Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows

The purpose of this study was to compare embryo quality of lactating Holstein Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH) and Belgian Blue beef cows (BB) and to identify factors that are associated with embryo quality in LHFC and NLHFH. After superovulation and embryo recovery at Day 7, embryos (n=727 from 47 LHFC, 27 NLHFH and 50 BB) were scored morphologically for quality, colour and developmental stage. Blood samples and data concerning parity, age, milk production and management were collected. Data were compared univariably between the three groups. A multivariable regression model was built with quality and colour of the LHFC and NLHFH embryos as dependent variables. Only 13.1% of LHFC embryos were categorized as excellent compared to 62.5% and 55.0% of the embryos in NLHFH and BB, respectively. Almost none of the NLHFH or BB embryos displayed a dark appearance of the cytoplasm compared to 24.1% of the LHFC embryos. Only 4% of all LHFC embryos reached blastocyst stage compared to 23.2% and 17.3% in NLHFH and BB. Based on the multivariable regression analysis, "physiological status" (lactating or not) together with the serum total protein concentration of LHFC and NLHFH, was significantly associated with embryo quality and colour. Thus, LHFC display an inferior embryo quality compared to NLHFH and BB. Producing milk or not seems to be significantly associated with embryo quality. Therefore, reduced embryo quality on Day 7 following AI, could be an important factor in the subfertility problem in modern high-yielding dairy cows.     

Ovum pick up and in vitro embryo production in cows superstimulated with an individually adapted superstimulation protocol

The aim of this experiment was to apply an ovarian superstimulation protocol prior to ovum pick up (OPU), tailored to the individual donor response, to evaluate its advantages and disadvantages in terms of follicle numbers and diameters, the numbers of retrieved oocytes and day 7 cultured blastocysts. Ten adult non-lactating dairy cows were superstimulated with pFSH and subjected to ovum pick up-in vitro fertilisation (OPU-IVF) 6 times at 2-week intervals. On day 0 of each 2-week period, all follicles >8mm were ablated and an ear implant (Crestar, Intervet, Belgium) was inserted. On day 2, 48 h after follicle ablation the animals were administered six equal doses of pFSH, divided into morning and evening doses for 3 days. On day 7, 48 h following the last pFSH injection, follicle diameters were measured by ultrasound and all follicles were subjected to OPU. All cumulus-oocyte complexes (COC), regardless of their quality, were subjected to in vitro maturation-in vitro fertilisation-in vitro culture (IVM-IVF-IVC). The total dose of pFSH prior to the first OPU session was 300 microg per animal. During the following OPU sessions, the total pFSH dose was either kept unchanged, increased or reduced (+/-50 microg), according to the percentage of follicles of more than 11 mm in diameter, present in the previous session of that particular donor. The mean number of punctured follicles per session was 11.9 +/- 7.7 (mean +/- S.D.), with 16% of follicles exceeding 11 mm. These follicles yielded a mean of 5.6 +/- 4.1 cumulus oocyte complexes (COC), 32% of which had >/=3 layers of cumulus cells (quality 1 and 2). The recovery rate was 47%. Finally, all COC were subjected to IVM-IVF-IVC, which resulted in a mean of 2.0 +/- 2.3 blastocysts on day 7 postinsemination. The subtle changes in pFSH dose influenced the sizes but not the numbers of follicles, the latter parameter was influenced by the individual donor and the OPU session.     

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.     

Retinoic acid potentiates TNF-alpha-induced ICAM-1 expression in normal human epidermal keratinocytes

ICAM-1 protein in keratinocytes is thought to contribute to cutaneous inflammatory reactions. Its induction depends-among others-on cytokines such as TNF-alpha, IFN-gamma, IL-1 or on retinoic acid (RA), a key regulator of epidermal homeostasis. We investigated the effect of treatments with TNF-alpha, RA or their combination on ICAM-1 expression on proliferative or differentiating keratinocytes over an 8 day culture period. Basal ICAM-1 levels were undetectable at low (30 microM) and standard (88 microM) Ca2+ and RA alone did not induce ICAM-1. However, at high Ca2+ (1500 microM), ICAM-1 levels were augmented in response to RA-treatment. TNF-alpha induced a transient ICAM-1 increase in NHK, which reached peak-levels 2-4 days post cytokine stimulus. RA potentiated the TNF-alpha-induced ICAM-1 response in all Ca2+-concentrations. This potentiating effect of RA was confirmed at the mRNA level. In summary, our results establish retinoic acid as an enhancer of TNF-alpha-induced ICAM-1 levels in NHK.     

Media for hybridoma growth and monoclonal antibody production

For the economical production of monoclonal antibodies (MAbs), the cell-culture medium must be optimized for three different phases: growth of the hybridomas, MAb productivity of the hybridomas, and MAb purification or downstream processing. Medium improvements are necessary to meet these requirements for large-scale MAb production. Information bearing on this issue is being addressed in two research areas, cell biology and biochemical engineering, and is reviewed in this article.     

Occlusion immobilization of hybridoma cells in chitosan

Summary The use of chitosan fibers as a matrix for immobilizing hybridoma cells was investigated. Optimal cell entrapment within fibrous chitosan occurred at pH values below 7.2. Chitosan fibers were found to be more effective than chitosan beads in the occlusion of the hybridoma cells. Maximum total cell concentrations of 5 to 5.8 × 10⁶ cells/mL were obtained in the chitosan fractions of cultures containing 25% to 50% chitosan volume fractions. The viability of the cells was found to be unaffected by the presence of chitosan in culture.     

Effects of long-term treatment with bovine somatotropin on follicular dynamics and subsequent oocyte and blastocyst yield in an OPU-IVF program

Fourteen Holstein-Friesian heifers between 15 and 22 months of age with normal reproductive tracts were used in an experimental set up to investigate the effect of a long term rBST-treatment on the follicular population prior to transvaginal ovum pick-up (OPU). The estrous cycle of the animals was synchronized by means of a double injection of 2 ml cloprostenol 11 days apart. The heifers were divided in 2 equal groups (n = 7) in which the animals had the same average body weight, one group receiving a weekly subcutaneous injection of 640 mg recombinantly derived bovine somatotropin (rBST) on Mondays (rBST-treatment group) and a control group, being injected with 10 ml of saline. Heifers in both groups were submitted to OPU twice a week on Mondays and Thursdays using a 5 Mhz transducer and a disposable, 55 mm long, 20-g short bevelled needle at a vacuum pressure corresponding to approximately 13 ml water/min. The experimental period lasted for 10 weeks (April to June), each animal receiving a total of 10 injections and being submitted to OPU for 20 times. Oocytes were subsequently matured and cultured in a separate drop per cow following conventional IVF procedures. A blood sample was taken on heparin immediately after each OPU session, for determination of blood progesterone concentrations to assess the influence of treatment and OPU procedure on the cow's estrous cycle. Although results show a significant increase in the total number of follicles and medium sized follicles in the rBST-treated group, no statistically significant different number of retrieved oocytes between the rBST-treated and nontreated group could be detected. The average number of retrieved oocytes per session per cow was comparable, being 6.4 for the treated and 6.0 for the control group. Additionally, the average number of blastocysts per cow per session did not differ significantly between groups, being 1.41 in the rBST-treated group and 1.53 in the control group. The number of cultured oocytes which developed to the blastocyst stage was 22% in the rBST-treated group, which was not significantly different from 25% in the control group. Repeated OPU appeared to induce a certain degree of acyclicity in both treated and nontreated animals.     

Transvaginal ovum pick-up (OPU) in the cow: a new disposable needle guidance system

A study was conducted to modify the routinely used ovum pick up (OPU) devices to permit use of disposable needles and to simplify the technique and to make it more economical and practical to use. Long nondisposable needles are commonly used in transvaginal OPU despite several disadvantages. A new OPU device was developed using 19-g disposable needles to eliminate these disadvantages and to make the technique more successful. The disposable needle was connected to silicone tubing by means of a stainless steel connector. The system was inserted into a stainless steel tube, creating a rigid structure within which to move the needle back and forth. A blunt needle can be changed simply by replacing it with a new one, even while the device is in the vagina of a cow. The needle guidance system is incorporated into a new OPU device together with the transducer of an ultrasonographic scanner with an unilateral orientated scanning field, making it possible to utilize the needle length to its maximum. This combination permits easy manipulation of the ovaries, easy positioning of follicles on the puncture line, and enables the use of shorter needles which directly enter the scanned area without loss of useful needle length. As a preliminary result we obtained an overall oocyte recovery rate of 42%. Although this is promising, additional puncture sessions are needed to establish more consistent recovery rates. When OPU is used routinely, application of short disposable needles is more practical and economical.     

Biotechnology and aquaculture: the role of cell cultures

Cell culturing complements recombinant DNA technology in the application of biotechnology to aquaculture. Cell cultures can be prepared from the three main groups of multicellular organisms in aquaculture: fish, shellfish, and seaweeds. These cultures can contribute indirectly to the successful farming of these organisms by providing basic insights into how their growth, reproduction, and health can be understood and manipulated. Finally, they can be a direct source of diverse biochemical products for use in aquaculture, medicine and the food industry.     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.