Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig.

Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to several morphological and cell biological properties, and evaluated the survival of in vitro produced, morphologically classified, blastocysts following non-surgical transfer. In vitro and in vivo produced blastocysts were allocated to two groups (classes A and B) on the basis of morphological characteristics. The quality of their actin cytoskeleton, their total cell number, their ability to re-expand after cytochalasin-B treatment and the occurrence of numerical chromosome aberrations were studied and compared. In vivo produced blastocysts were used as a control. Our results indicate that the ability of blastocysts to re-expand after cytochalasin-B-induced actin depolymerization was positively correlated with the morphology of the blastocyst, and associated with the quality of the actin cytoskeleton. Chromosome analysis revealed that mosaicism is inherent to the in vitro production of porcine embryos, but also that in vivo produced blastocysts contained some non-diploid cells. In non-surgical embryo transfer experiments more recipients receiving class A blastocysts were pregnant on Day 20 than those receiving class B blastocysts. One recipient gave birth to six piglets from class A in vitro produced blastocysts, providing a verification of the enhanced viability of blastocysts that were scored as 'good' on the basis of their morphology.

Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study.

The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I) blastocysts were compared for the occurrence of apoptosis in blastomeres. The total number of cells per embryo and the number of cells with damaged plasma membranes, fragmented DNA and fragmented nuclei per embryo were assessed in selected blastocysts by combining Ethidium homodimer (EthD-1), terminal dUTP nick end labeling (TUNEL) and Hoechst 33342 staining. In general, the level of blastomere apoptosis was low. A higher level of apoptosis was observed in in vitro produced equine, porcine and bovine blastocysts compared to their in vivo counterparts. Interestingly, 4 of the initially selected 29 bovine in vitro produced blastocysts exhibited extensive signs of apoptosis affecting the inner cell mass (ICM), which is not compatible with a viable conceptus. Repeated occurrence of this observation may explain the lower ET outcome of in vitro produced bovine embryos compared to in vivo produced embryos. It is concluded that, although in morphologically high quality blastocysts of several farm animal species a significant difference exists in the percentages of apoptotic cells between in vivo and in vitro produced embryos, the incidence of apoptosis at the blastocyst stage is at such a low level that it cannot reflect the substantial differences in embryo viability that have been described between in vivo and in vitro produced blastocysts following ET.

Quality of porcine blastocysts produced in vitro in the presence or absence of GH.

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro.

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.

Role of myometrial activity in sperm transport through the genital tract and in fertilization in sows.

The effects of stimulation and suppression of uterine contractility at about the time of insemination on sperm distribution and fertilization in multiparous sows are described. For assessment of fertilization, sows were inseminated about 28 h before (synchronized) ovulation and killed at day 5 after ovulation (n = 53). For assessment of sperm distribution, sows were inseminated about 20 h before expected ovulation and were killed 12 h later (n = 26). At 10 min before insemination, sows received an intrauterine infusion of one of three solutions: (i) saline (control); (ii) 0.60 mg clenbuterol hydrochloride to suppress contractility; or (iii) 1 mg cloprostenol to stimulate contractility. Both clenbuterol and cloprostenol reduced median fertilization rate (P < 0.05) and median number of accessory sperm cells (P < 0.05). Distribution of sperm cells was also affected by treatments. Clenbuterol increased, and cloprostenol decreased, the number of sperm cells (P < 0.05) in the proximal 20 cm of the uterine horn and in the uterotubal junction. In addition, clenbuterol tended to increase and cloprostenol tended to decrease the number of sperm cells in the isthmus, although these effects were not significant. However, relative to the number of sperm cells in the uterus, clenbuterol treatment reduced the number of sperm cells in the uterotubal junction and oviduct, in contrast to cloprostenol. Cloprostenol increased the reflux of semen during insemination. It is hypothesized that suppression of uterine contractility increases transuterine transport time, reducing the ability of sperm cells to enter the uterotubal junction and the oviduct. Stimulation of uterine contractility above a certain level probably increases reflux and impedes transuterine transport of sufficient numbers of sperm cells.

Zona pellucida mediated acrosome reaction and sperm morphology.

Sperm samples from 29 men randomly selected from the andrology laboratory, were used to evaluate acrosome reaction response to solubilized human zona pellucida. Capacitated sperm samples were exposed to a solution containing 2 zona pellucidae (ZP) per microl for 60 min, after which acrosomal status were recorded using a PSA-FITC technique. Controls included samples supplied by fertile sperm donors. After completion of acrosome reaction studies, patient samples were divided according to the percentage of morphologically normal spermatozoa. Three basic groups were identified, namely, fertile donors, teratozoospermic (normal sperm morphology 5-14%; n = 25) and severely teratozoospermic (normal sperm morphology < 4%; n = 4) groups. The mean percent normal sperm were 15.8 +/- 0.9, 10.4 +/- 0.7 and 2.7 +/- 0.7, respectively, for normozoospermic donors, teratozoospermic and severely teratozoospermic men. The mean percentage (+/-SE) ZP mediated acrosome reacted sperm among teratozoospermic and severely teratozoospermic cases was 25.8% +/- 0.9 and 19.0% +/- 0.9 (P = 0.001), compared to 36.8% +/- 0.9 for the donor controls. Results were analysed and expressed as correlations between sperm morphology and acrosomal response to human solubilized zona pellucida, spontaneous and calcium ionophore induced acrosome reaction. Predictive values for acrosome responsiveness were depicted with ROC curve analyses. Sperm morphology evaluated by strict criteria correlated positively and highly significantly with the responsiveness of the acrosome reaction (r = 0.91, P = 0.0001). At a morphology cut-off value of 4%, the ROC curve analysis showed sperm morphology to be highly predictive of zona pellucida induced acrosome responsiveness with a sensitivity of 100% and negative predictive value of 100%. Spontaneous and calcium ionophore induced acrosome reactions revealed no correlation with sperm morphology. It was concluded that (i) morphological features of human spermatozoa are indicative of specific functional characteristics; (ii) zona pellucida induction of the acrosome reaction is superior, as a predictor of sperm morphology, compared to calcium ionophore induced and spontaneous acrosome reactions.

A novel presentation of saline for wound irrigation.

Laboratory studies were carried out to investigate whether a multiuse container of sodium chloride 0.9% presents a cross-infection risk when used for wound irrigation. The container was subjected to bacterial contamination well in excess of that normally encountered during wound dressing procedures. It was concluded that this product is appropriate for community and hospital use provided that normal, sensible precautions are observed.

The use of alcoholic wipes for disinfection of injection sites.

An evaluation of a proprietary skin disinfection wipe - paper impregnated with 70% isopropanol - with a discussion of pre-inoculation disinfection methods used.

An adherent semipermeable film dressing for burns.

A preliminary report of a study comparing Omiderm with chlorhexidine-medicated tulle.

Evaluation of phenoxetol-chlorhexidine cream as a prophylactic antibacterial agent in burns.

A controlled clinical trial was conducted to compare the value of a cream containing 2% phenoxetol and 0.2% chlorhexidine as a prophylactic agent against wound infection in patients with burns affecting up to 15% total body surface area. The acquisition of bacteria was similar in the two treatment groups but the incidence of Staphylococcus aureus in the burns treated with phenoxetol-chlorhexidine cream significantly lower. The incidence of gram-negative bacilli was low in the two treatment groups, and no wound yielded Pseudomonas aeruginosa. Unlike preparations containing silver, phenoxetol-chlorhexidine does not cause electrolyte imbalance or stain materials with which it comes into contact, and it did not produce adverse effects during this trial.

Gentamicin- and silver-resistant pseudomonas in a burns unit.

In 1977-8 gentamicin-resistant strains of Pseudomonas aeruginosa became very common in a burns unit, over 90% being resistant at the peak of the outbreak. Some strains were also resistant to silver nitrate, though silver resistance was not found in any other strains of Ps aeruginosa isolated. Unlike the gentamicin resistance, the silver resistance was unstable, and strains became sensitive on repeated subculture. All the gentamicin-resistant strains of Ps aeruginosa were of the same serotype (O:11, H:2,5). Though gentamicin resistance could be transferred in vitro from resistant strains of Ps aeruginosa to one sensitive strain of Ps aeruginosa, there was no evidence of in-vivo transfer of gentamicin resistance between strains of pseudomonas in the patients' burns, nor was there evidence of transfer of gentamicin resistance between Ps aeruginosa and enterobacteria. Carbenicillin-resistant and gentamicin-resistant Ps aeruginosa were sometimes found in the same burns, but no gentamicin-carbenicillin (doubly) resistant strains were found among the 986 strains tested during the outbreak. The outbreak of gentamicin-resistant Ps aeruginosa from burns was not reduced by stopping treatment with gentamicin and its analogues but only by segregating all patients with Ps aeruginosa in one of the two wards of the unit and admitting new patients only to the other ward.

Prophylaxis against tetanus in non-immune patients with wounds: the role of antibiotics and of human antitetanus globulin.

The potential value of oral erythromycin for antitetanus prophylaxis in non-immune patients with open wounds was assessed. Serum obtained by venepuncture from health persons 2 h after an oral dose of an erythromycin preparation was used as a culture medium rendered anaerobic by addition of cooked meat. Strains of Clostridium tetani inoculated into these sera failed to multiply when the donor had taken 500 mg of erythromycin estolate before a meal; other erythromycin preparations and the estolate at a dosage of 250 mg were ineffective or inconsistent in their inhibition of the growth of Cl. tetani. Human antitetanus globulin (ATG) was given to 12 patients, 9 with severe injuries and 3 with extensive burns, all of whom were judged, from their history, to be non-immune (or with expired immunity); all except one had received large intravenous infusions of blood and/or other fluids. Serum antitoxin assays by a mouse protection technique on days 0, 1--2, 3--5, 6--10 and 14+ showed no detectable antitoxin (less than 0.01) unit/ml) in the initial (pre-ATG) sample from three patients with severe injuries and in one with extensive burns. All the patients in the severely injured group showed an early appearance or increase in tetanus antitoxin to protective titres. Two of the three severely burned patients showed, respectively, a delayed appearance or an increase in antitoxin; the other burned patients showed a reduction from the initial pre-ATG titre, followed by a return to that titre after day 5.

"Methicillin-resistant" Staphylococcus aureus: reassessment by controlled trial in burns unit.

A controlled trial of oral flucloxacillin (250 mg six-hourly for four days) was performed in 34 patients treated by the covered method whose burns had yielded a heavy or moderate growth of Staphylococcus aureus resistant to methicillin at 30 degrees C but moderately sensitive at 37 degrees C. Staph aureus was eliminated in nine of the 17 patients treated with flucloxacillin but in none of the 17 controls; the proportion of patients from whose burns sensitive Staph aureus was eliminated in an earlier trial of cloxacillin was greater than this. Strains of Staph aureus commonly described as methicillin-resistant and showing heterogeneous growth at 37 degrees C of many sensitive and very few resistant bacterial cells should, in the light of these findings, be called moderately sensitive to flucloxacillin. Such "heteroresistant" strains showed consistent moderate sensitivity in replicate diffusion sensitivity tests at 37 degrees C, but very inconsistent results in replicate dilution tests, especially with flucloxacillin. These studies showed that 18-hour diffusion sensitivity tests indicate the clinical value of treatment with flucloxacillin for staphylococcal infections of moderate severity more correctly at 37 degrees C than at 30 degrees C.