The vitrification of in vitro fertilized cow blastocysts by the open pulled straw method

In 6 replicates, a total of 450 immature oocytes recovered from 144 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in tissue culture medium 199 (TCM-199) supplemented with fetal calf serum. Of 126 blastocysts (28% of oocytes cultured), 117 (26% of oocytes cultured) were vitrified in Hepes/bicarbonate-buffered TCM-199 medium and 20% fetal calf serum, with ethylene glycol and dimethylsulfoxid as the cryoprotectants. After thawing in 1.2 mL holding medium with 0.25-M sucrose and after 1 min in holding medium with 0.15-M sucrose, blastocysts were cultivated in vitro for 24 h. The re-expansion rate of blastocysts was 69.2% (81 blastocysts), and 39.5% (32 blastocysts) were hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on 7 and 8+9 days (74.6% and 46% vs. 62% and 29%, respectively). After transfer to recipient cows, 3 out of 6 were diagnosed by ultrasonography as pregnant. Three calves were born from 18 transferred embryos (16.7%). The open pulled straw (OPS) method seems to be a convenient, simple and effective method for cryopreservation of 7 to 9 d bovine embryos.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

12q13 fragility in a family with recurrent spontaneous abortions: expression of the fragile site under different culture conditions

A fragile site at the 12q13 band was found in metaphases from lymphocyte cultures of three members of a family. A comparison of the frequency and expression of the fragile site was carried out on cells cultured in RPM-I 1640 with and without BrdU and in 199 media. The fragile site was not typically folate-sensitive, being expressed in standard medium as well as in cultures after exposure to BrdU.     

Development of porcine blastocysts from in vitro- matured and activated haploid and diploid oocytes

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 rhojusec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 micro/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat- treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whittenk medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electro-activation (P < 0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro- activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P < 0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P < 0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.     

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the >or= 2-cell (51 to 60%), >or= 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

Long-term viability and differentiation of bovine oviductal monolayers: Bidimensional versus three-dimensional culture

Different in vitro models have been developed to study the interaction of gametes and embryos with the maternal tract. In cattle, the interaction of the oviduct with gametes and embryos have been classically studied using oviductal explants or monolayers (OMs). Explants are well differentiated but have to be used within 24 h after collection, whereas OMs can be used for a longer time after cell confluence but dedifferentiate during culture, losing cell polarity and ciliation. Herein, OMs were cultured either in M199 plus 10% fetal calf serum or in a semidefined culture medium (Gray's medium), in an immersed condition on collagen-coated coated microporous polyester or polycarbonate inserts under air-liquid interface conditions. The influence of culture conditions on long-term viability and differentiation of OMs was evaluated through scanning electron microscopy, localization of centrin and tubulin at the confocal laser scanning microscope, and assessment of maintenance of viability of sperm bound to OMs. Findings demonstrated that OMs cultured in an immersed condition with Gray's medium retain a better morphology, do not exhibit signs of crisis at least until 3 wks postconfluence, and maintain the viability of bound sperm significantly better than parallel OMs cultured in M199 plus 10% fetal calf serum. OM culture with Gray's medium in air-liquid interface conditions on porous inserts promotes cell polarity, ciliation, and maintenance of bound sperm viability at least until 3 wks postconfluence. In conclusion, oviduct culture in Gray's medium in an immersed or air-liquid condition allows long-term culture and, in the latter case, also ciliation of bovine OMs, and may represent in vitro systems that mimick more closely the biological processes modulated by the oviduct in vivo.     

Variant endothelial cells from human carotid artery in culture

Summary Endothelial cells were cultured from the carotid artery with thickened intima comprised of two to five layers of smooth muscle cells, isolated from a 19-yr-old female, who died from a accident. The cells were grown and subcultured in Medium 199 supplemented with 20% heat inactivated fetal bovine serum. The cells are still viable at present, the 22nd passage. The cultured cells were found to have the following characteristics: existence of Factor VIII-related surface angiten and prostacyclin synthesis slightly less than that for typical endothelial cells. The most outstnading feature was the formation by an individual cell of a single ring, and composite ring formed by two to five cells. Neither the synthesis of an angiotensin converting enzyme nor that of a Weibel-Palade body could be detected by electron microscopy. The cultured cells possessed only a few characteristics specific for typical endothelial cells and were designated as variant endothelial cells.     

Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.     

Nuclear maturation of canine oocytes cultured in protein-free media

The objective of this study was to determine the ability of canine oocytes to complete nuclear maturation in a protein-free medium. Oocytes obtained from ovaries of bitches aged 6 months to 2 years were cultured either in TCM199 or CMRL1066 medium without protein supplementation in 5% or 20% O(2). Sixteen of 121 (13%) oocytes cultured in TCM199 reached metaphase II, but only 1 of 135 oocytes cultured in CMRL1066 did so (P < 0.05). Oxygen concentration did not affect nuclear maturation. An additional 103 oocytes were cultured in TCM199 for 48 hr, inseminated with chilled ejaculated spermatozoa, fixed in 1:3 acetic acid-ethanol and then stained with aceto-orcein; 34% of these oocytes were penetrated by spermatozoa. To determine developmental competence of oocytes cultured in a protein-free medium, 85 oocytes were cultured in TCM 199 for 48 hr, inseminated and then cultured; 7 early stage embryos were produced. The effects of growth hormone, beta-mercaptoethanol (betaME), luteinizing hormone (LH) and energy substrates, alone or in combination, on nuclear maturation of oocytes cultured in a protein-free medium were also determined. Growth hormone enhanced cumulus expansion, but did not improve nuclear maturation. beta-mercaptoethanol had no effect on nuclear maturation. However, percentages of MII oocytes significantly decreased when the oocytes were cultured for 48 hr in the medium containing LH or a high concentration of glucose (P < 0.05). In conclusion, canine oocytes are able to complete nuclear maturation in a protein-free medium. The specific type of medium and other supplements significantly influence the meiotic maturation of canine oocytes.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrient deprivation increases vulnerability of endothelial cells to proinflammatory insults

Nutrient deprivation is a stimulus for oxidative stress and is an established method for induction of cell autophagy and apoptosis. The aims of this study were to identify conditions that evoke superoxide production in cultured human umbilical vein endothelial cells (HUVECs), determine the mechanism of action for this response, and examine whether the stimulus might facilitate the adhesion of human isolated neutrophils to the HUVECs. HUVECs were incubated in M199 medium under conditions of serum starvation (serum-free M199 medium), low serum (medium containing 2% fetal calf serum), and high serum (medium containing 20% fetal calf serum). HUVECs were also incubated under proinflammatory conditions, in medium supplemented with 50 ng/ml tumor necrosis factor-α (TNF-α) or neutrophils preactivated with 10 nM phorbol 12-myristate 13-acetate (PMA). Superoxide production was increased fourfold in serum-starved HUVECs compared to cells incubated in 20% medium, and this was reduced by inhibitors of the mitochondrial electron transport chain and mitochondrial Ca²⁺ uniporter. Superoxide production was 23.6% higher in HUVECs incubated with TNF-α in 2% medium compared to 2% medium alone, but unchanged with TNF-α in 20% medium. PMA-activated neutrophils adhered to morphologically aberrant HUVECs, which were mainly evident under the low-serum condition. The findings show a role of mitochondrial enzymes in superoxide production in response to nutrient deprivation and suggest that proinflammatory responses in HUVECs become manifest when HUVECs are in an already-compromised state.     

The effect of human amniotic fluid in supporting long-term survival of pancreatic islets in tissue culture

Pancreatic islets obtained from newborn Lewis rats were kept free-floating in TCM 199 either in the presence of 50% human amniotic fluid (HAF), 50% Hank's balanced salt solution (HBSS) or 10% fetal calf serum (FCS) for up to 14 days. The culture, in the presence of HAF, resulted in a good islet viability as demonstrated by islet number, islet insulin content and insulin release into the medium. Contradictory results were obtained when HBSS was used as the medium supplement. Moreover, the islets cultured in HAF-supplemented medium are characterized by a marked replicatory activity as reflected by the incorporation of labeled thymidine into islet DNA and gradual increase in DNA content with the progression of culture time. Even if compared to DNA synthesis of islets cultured under so called standard culture conditions as e.g. supplementation of 10% FCS to the medium, 50% HAF but not yet 10% HAF was found to stimulate the islet replication twofold already after 4 days of culture. It was also demonstrated that FCS has no additional effect on HAF-stimulated DNA synthesis. These findings support the view that HAF-enriched culture medium may have a use in supporting the long-term survival of well preserved endocrine pancreatic tissue.     

Oxygen tension and medium type actions on blastocyst development and interferon-tau secretion in cattle

Most current in vitro production systems terminate at the blastocyst stage in cattle. The goal of the present research was to identify culture conditions that support individual blastocyst survival and interferon-tau (IFNT) production in cattle. In the first study, two media (medium 199 [M199] and potassium simplex optimized medium [KSOM]) and two oxygen tensions (5 and 20%) were compared for their ability to sustain blastocyst survival and IFNT production from days 8 to 11 post-insemination. Survival and total cell numbers were greater (P<0.05) for blastocysts cultured in M199 in a 5% oxygen environment compared with other medium and oxygen treatment combinations. Serum supplementation was required for blastocyst survival and IFNT production. IFNT concentrations in conditioned medium were similar for blastocysts cultured in M199 or KSOM, but blastocysts incubated in 5% oxygen produced less (P<0.001) IFNT than their 20% oxygen counterparts. Oxidative stress was not responsible for the increase in IFNT concentrations. Supplementation with fibroblast growth factor 2 did not affect cell numbers but increased (P<0.02) IFNT concentrations for blastocysts cultured in 5% oxygen but not those cultured in 20% oxygen. In conclusion, culturing blastocysts of cattle in a 5% oxygen environment with M199 containing serum sustains embryo viability and permits constitutive and inducible IFNT production. Incubation in 20% oxygen increases IFNT production. The mechanism responsible for this event and its physiological relevance to conceptus development in utero remain unknown.     

Chemically defined and serum supplemented media effects on mouse embryo development

Mouse embryos at various stages of development were used to test three types of media: TC199, CMRL-1066 and TALP. The effect of 20% human cord serum (HCS) and fetal calf serum (FCS) were also compared in TC199 and CMRL-1066 media. Embryos were recovered at the two, four and eight cell stages and assessed for at least one cleavage progression in 24 hours. There was no difference in embryo growth rates between the media for four and eight cell embryos, however TALP significantly increased the proportion of two cell embryos which underwent at least one cleavage division. HCS significantly promoted a greater number of cleavage divisions compared with FCS. This study indicates that the defined medium (TALP) can be employed for equal or increased growth rates of early mouse embryos cultured in vitro compared to two serum supplemented media (TC199 and CMRL-1066).     

Interaction of blood lymphocyte Fc receptors with IgG during in vitro cultivation

Bovine blood lymphocytes taken from normal cows and those suffering from chronic lymphocytic leukemia were cultured in complete medium 199 with 10% of heat-inactivated fetal bovine serum. After a 48 hour culturing an enhanced quantity of the Fc-receptor bound fluoresceinated immunoglobulin G (IgG) was established. When lymphocyte fractions enriched with T- or B-cells were cultured, the binding capacity of Fc-receptors for IgG (48 hours after culture) increased in both the cell populations. A study of the kinetics of interactions of Fc-receptors with IgG showed that the increased number of Fc-receptors after culturing was followed by an enhanced affinity of Fc-receptors towards IgG. The affinity of Fc-receptors of blood lymphocytes of cows with chronic lymphocytic leukemia was lower than that of normal lymphocytes.     

Relaxin, oxytocin, and prostaglandin effects on progesterone secretion from bovine luteal cells during different stages of gestation

To determine the effects of relaxin, oxytocin, and prostaglandin F2 alpha on progesterone secretion, bovine luteal cells from different stages of gestation were dispersed in Medium 199 with 200 units/ml penicillin, 1.0% kanamycin, 0.5% bovine serum albumin, and 400 units/ml collagenase. Cells (10(5) were cultured in 400 microliters of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing fetal bovine serum and antibiotics, in Falcon multiwell plates, in a humidified environment of 95% O2 and 5% CO2 at 37 degrees C. Cells were cultured for 24 hr without treatment and thereafter with medium-hormone replacement every 24 hr. Progesterone was quantified from unextracted media by radioimmunoassay. Basal progesterone secretion after 24 hr was 1.81 +/- 0.14, 1.76 +/- 0.17, 0.54 +/- 0.49, and 0.57 +/- 0.21 pg/ml per viable luteal cell from 145-, 165-, 185-, and 240-day-old corpora lutea, respectively. Basal progesterone secretion increased (P less than 0.05) with time in culture. Relaxin induced a dose-dependent (greater than 100 ng/ml) increase in progesterone release, compared with the controls. Oxytocin and prostaglandin F2 alpha induced greater release (P less than 0.05) of progesterone than relaxin at all stages of gestation, but progesterone release was dependent on the stage of gestation and the duration in culture. Luteinizing hormone (100 ng/ml) stimulated whereas 17 beta-estradiol (50 ng/ml) inhibited progesterone secretion by luteal cells at all stages of gestation examined. Relaxin obliterated the prostaglandin- and oxytocin-induced progesterone secretion by bovine luteal cells from 145 to 214 days of gestation. Thus, relaxin, cloprostenol, and oxytocin regulate progesterone production by cultured bovine luteal cells, but hormone secretion was dependent on the stage of gestation.     

Primary mantle tissue culture from the bivalve mollusc Mytilus galloprovincialis: investigations on the growth promoting activity of the serum used for medium supplementation

The main objective of the present work was to evaluate with a quantitative approach the effects of different chicken serum (CS) concentrations in the medium used for mussel mantle tissue culturing. Our results showed that a CS level of 20% was optimal. Under these conditions, the cultures reached a maximum mean number of mitotic figures per slide exceeding widely 100. Cultures were also achieved to test whether CS must be heat treated to inactivate complement components before use for medium enrichment. We demonstrated that heat inactivation did not significantly change the promoting activity of the CS. Finally, the growth-stimulatory properties of CS were compared to those of fetal calf serum (FCS). The best results were obtained with 30% FCS. The difference with 20% CS was not statistically significant, but the FCS yielded a much higher level of polyploid metaphases than the CS. Since it had no adverse effect such as polyploid metaphases induction, was readily commercially available and relatively less expensive than other additives, CS at the concentration of 20%, without heat-decomplementation, is routinely used as growth medium supplement for mussel mantle cell culturing in our laboratory. Even though our primary cultures do not have the potency of continuous cell lines, it is possible to use such cultures for in vitro experiments.