP transcripts. RHAMM/IHABP mRNA levels decreased from the 2-cell to the 16-cell stage, increased again at the morula stage, and reached their highest level at the expanded blastocyst stage. RHAMM/IHABP mRNA abundance was significantly (P < 0.05) lower in embryos recovered in serum-containing medium than in embryos from serum-free media. Immunohistochemistry revealed the presence of RHAMM/IHABP first in 8-cell stages. Whereas RHAMM staining in 8-cell and morula stages was intense, it was weaker in blastocysts. Embryonic secretion of HA increased from the 2-cell stage until the 8-cell stage and then decreased in 16-cell embryos. After this, HA secretion increased in expanded and hatched blastocyst stages. These data suggest that the positive effects of HA on in vitro-produced bovine embryos may be mediated at least in part by RHAMM/IHABP.

Growth, morphology, morphometry and keratin patterns of bovine corneal epithelial cells cultured in vitro

In this study, the effects of different culture systems on bovine corneal epithelial cells were analysed in order to better understand the influence of bovine keratocytes on epithelial cells. Growth, morphological, morphometrical analyses of cells and keratin patterns were evaluated. The aim was to improve the culture technique in order to obtain a good in vitro proliferation of these cells for their employment in clinical and toxicological situations. The bovine corneal epithelial cells were cultured under different conditions: on keratocyte or 3T3-J2 fibroblast feeder layers, with media conditioned either by the two feeder layers or with a basal medium. The epithelial cells cultured on a keratocyte feeder layer as compared to those grown under the other conditions, proved to have a higher growth rate as well as to be smaller in the cytoplasmic and nuclear area; moreover, after 21 days of culture they expressed 64-kDa keratin, designed as a marker for corneal epithelial cell differentiation. To sum up, the keratocyte feeder layer is the most effective for stimulating the growth and differentiation of corneal epithelial cells, resembling the in vivo situation. It might also be successfully employed for clinical and toxicological purposes.

Fertilization and early embryology: Effect of different co-culture systems in early human embryo development

The objective of this study was to examine the effects of different culture systems on the development of early human embryos in vitro. A total of 460 fertilized oocytes from 82 cycles of patients was transferred into one of four systems: (1) into droplets of Ham's F10 medium + 12% normal human serum (NHS); (2) co-cultured on a human granulosa monolayer; (3) co-cultured with bovine oviductal epithelial cells (BOEC); or (4) co-cultured with bovine uterine epithelial cells (BUEC). The percentage of cleavage and the morphological appearance of embryos were recorded daily for 72 h in each system using an inverted phase-contrast microscope. The results showed that the proportions of the fertilized oocytes which developed to the four-cell stage 48 h after retrieval were, by culture system: (1) 70% (84/120); (2) 74% (85/115); (3) 78% (91/117); and (4) 76% (82/108). At 72 h after retrieval, the proportions of the eight-cell stage were, by culture system: (1) 45% (38/84); (2) 62% (53/85); (3) 75% (68/91); and (4) 70% (57/82). We concluded that a higher proportion of fertilized oocytes developed to embryos at the eight-cell stage in systems 2, 3 and 4 than in system 1. This indicates the beneficial effect of co-culture of human embryos with granulosa cell, BOEC and BUEC monolayers, which may be due to various factors.