Children’s Phthalate Intakes and Resultant Cumulative Exposures Estimated from Urine Compared with Estimates from Dust Ingestion,Inhalation and Dermal Absorption in Their Homes and Daycare Centers

Total daily intakes of diethyl phthalate (DEP), di(n-butyl) phthalate (DnBP), di(isobutyl) phthalate (DiBP), butyl benzyl phthalate (BBzP) and di(2-ethylhexyl) phthalate (DEHP) were calculated from phthalate metabolite levels measured in the urine of 431 Danish children between 3 and 6 years of age. For each child the intake attributable to exposures in the indoor environment via dust ingestion, inhalation and dermal absorption were estimated from the phthalate levels in the dust collected from the child’s home and daycare center. Based on the urine samples, DEHP had the highest total daily intake (median: 4.42 µg/d/kg-bw) and BBzP the lowest (median: 0.49 µg/d/kg-bw). For DEP, DnBP and DiBP, exposures to air and dust in the indoor environment accounted for approximately 100%, 15% and 50% of the total intake, respectively, with dermal absorption from the gas-phase being the major exposure pathway. More than 90% of the total intake of BBzP and DEHP came from sources other than indoor air and dust. Daily intake of DnBP and DiBP from all exposure pathways, based on levels of metabolites in urine samples, exceeded the Tolerable Daily Intake (TDI) for 22 and 23 children, respectively. Indoor exposures resulted in an average daily DiBP intake that exceeded the TDI for 14 children. Using the concept of relative cumulative Tolerable Daily Intake (TDI_cum), which is applicable for phthalates that have established TDIs based on the same health endpoint, we examined the cumulative total exposure to DnBP, DiBP and DEHP from all pathways; it exceeded the tolerable levels for 30% of the children. From the three indoor pathways alone, several children had a cumulative intake that exceeded TDI_cum. Exposures to phthalates present in the air and dust indoors meaningfully contribute to a child’s total intake of certain phthalates. Such exposures, by themselves, may lead to intakes exceeding current limit values.     

Ultrastructure of in-vivo fertilization in superovulated cattle

Heifers were induced to superovulate by treatment with PMSG or FSH. Subsequently, oestrus was induced with prostaglandins and artificial insemination was performed. Ova were collected from the oviducts and their ultrastructural features were related to an estimated time of ovulation based on the time of the LH peak. With the insemination schedule used, the estimated time of ovulation defined the time at which fertilization was expected to occur. The ova were characterized as unfertilized, fertilized or possibly fertilized, and a sequence of nuclear and cytoplasmic changes associated with fertilization was revealed. Within 4 h after the estimated time of ovulation formation of the female and male pronucleus was initiated, and at 5-7 h swelling of the pronuclei occurred. At 19 h the pronuclei were closely apposed and synkaryosis was seen, and at 23 h the first two-cell stage was obtained. Within 2-3 h after the estimated time of ovulation cortical granule release, development of conspicuous Golgi complexes, and transformation of the smooth endoplasmic reticulum occurred. At approximately 7 h parallel arrays of annulate lamellae appeared. In one third of the unfertilized ova deviant oocyte maturation was noticed.     

Preovulatory plasma estradiol-17beta concentrations and ovulation rates in PMSG/anti-PMSG treated heifers

Possibilities for early characterization of the superovulatory response were studied in 41 PMSG/PG-treated dairy heifers, of which 21 received an additional treatment of PMSG-antiserum. Plasma was obtained at 33, 36, 41, 47 and 51 h after PG for hormone analyses. After slaughter at 6 or 7 d after insemination, the number of follicles and corpora lutea (CL) were recorded, and ova were recovered for morphological evaluation. Significant correlations were demonstrated between plasma concentrations of estradiol-17beta (E2) at 33, 36 and 41 h after PG and the ovulation rate (number of CL). Each of these correlations was equal to the one found by using the peak concentration of E2 achieved during the preovulatory E2 surge. In heifers with preovulatory E2 surges, as determined with the blood sampling scheme used, both the ovarian response (number of CL and follicles) and the quality of ova recovered (number of transferable embryos) was clearly better compared to heifers without this surge. None of the parameters studied was affected significantly by treatment with PMSG-antiserum. It is concluded that plasma E2 determinations at fixed times in relation to prostaglandin treatment can be used to characterize the superovulatory response in donor cattle in terms of the ovulation rate and the quality of ova recovered. No evidence was found in favor of using PMSG-antiserum for improving either the superovulatory response or such characterization.     

Follicular dynamics prior to and during superovulation in heifers

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).     

Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system

The aim of this study was to establish an in vitro system that supports development and differentiation of bovine blastocysts. Agar gel tunnels were covered with modified synthetic oviduct fluid medium supplemented with 10% fetal calf serum and 3g/l D-glucose. Of the total 67 blastocysts loaded individually into the tunnels, 46 continued expansion to 1mm and reached the walls of the gel on Day 10. On Day 12, 35 blastocysts elongated to minimum 1.6 mm while filling completely the space between the walls of the tunnel, and 16 still continued growth and reached an average of 4.3 mm length on Day 14. The largest blastocyst on Day 16 was 12 mm long. On Day 12, in 31 of the 35 elongated blastocysts a second cell layer occurred beneath the trophoblast and formed a complete cover in surviving Day 14 embryos. In most proliferating embryos the inner cell mass was prominent, however, the detection of signs of embryonic disc formation will require further studies. The established system was suitable to induce in vitro elongation, rapid growth and further differentiation, and may have considerable theoretical and practical value for studies of development and differentiation of bovine embryos.     

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.     

Application of the zona-free manipulation technique to porcine somatic nuclear transfer

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.     

Ease of calving, blood chemistry, insulin and bovine growth hormone of newborn calves derived from embryos produced in vitro in culture systems with serum and co-culture or with PVA

Blood chemistry (pH, pCO2, pO2, glucose, lactate) as well as plasma insulin and growth hormone of calves derived from embryos produced under 2 different in vitro culture systems (modified SOFaa with 20% serum and co-culture with bovine oviduct epithelial cells [IVP serum, n=8] or with 3 mg/mL PVA [IVPdefined, n=6]) were compared with those of calves derived from AI (n=5). Calvings were classified according to the ease (unassisted, light traction, heavy traction). Blood samples were taken from the jugular vein of calves at 5, 15, 30 and 60 min, and at 2, 3, 6, 12, 18 and 24 h after delivery, then daily for 6 d. At the second day of life after 4 feedings and a 4-h fasting period, a glucose tolerance test was performed to evaluate glucose metabolism and insulin secretion. Calves in the IVP serum group had higher birth weights than AI calves (LS mean +/- SEM, IVP serum: 45.2 +/- 1.4 kg vs AI: 40.4 +/- 1.7 kg; P < 0.05), while the birth weights of calves in the IVP defined group were in between (IVPdefined: 41.9 +/- 1.6 kg). More IVP serum calves (75%) needed assistance than IVP defined (33%) or AI (40%) calves. The effect of ease of calving vs the effect of embryo culture was compared in relation to blood parameters at birth. There was an effect of ease of calving but not of embryo culture conditions on blood pH, lactate and PCO2. Calves requiring heavy traction had lower pH during the first 3 h after calving, a higher lactate during the first 60 min after calving and a higher pCO2 the first 2 h after calving than calves born unassisted. Calves requiring heavy traction also had lower pH the first 2 h and higher lactate the first 3 h after calving than calves born by light traction. IVP defined calves had lower lactate than IVP serum calves the first 60 min after calving. At 6 h after delivery, all blood parameters had stabilized. There was no effect of either embryo culture or ease of calving on basal insulin and growth hormone level, or the ability of the calves to handle glucose postnatally and during a glucose tolerance test.