Heat-treated Fetal Calf Serum Research Articles

Inherent inferior quality of follicular fluid in repeat breeder heifers as evidenced by low rates of in vitro production of bovine embryos

The aims of the present study were to determine the effect of follicular fluid obtained from the ovulatory follicle of repeat breeder heifers on in vitro oocyte maturation (Experiment 1), fertilization (Experiment 2) and production of bovine embryos (Experiment 3). Holstein virgin heifers (VH, n = 5) with normal fertility or repeat breeder syndrome (RBH, n = 5) were used in the present study. Follicular fluid of VH and RBH was aspirated from ovulatory follicles and used as maturation medium. Bovine oocytes were aspirated from follicles of slaughterhouse ovaries and randomly allocated in three groups; in Group 1, oocytes cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU/mL hCG plus 0.1 IU/mL rFSH); in Group 2, oocytes cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in Group 3, oocytes cultured in TCM-199 supplemented with 10% filtered follicular fluid of RBH without hormones. The mean (±SEM) percentage of matured oocytes was different between VH and RBH groups (72.2 ± 4.0 vs 56.4 ± 4.6%; P < 0.05, respectively). Further, the mean (±SEM) percentage of normal oocyte fertilization was higher in the VH than the RBH group (49.3 ± 2.1 vs 32.0 ± 4.2; P < 0.05, respectively). The mean percentage of embryos developed to the blastocyst stage was higher in the VH than the RBH group (12.0 ± 1.3 vs 7.0 ± 1.6; respectively; P < 0.05). In conclusion, our findings support our hypothesis that the ovulatory follicle microenvironment of Holstein repeat breeder heifers places their oocytes at a developmental disadvantage compared with Holstein fertile virgin heifers and that this suggest the existence of an inherent inferior quality of the ovulatory follicle microenvironment in repeat breeding Holstein heifers.

Effect of follicular size on in vitro developmental competence of oocytes and viability of embryos after transfer in the dromedary ( Camelus dromedarius)

The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3–6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 °C, under 5% CO 2 and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 μM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs ( n = 30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 × 10 6 spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O 2 and 90% N 2. The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively ( P > 0.1). The rate of total cleavage (two cells to blastocyst stage) was greater ( P < 0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively ( P < 0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6 mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% ( n = 8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.

397 LONG-TERM CULTURE OF BOVINE SECONDARY FOLLICLES IN MEDIA CONTAINING BSA, FCS, PLASMA, AND FOLLICULAR FLUID

Bovine oocytes within early antral follicles 0.4–0.7 mm in diameter (oocyte: 90–99 �m in diameter) grow to a final size of 120 �m after culture for 2 weeks. However, there has been little success in culturing oocytes in secondary follicles. The aim of this study was to establish a long-term culture system to support the growth of bovine oocytes within secondary follicles. We examined the effect of bovine serum albumin (BSA), heat-treated fetal calf serum (FCS), bovine plasma (Pl), and bovine follicular fluid (FF) on follicular development and oocyte growth. Bovine secondary follicles 150–200 �m in diameter were collected mechanically from bovine ovaries. In the first experiment, secondary follicles were embedded in collagen gels and cultured in �MEM supplemented with 0.1 mg mL-1 sodium pyruvate, 0.08 mg mL-1 kanamycin, 0.05 mM β-mercaptoethanol, 25 or 50 ng mL-1 FSH, and 3 mg mL-1 BSA, 5% FCS, 5% Pl, or 5% FF for 4 weeks. Secondary follicles formed an antrum and the mean diameters of follicles increased significantly in all groups (P &amp;lt; 0.05; Student&amp;apos;s t-test) other than the BSA-supplemented one. Integrity of the follicles cultured in BSA- and Pl-supplemented media was maintained better than in other groups, and supplementation with 25 ng mL-1 FSH was more effective than 50 ng mL-1 FSH (P &amp;lt; 0.05; chi-square test). Moreover, the antra were maintained in the Pl-supplemented medium. In the BSA- and Pl-supplemented groups with 25 ng mL-1 FSH, 46% (11/24) and 48% (11/23) of normal-looking oocytes were recovered, and their mean diameters were 75.3 � 3.8 and 91.9 � 2.7 �m, respectively, which were significantly larger than before culture (59.8 � 3.5 �m (P &amp;lt; 0.05); Student&amp;apos;s t-test). On the other hand, only 0–7% of the oocytes showed normal morphology in FCS- and FF-supplemented media after 4 weeks. In the second experiment, secondary follicles were cultured in BSA- or Pl-supplemented medium with 25 ng mL-1 FSH for 8 weeks to produce fully grown oocytes. About 30% of the oocytes showed normal morphology in both groups, although they had not reached full size. The diameters of oocytes cultured in BSA- or Pl-supplemented media were 98.8 � 2.1 and 97 � 1.8 �m, respectively. Some of the oocytes from BSA-supplemented medium were denuded, whereas the oocytes were enclosed by cumulus cells in Pl-supplemented medium. These results suggest that bovine secondary follicles can be efficiently cultured in BSA- or Pl-supplemented medium with 25 ng mL-1 FSH in collagen gels over one month.

The first dromedary ( Camelus dromedarius) offspring obtained from in vitro matured, in vitro fertilized and in vitro cultured abattoir-derived oocytes

Dromedary offspring have never been produced fully in vitro. We have previously demonstrated that embryos obtained by culture in semi-defined medium (mKSOMaa) have better in vitro development ability than those cultured with oviductal epithelial cells. The aim of the present experiment was to study the pregnancy rate after embryo transfer of in vitro-produced (IVP) dromedary embryos cultured in semi-defined modified medium (mKSOMaa). IVM/IVF procedures were conducted on six hundred and sixty four (664) cumulus oocytes complexes (COCs) aspirated from ovaries collected at a local slaughterhouse and cultured in vitro (38.5 °C; 5% CO 2, and maximum humidity >95%). Maturation was completed by incubation in TCM-199 medium supplemented with 10% heat-treated Fetal Calf Serum (FCS), 10 ng/mL EGF, 1 μg/mL FSH, 1 μg/mL E2 and 500 μM cysteamine for 30 h. In vitro fertilization was performed using fresh semen (0.5 × 10 6 spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 38.5 °C, 5% CO 2 and 90% N 2 with maximum humidity (>95%). All IVC steps were done in seven replicates. The cleavage rate (two cells to blastocyst stage) was 64% (425/664) and the percentage of oocytes reaching the blastocyst stage was 23% (155/664). The hatching rate of blastocyst obtained after culture was 46% (71/155). Good quality hatched blastocysts ( n = 66) were transferred individually to synchronized recipients. Pregnancy rates, determined by ultrasonography at 15, 60 and 90 days after embryo transfer (ET), were 38%, 32% and 27%, respectively. Out of 18 pregnant females 5 aborted between the fifth and seventh month of pregnancy and 13 females (20%) remained pregnant. After 385 days of pregnancy, the first healthy and normal male-dromedary offspring produced fully in vitro was born at a birth weight of 38 kg. More dromedary calves ( n = 4) were born later on. The remaining pregnant females ( n = 8) are due to calf within the next months. In conclusion, this is the first reported offspring in camelids obtained by transfer of embryos produced by IVM, IVF and IVC using abattoir-derived oocytes, fresh semen and culture in a semi-defined medium.

230 IN VITRO MATURATION OF OOCYTES FROM ENDANGERED DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA)

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO2/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring. This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).

Production of dromedary ( Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 °C, 5% CO 2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 μg/ml FSH, and 500 μM cysteamine. Matured COCs ( n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 × 10 6 spermatozoa/ml in modified TALP-solution). Inseminated oocytes ( n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes ( n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively ( P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.

Cytotoxicity of cholestane 3 beta,5 alpha,6 beta-triol on cultured intestinal epithelial crypt cells (IEC-6).

The effects of cholestane 3 beta,5 alpha,6 beta-triol on intestinal epithelial crypt cells were investigated using the IEC-6 cell line. Cholestane 3 beta,5 alpha,6 beta-triol decreased SH groups (glutathione and protein SH) in the cell, and showed cytotoxicity in a time-dependent manner. Although the concentration of cholestane 3 beta,5 alpha,6 beta-triol used in this study (100 microM) was very high compared with that in plasma of experimental animals, cholestane 3 beta,5 alpha,6 beta-triol did not show any cytotoxicity on IEC-6 cells without fetal calf serum (FCS). The level of cytotoxicity was dependent upon the concentration of FCS in the culture medium. Unknown components in FSC (not VLDL or LDL) were suggested to be associated with the cytotoxicity of cholestane 3 beta,5 alpha,6 beta-triol. Moreover, the fact that even heat-treated FCS (100 degrees C for 30 min) still mediated the cytotoxicity suggested the participation of non-protein components.

Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medium

The effects of fetal calf serum (FCS) or serum fractions on the development of bovine embryos was investigated. Bovine zygotes were produced in vitro and were cultured in a semi-defined culture medium (mSOF). In the first experiment, blastocysts produced in mSOF supplemented with 10% whole heat-treated FCS or desalted FCS appeared about 1 day earlier, their proportion was significantly (P < 0.05) higher (whole: 30%, desalted: 29%) and they had significantly (P < 0.05) more cells at day 8 (119 cells, 127 cells) than did blastocysts produced in mSOF without any supplement (16%, 98 cells) or mSOF supplemented with a glucose concentration equivalent to that of serum (15%, 88 cells). Our results indicate that high molecular mass components (> 5 kDa) of serum are responsible for the effects of FCS on the kinetics of development, on the percentage of blastocysts obtained and the total number of cells in blastocysts. A further analysis using time-lapse microcinematography showed that the acceleration of development induced by serum occurred between the 9-16-cell and morula stages. Finally, in an experiment designed to analyse by microcinematography the effect of the addition of FCS using semen from a different bull to inseminate the oocytes, a different batch of serum and adding mSOF at a different time (42 h after insemination), acceleration was similarly observed between these two stages. Our microcinematographic studies demonstrate that the addition of FCS at two developmental stages (three-four-cell and five-eight-cell) before the 8-16-cell stage accelerates development just after this critical blocking stage.

Effects of amino acids on development of IVM/IVF bovine embryos in a simple protein-free medium

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 °C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 μg/ml FSH, and 500 μM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 × 106 spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.

Activation of the g protein in porcine oocytes

Fertilization in most animal species involves a transient increase in the intracellular free Ca” concentration of the oocyte in the form of a CaPt wave starting from the site of sperm attachment. It is thought that the origin of this Ca” is inositol triphosphate (IP,) induced Ca” release from the endoplasmic reticulum. IP, is derived from the hydrolyses of phosphatidylinositol4,5biphosphate (PIP,) which is catalyzed by the enzyme phospholipase C under regulation of a G protein. Thus the G protein mediated PIP, hydrolysis seems to play an important role in oocyte activation. G proteins or guanine nucleotide binding proteins are a class of membrane proteins that couple membrane receptors to membrane effector enzymes. They are present in a variety of cells, and in oocytes their activation by GTP-7-S (a hydrolysisresistant analog of GTP) causes resumption of meiosis in many species. The present study was undertaken to determine the possibility of porcine oocyte activation through a G protein. Oocytes from slaughterhouse-obtained porcine ovaries were matured in Waymouth medium supplemented with 2.5 mM pyruvate, 10 IU/ml PMSG, 10 IU/ml hCG, 10% (v/v) heat-treated fetal calf serum (Sigma Chemical Co., St. Louis, MO) and 10% (v/v) porcine follicular fluid for 20 h, then in the same medium without hormonal supplement for an additional 16 h. Mature oocytes were stripped of their cumulus cells by vortexing in 0.3 M mannitol solution containing 0.3 mg/ml hyaluronidase. Then 40 pl of 1 mM GTP-7-S (Sigma Chemical Co., St. Louis, MO) was injected near the center of the oocytes using a microinjector (Narishige Co. Ltd, Tokyo, Japan). The carrier medium in which GTP-7-S was dissolved consisted of 100 PM EGTA and 10 mM Hepes buffered at pH 7.0. Control oocytes were injected with either 40 pl of 1 mM GDP-p-S, a metabolically stable analog of GDP (which inactivates G proteins) or carrier medium. Injected oocytes were incubated in Hepes-buffered Tyrodes medium (HbT) for six hours then they were evaluated for some of the early and late events of oocyte activation. Transmission electron microscopy revealed that in contrast to GDP-p-S, GTP-rS caused the release of the vast majority of the cortical granules. It also triggered changes in the protein profiles (i.e., dephosphorylation of a 25 kDa protein) as determined by one-dimensional polyacrylamide gel electrophoresis (1D SDS PAGE). Following GTP-7-S injection 71.4% (190/266) of the mature oocytes formed pronuclei, which was significantly higher (P<O.OOl) than the rates of pronuclear formation in oocytes injected with GDP-p-S (23.7%; 67/282) or carrier medium (25.9%; 52/201). Furthermore, following 7 d of incubation in ligated porcine oviducts 13.1 ok ( 10/76) of the transferred injected oocytes developed to the compact morula/blastocyst stage. We conclude that a signal transducing pathway involving a G protein does exist in the porcine oocyte, and by stimulating the G protein with GTPq-S porcine oocytes can readily be activated.

Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of ≥-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of ≥-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.

In vitro maturation and fertilization of sheep oocytes collected by laparoscopic folliculocentesis

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 °C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 μg/ml FSH, and 500 μM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 × 106 spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.

Expression of 6WTK-LacZ gene construct in in vitro produced bovine embryos following microinjection into pronuclei or cytoplasm

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.

In vitro production of cattle embryos: review and Belgian results.

This paper reviews the overall process of in vitro production and cryopreservation of bovine embryos in Belgium. Three laboratories are involved in this field, one at the University of Liège, one at the University of Ghent and ours at the University of Louvain-La-Neuve. Each one uses this technology as a tool to reach different goals. This paper refers mainly to the work done in Louvain-La-Neuve. Oocytes are obtained by punction of 2-4 mm follicles on slaughtered cow ovaries. They are matured in hormone-supplemented TCM199 containing 10% heat-treated fetal calf serum. In vitro fertilization by Percoll-selected spermatozoa is followed by in vitro culture in oviduct-conditioned medium for 7-9 days. Six calves have been born from in vitro produced blastocysts. Recently, full development was obtained in conditioned medium without protein supplementation. This finding will allow further investigations on oviduct/embryo molecular communication and research of oviduct-secreted embryotrophic proteins which were impaired in previous culture systems using serum-supplemented media. In vitro produced blastocysts were frozen-thawed and non-surgically transferred: 7/19 recipients remained pregnant beyond 2 months. Embryo loss was high between day 21 and 35 (31%).

Mitogenesis in Cultured Vascular Smooth Muscle Cells from Two Rat Models of Hypertension in Response to Fetal Calf Serum and Angiotensin II

Hypertension may result from vascular hypertrophy or hyperplasia due to enhanced growth of vascular smooth muscle cells (VSMCs), which has been demonstrated in VSMCs from spontaneously hypertensive rats (SHRs) compared to Wistar-Kyoto (WKY) rats. To determine whether this enhanced mitogenesis is peculiar to SHRs or a general phenomenon in genetic models of hypertension, we have measured indices of cell growth [3H]-thymidine uptake in VSMCs from SHRs and New Zealand genetically hypertensive (GH) rats and controls [WKY and normal Wistar (N) rats] cultured in fetal calf serum (FCS) or angiotensin II (Ang II, 0.1 microM) in either 3% heat-treated FCS or serum-free medium. SHR cell numbers increased faster in response to both mitogens compared to WKY rats. However, GH and N rat responses to FCS were the same. Ang II caused a significant but similar increase in cell numbers in both GH and N rat cells (i.e., Ang II caused hyperplasia in all four strains) but [3H]thymidine uptake was significantly greater in GH rat cells. Ang II increased the total well protein content but not protein normalized on cell number, i.e., no hypertrophic effect of Ang II was seen in these actively dividing cells. We conclude that (a) growth properties of VSMCs from rats with genetic hypertension vary between strains; the differences in growth may reflect strain-specific variation in the activity of intracellular signalling systems subserving mitogenesis; and (b) Ang II causes VSMC hyperplasia.

Laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocytes

The objective of this study was to obtain normal pregnancy following laparoscopic oviductal transfer of in vitro matured and fertilized bovine oocytes. Methods for in vitro maturation and in vitro fertilization were similar to those previously reported (1). Primary oocytes judged to be potentially viable were cultured for 26 h in modified TCM 199 supplemented with heat-treated fetal calf serum (20% v/v), 5μg/ml FSH (USDA-bFSH-B-1), and 1μg/ml estradiol 17-β. Oocyte cumulus complexes were microscopically evaluated for maturation (first polar body formation) following a brief treatment with hyaluronidase. Mature oocytes were inseminated with heparin-treated spermatozoa and incubated at 39°C under paraffin oil and moist 5% CO 2, 5% O 2, 90% N 2. In this work, 450 oocytes were recovered at slaughter from ovaries of 42 random cows of unknown reproductive status and 336 oocytes (74.7%) with compact cumulus were selected for culture. Of these, 322 (95.4%) matured in vitro. Of 218 inseminated oocytes, 198 (90.8%) were penetrated by sperm and 83 (38.1%) cleaved, with 102 (46.6%) of the embryos reaching four- to eight-cell stages. None of 40 oocytes not exposed to sperm and none of 30 oocytes inseminated with untreated sperm showed signs of activation. In a control experiment with hormones added, 105 of 115 (91.3%) oocytes matured in vitro and 20 of 105 (19.5%) cleaved following in vitro insemination. Laparoscopy was performed on four synchronized recipients under local anesthesia. A catheter containing three embryos in the two to four cell stages was passed through the operating channel of a direct viewing bronchoscope for deposition in the oviduct ipsilateral to the recipients developing corpus luteum while the fimbria and the mesovarium were manipulated with Semm's forceps. A normal term pregnancy confirmed in vitro fertilization and provides feasibility data for use of laparoscopic methodology developed in this work for testing viability of bovine oocytes and embryos. These results are encouraging for the application of in vitro maturation and in vitro fertilization for overcoming infertility in domestic and endangered species.

Timing of Nuclear Progression and Protein Synthesis Necessary for Meiotic Maturation of Bovne Oocytes1

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The culture of bovine oocytes to obtain developmentally competent embryos.

Bovine cumulus-oocyte complexes (COC) (n = 4230) were used in this study to assess the effects of culture method, hormonal supplementation, and cumulus cell concentration on maturation, fertilization and development of resulting embryos. Five treatments were evaluated. 1) 10 COC/50-microliter drops under oil in TCM 199 supplemented with 10% heat-treated fetal calf serum, follicle-stimulating hormone (0.5 microgram/ml), luteinizing hormone (5 micrograms/ml), and estradiol-17 beta (1 microgram/ml); 2) as in 1 without hormones; 3) as in 1 but in 3 ml TCM-199 in petri dishes without paraffin oil; 4) as in 2 but only 1 COC/50-microliter drop; and 5) as in 1 but with denuded oocytes. After 24 h maturation, the frequencies of oocytes reaching metaphase II were 98, 84, 92, 93, and 87%, respectively, for the five treatments. In the same order, percentages of normal fertilization were 73, 70, 62, 81, and 62%, and the frequencies of embryos containing two or more blastomeres at 65 h postinsemination were 69, 82, 66, 51, and 43%. The same five treatments were used in a second study in which 3,199 oocytes were fertilized, allowed to cleave in vitro to the 2- to 3-cell stage (42 h postinsemination), and transferred to oviducts of sheep (one treatment/oviduct) for 4 days. The frequencies of morulae or blastocytes obtained were 28, 18, 23, 24, and 11% for the five treatments, respectively. After nonsurgical transfer to bovine recipients (n = 8) using fresh or frozen-thawed embryos, three pregnancies past 50 days were obtained. Only one went to term with the birth of a live heifer calf.

In vitro development of porcine embryos in coculture with endometrial cell monolayers or culture supernatants.

The objective of this study was to determine if porcine endometrial cell contact would enhance the in vitro development of porcine embryos relative to endometrial cell culture supernatants or ovarian fibroblast contact. One hundred forty-nine embryos of various developmental stages were collected from 10 excised gilt reproductive tracts. Embryos were washed three times and placed randomly into one of four treatments: Minimal Essential Medium + 5% heat-treated fetal calf serum (complete MEM), porcine endometrial cell coculture (pEM), porcine endometrial cell supernatant (pEMS) and porcine ovarian fibroblast coculture (pOF). All cultures were conducted in 96 well microtiter plates, one embryo per well, at 37 C in a 5% CO2 in humidified air atmosphere. Observations for stage of development were made every 12 h by phase contrast microscopy (100X). Embryos in the pEM and pOF treatments had significantly greater (P less than .05) mean embryonic development scores (1.59 +/- .22 and 1.44 +/- .18, respectively) than embryos cultured in complete MEM only or pEMS (.63 +/- .13 and .48 +/- .15, respectively). The results of this study indicate that cell to embryo contact enhances in vitro development of porcine embryos.