Applying embryo cryopreservation technologies to the production of domestic and black-footed cats.

Our objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled-rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black-footed cat (BFC, Felis nigripes) embryos after intra- and inter-species transfer. In vitro-derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled-rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22-26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies--three of six (50%) and one of two (50%) that received embryos from in vivo- and in vitro-matured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61-65 of gestation. In BFC, four intra-species embryo transfer procedures were carried out--two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2-year-old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra- and inter-species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species, the black-footed cat.

Characterization of mitochondrial and actin patterns in cat oocytes and blastocysts.

Actin microfilaments and mitochondria distribution are considered useful markers of cytoplasmic maturation, but no information is available regarding their distribution in cat oocytes and embryos. Thus, the purpose of this study was to (i) assess cytoplasmic characteristics of the oocyte by mitochondria and actin staining in immature and in vitro/in vivo matured cat oocytes and (ii) characterize mitochondria and actin distribution in in vitro produced blastocysts by confocal laser scanning microscopy. Additionally, in vivo matured oocytes were collected to assess mitochondria and actin. Transzonal cumulus cell projections were more abundant in immature oocytes than in matured oocytes. A relocation of mitochondria throughout meiosis was not clearly observed. However, most in vitro produced blastocysts were of good quality, according to their actin cytoskeleton integrity and mitochondria distribution. The functional significance of mitochondria distribution in cat oocytes in relation to their developmental competence requires further research. This study represents the original description of actin and mitochondrial patterns in cat oocytes and embryos.

The evolution and distribution of phage ST160 within Salmonella enterica serotype Typhimurium.

Salmonellosis is an internationally important disease of mammals and birds. Unique epidemics in New Zealand in the recent past include two Salmonella serovars: Salmonella enterica subsp. enterica serovar Typhimurium definitive type (DT) 160 (S. Typhimurium DT160) and S. Brandenburg. Although not a major threat internationally, in New Zealand S. Typhimurium DT160 has been the most common serovar isolated from humans, and continues to cause significant losses in wildlife. We have identified DNA differences between the first New Zealand isolate of S. Typhimurium DT160 and the genome-sequenced strain, S. Typhimurium LT2. All the differences could be accounted for in one cryptic phage ST64B, and one novel P22-like phage, ST160. The majority of the ST160 genome is almost identical to phage SE1 but has two regions not found in SE1 which are identical to the P22-like phage ST64T, suggesting that ST160 evolved from SE1 via two recombination events with ST64T. All of the New Zealand isolates of DT160 were identical indicating the clonal spread of this particular Salmonella. Some overseas isolates of S. Typhimurium DT160 differed from the New Zealand strain and contained SE1 phage rather than ST160. ST160 was also identified in New Zealand isolates of S. Typhimurium DT74 and S. Typhimurium RDNC-April06 and in S. Typhimurium DT160 isolates from the USA. The emergence of S. Typhimurium DT160 as a significant pathogen in New Zealand is postulated to have occurred due to the sensitivity of the Salmonella strains to the ST160 phage when S. Typhimurium DT160 first arrived.

Changes in fecal microbiota with CFTR modulator therapy: A pilot study.

Studies have demonstrated that people with CF with pancreatic insufficiency (PI) have fecal dysbioses. Evidence suggests the causes of these dysbioses are multifactorial, and that important drivers include antibiotic exposure, dietary intake, and CF gastrointestinal tract dysfunction, including nutrient malabsorption. In this pilot study, we tested whether initiation of the CFTR modulator treatments ivacaftor (in a cohort of pancreatic sufficient (PS) people with CF and an R117H CFTR variant) or lumacaftor/ivacaftor (in a cohort of PI people with CF and an F508del variant) changed fecal measures of malabsorption or fecal microbiomes. While we identified no statistically significant fecal changes with either treatment, we detected trends in the PI cohort when initiating lumacaftor/ivacaftor towards decreased fecal fat content and towards fecal microbiomes that more closely resembled the fecal microbiota of people without PI. While these findings support a model in which nutrient malabsorption resulting from CF-induced PI drives fecal dysbiosis, they must be validated in future, larger studies of fecal microbiome and malabsorption outcomes with highly effective CFTR modulator therapies.

Birth of domestic cat kittens of predetermined sex after transfer of embryos produced by in vitro fertilization of oocytes with flow-sorted sperm.

Our goals were to: (1) determine if domestic cat sperm could be sorted to high purity by flow cytometry after overnight shipment of cooled samples; (2) evaluate the efficiency with which sorted sperm could be used to generate cat embryos in vitro; and (3) determine if live kittens of predetermined sex could be produced after transfer of embryos derived by IVF using sorted sperm. Semen samples (n=5) from one male were extended in electrolyte-free solution and shipped overnight at 4 degrees C to the sorting facility. Samples were adjusted to 75x10(6)sperm/mL and stained with Hoechst 33342. After 1h at 34.5 degrees C, samples were adjusted to 50x10(6)sperm/mL with 4% egg yolk TALP+0.002% food dye and sorted by high-speed flow cytometry. Later resort analysis confirmed purities of 94% and 83% for X- and Y-chromosome bearing sperm, respectively. Sorted sperm were centrifuged, re-suspended in TEST yolk buffer and shipped overnight to the IVF laboratory. After IVF of in vivo matured oocytes with X-chromosome bearing sperm, cleavage frequency was 62% (54/87). After IVF of IVM oocytes with control, X- or Y-chromosome bearing sperm, the incidence of cleavage was 42% (48/115), 33% (40/120), and 35% (52/150), respectively, and blastocyst development was 53% (21/40), 50% (11/22), and 55% (23/42), respectively (P>0.05). On Day 2, 45 embryos produced by IVF of in vivo matured oocytes with X-chromosome bearing sperm were transferred to the oviduct of four Day 1 recipients, three of which subsequently delivered litters of one, four, and seven female kittens, respectively. In conclusion, we confirmed that sperm sorting technology can be applied to domestic cats and established that kittens of predetermined sex can be produced.

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality.

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR((R))14-PI) and DNA fragmentation (TUNEL). After addition of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 degrees C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P<0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.

Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope.

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams.

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.

In vitro embryo production and embryo transfer in domestic and non-domestic cats.

Over a 5-year interval, multiple laparoscopic oocyte retrievals were done in fishing cats (Prionailurus viverrinus), caracals (Caracal caracal) and domestic cats after ovarian stimulation with gonadotropins. From 21 retrievals in five fishing cats, 579 preovulatory oocytes (mean = 27.6) were recovered and 348 embryos were produced in vitro (mean = 16.6). A total of 452 preovulatory oocytes (mean = 25.1) were recovered from 18 of 24 retrievals in six caracals and 297 (mean = 16.6) embryos were produced. An additional 16 caracal embryos (19%) were produced after in vitro maturation of 83 oocytes, 59 of which came from six retrievals producing only immature oocytes. The presence of corpora lutea at oocyte retrieval occurred in each species (1) at a similar frequency (33%) and (2) more frequently during January through May (11 of 15 retrievals) than during the latter half of the year (4 of 30 retrievals). Of the 12 embryo transfer procedures done in fishing cats, one pregnancy (8%) was obtained and one live kitten born after the auto-transfer of 10 Day-6 embryos. In caracals, a total of 46 Day-4 or Day-5 embryos were auto-transferred to six recipients, one of which delivered two live kittens. Then, 109 caracal embryos were cryopreserved before thawing and transferring to nine recipients (mean = 12.1) on Days 5 or 6. From three pregnancies established (33%), a total of three kittens were born. Two to six gonadotropin treatments/oocyte retrievals were done in domestic cats during 1999 through 2003; an average of 24.9, 23.5, 22.0, 23.1, 23.5 and 40.9 oocytes (P > 0.05) were recovered at the first through the sixth treatment cycles from 138, 138, 97, 49, 22, and seven retrievals, respectively.

Nuclear transfer in cats and its application.

Nuclear transfer (NT) technology is typically used for generating identical individuals, but it is also a powerful resource for understanding the cellular and molecular aspects of nuclear reprogramming. Most recently, the procedure has been used in humans for producing patient-specific embryonic stem cells. The successful application of NT in cats was demonstrated by the birth of domestic and non-domestic cloned kittens at a similar level of efficiency to that reported for other mammalian species. In cats, it has been demonstrated that either in vivo or in vitro matured oocytes can be used as donor cytoplasts. The length of in vitro oocyte maturation affects in vitro development of reconstructed embryos, and oocytes matured in vitro for shorter periods of time are the preferred source of donor cytoplasts. For NT, cat somatic cells can be synchronized into the G0/G1 phase of the cell cycle by using different methods of cell synchronization without affecting the frequency of in vitro development of cloned embryos. Also, embryo development to the blastocyst stage in vitro is not influenced by cell type, but the effect of cell type on the percentage of normal offspring produced requires evaluation. Inter-species NT has potential application for preserving endangered felids, as live offspring of male and female African wildcats (AWC, Felis silvestris lybica) have been born and pregnancies have been produced after transferring black-footed cat (Felis nigripes) cloned embryos into domestic cat (Felis silvestris catus) recipients. Also, successful in vitro embryo development to the blastocyst stage has been achieved after inter-generic NT of somatic cells of non-domestic felids into domestic cat oocytes, but no viable progeny have been obtained. Thus, while cat cytoplasm induces early nuclear remodeling of cell nuclei from a different genus, the high incidence of early embryo developmental arrest may be caused by abnormal nuclear reprogramming. Fetal resorption and abortions were frequently observed at various stages of pregnancy after transfer of AWC cloned embryos into domestic cat recipients. Abnormalities, such as abdominal organ exteriorization and respiratory failure and septicemia were the main causes of death in neonatal cloned kittens. Nonetheless, several live domestic and AWC cloned kittens have been born that are seemingly normal and healthy. It is important to continue evaluating these animals throughout their lives and to examine their capability for natural reproduction.

In vitro production and transfer of cat embryos in the 21st century.

Appreciable progress has been made in the development of assisted reproductive technology (ART) for creating in vitro embryos in cats. Moreover, the extent of advancement in the last decade has been similar, albeit of more modest magnitude, to that seen in some other domestic and laboratory species, particularly when the disparities in financial, and, hence, scientific, resources are considered. The recent progress in domestic felid ART has made it possible to envisage their potential role in supporting the conservation of endangered felid species, which, in reality, is a multifarious process requiring wide-ranging, yet coordinated approaches. The prospect of incorporating ART into that intricate domain, with limited exceptions, remains a long-term, but highly motivating objective. Meanwhile, the straightforward accessibility and abundant supply of domestic cat gametes from local veterinary clinics provides a valuable and practical source of material for further research on the basic aspects of in vitro oocyte maturation, fertilization and early embryo development. Furthermore, extrapolating the domestic biotechniques to non-domestic felids has produced encouraging results in some species.

The microbiota in bronchoalveolar lavage from young children with chronic lung disease includes taxa present in both the oropharynx and nasopharynx.

BACKGROUND: Invasive methods requiring general anaesthesia are needed to sample the lung microbiota in young children who do not expectorate. This poses substantial challenges to longitudinal study of paediatric airway microbiota. Non-invasive upper airway sampling is an alternative method for monitoring airway microbiota; however, there are limited data describing the relationship of such results with lung microbiota in young children. In this study, we compared the upper and lower airway microbiota in young children to determine whether non-invasive upper airway sampling procedures provide a reliable measure of either lung microbiota or clinically defined differences. RESULTS: The microbiota in oropharyngeal (OP) swabs, nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) from 78 children (median age 2.2 years) with and without lung disease were characterised using 16S rRNA gene sequencing. Permutational multivariate analysis of variance (PERMANOVA) detected significant differences between the microbiota in BAL and those in both OP swabs (p = 0.0001, Pseudo-F = 12.2, df = 1) and NP swabs (p = 0.0001; Pseudo-F = 21.9, df = 1) with the NP and BAL microbiota more different than the OP and BAL, as indicated by a higher Pseudo-F value. The microbiota in combined OP and NP data (upper airways) provided a more comprehensive representation of BAL microbiota, but significant differences between the upper airway and BAL microbiota remained, albeit with a considerably smaller Pseudo-F (PERMANOVA p = 0.0001; Pseudo-F = 4.9, df = 1). Despite this overall difference, paired BAL and upper airway (OP and NP) microbiota were >50 % similar among 69 % of children. Furthermore, canonical analysis of principal coordinates (CAP analysis) detected significant differences between the microbiota from clinically defined groups when analysing either BAL (eigenvalues >0.8; misclassification rate 26.5 %) or the combined OP and NP data (eigenvalues >0.8; misclassification rate 12.2 %). CONCLUSIONS: Upper airway sampling provided an imperfect, but reliable, representation of the BAL microbiota for most children in this study. We recommend inclusion of both OP and NP specimens when non-invasive upper airway sampling is needed to assess airway microbiota in young children who do not expectorate. The results of the CAP analysis suggest lower and upper airway microbiota profiles may differentiate children with chronic suppurative lung disease from those with persistent bacterial bronchitis; however, further research is needed to confirm this observation.

The esophagus for the nonesophagologist.

The esophagus is a muscular tube (striated muscle at the top end, smooth muscle in the middle and lower portion) connecting the pharynx and the stomach. It is closed at both the top and bottom ends by specialized muscular sphincters. Deglutition begins as a programmed act, mediated by nerve fibers from the brain stem, and spreads aborally with a wave of inhibition preceding a moving-ring contraction that obliterates the lumen of the esophagus. Fluids fall by gravity in the upright position or are pushed down by the moving ring contraction when the subject is horizontal. There are several factors that prevent constant regurgitation of gastric contents back into the esophagus. Reflux of both gas and liquid does occur as a normal event. The esophagus usually performs its job description of transferring material from the pharynx to the stomach and venting the stomach when necessary with a minimum of fuss.

SP-I secretion by baboon embryos in vitro.

Baboon embryos cultured to postimplantation stages have been shown to secrete the placental protein SP-I into the culture medium in quantities of up to almost 5 micrograms/day, based on a rhesus monkey standard. Twelve embryos, for which spent media samples have been assayed, have been shown to secrete this protein, with measurable quantities usually being present on day four following attachment of the zonaless embryo to the culture dish. Secretion has continued for up to 14 days, with over 26 micrograms total SP-I secretion from one embryo. These observations further enhance the utility of the baboon embryo culture system as a model for studying early placental development in the primate.

Medical and surgical treatment of nonallergic asthma associated with gastroesophageal reflux.

Patients presenting to a chest clinic because of adult-onset wheezing with no history of allergy had a 90 percent prevalence of gastroesophageal reflux, even though reflux symptoms were mild or absent. Ninety patients were randomly assigned to receive cimetidine or an identical placebo or to undergo antireflux surgery. During a six-month period, all groups improved clinically; the cimetidine and surgical groups improved more than the placebo group. The intake of pulmonary medication decreased significantly in both cimetidine and surgical groups. Pulmonary function test results improved in the cimetidine- and surgically treated patients; improvement was not statistically significant. At long-term follow-up, the surgical group maintained clinical improvement and decreased pulmonary medication intake, whereas the placebo group worsened. We conclude that gastroesophageal reflux can play a significant role in some patients with nonallergic pulmonary disease and that its treatment can improve pulmonary symptoms and objective measurements of pulmonary function.

Pharyngeal pH monitoring in 222 patients with suspected laryngeal reflux.

To determine the existence of and characterize gastroesophagopharyngeal reflux in patients with symptoms of airway irritation, we monitored pharyngeal pH over a 24-hour period in 222 consecutive patients. Pharyngeal reflux was defined as a drop in pH to less than 4 at the pharyngeal sensor, which occurred simultaneously with acidification of the distal esophagus. Patients were divided into two groups: those with pharyngeal reflux (PR+) and those without (PR-). The Mann-Whitney U test and Student's t test were used to assess intergroup comparisons. Episodes of pharyngeal reflux (range 1 to 36, average 4.4) were identified in 90 PR+ patients (40%). No pharyngeal reflux was identified in the remaining 132 patients (PR-). Episodes of pharyngeal reflux were rapidly cleared (average duration 1.5 minutes), and occurred while in the upright position in 77 (86%) of 90 patients and while in the supine position in 11 (12%) of 90 patients. Twenty-three patients (25%) experienced symptoms in association with an episode of pharyngeal reflux. In the distal esophagus, the percentage of time the pH was below 4 during the upright position and the total percentage of time the pH was below 4 were greater in PR+ patients (6.4% and 5.8%, respectively) when compared to PR- patients (2.6% and 2.6%, respectively). Laryngoscopic findings did not distinguish PR+ from PR- patients. Pharyngeal reflux occurs most commonly in the upright position and can be identified in more than 40% of patients thought to have acid-induced laryngeal symptoms. Even though these episodes are short lived and rapidly cleared, symptoms occur concomitantly in 25% of patients with proven pharyngeal reflux. Patients with laryngeal symptoms and documented pharyngeal reflux have greater amounts of esophageal reflux when compared to patients with laryngeal symptoms and no demonstrable pharyngeal reflux.

Role of diagnostic tests in esophageal evaluation.

In the evaluation of esophageal disease, the appropriate question must be asked before the correct tests can be selected. Reflux can be demonstrated by radiologic methods, pH testing or radioisotopic techniques. Esophageal mucosal damage is best evaluated by x-ray, endoscopy or biopsy. Chest pain is demonstrated by acid infusion or by manometry. Two algorithms are presented for the evaluation of chest pain and reflux symptoms.

Pharyngeal pH measurements in patients with respiratory symptoms before and during proton pump inhibitor therapy.

BACKGROUND: Pharyngeal pH monitoring is a diagnostic tool used to identify Gastroesophageal reflux disease (GERD) as an etiology of respiratory symptoms. We performed pharyngeal pH monitoring on 14 patients with respiratory symptoms thought to be induced by GERD. METHODS: Symptoms and pH monitoring (esophageal and pharyngeal) were assessed prior to and 3 months after the initiation of double-dose proton pump inhibitor therapy. RESULTS: Symptoms included cough, hoarseness, and throat clearing. Ten patients had at least one episode of pharyngeal reflux (PR+) and 4 patients had no pharyngeal reflux (PR-). Pharyngeal reflux episodes in PR+ patients decreased from 3.5 to 0.9 (P <0.05) per day with 8 of 10 (80%) patients having elimination or reduction of such episodes. Eight of 9 PR+ patients (89%) with suppressed pharyngeal reflux on medical therapy had resolution of respiratory symptoms. Three of 4 PR- patients (75%) had persistent symptoms on medical therapy. CONCLUSIONS: Proton pump inhibitor therapy improves clinical symptoms and decreases pharyngeal reflux episodes in patients with respiratory symptoms related to GERD. Direct measurement of pharyngeal pH is helpful in the identification of patients likely to respond to antireflux therapy.

The abortifacient effect of synthetic androstane derivatives in the baboon.

Synthetic androstane derivatives have been tested for their ability to induce abortion during early pregnancy in primates. Two compounds were studied following intramuscular (IM) and oral treatment in fourteen baboons. A five-day treatment regimen was started at approximately day 20 of pregnancy in 12 baboons, with treatment delayed until after day 40 in two baboons. All seven baboons treated IM with either compound aborted following intramuscular treatment, although three required a second treatment series beginning on approximately day 40 of pregnancy. Two of five baboons treated orally aborted following the single treatment series initiated around day 20 of pregnancy. The two baboons treated only after day 40 continued to term and delivered healthy infants. These compounds are therefore effective at terminating pregnancy when given around the time of the missed menstrual period. Further studies are necessary to determine optimal dose and treatment schedule.

Births of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro.

In Experiment 1, cleavage frequency and in vitro development of domestic cat embryos produced after in vitro maturation of oocytes obtained from ovaries after ovariohysterectomy (in vitro) with that of oocytes retrieved from follicle-stimulating hormone-treated donors at 24 h after administration of luteinizing hormone (in vivo) and fertilization by intracytoplasmic sperm injection (ICSI) or IVF were compared. In each group presumptive zygotes were assessed for cleavage on IVC Days 1 and 4 and for development to blastocysts on IVC Day 7. In vitro matured oocytes had lower frequencies of meiotic maturation (59.2% v. 66.5%), cleavage at Day 1 (41.4% v. 64.9%) and development to the morula stage at Day 4 (65.8% v. 87.9%) than did in vivo matured oocytes, after ICSI and IVF. Development to the blastocyst stage was lower in in vitro matured oocytes (19.0%) than in vivo matured oocytes (29.5%) after ICSI. In Experiment 2, we evaluated the capacity of sperm injected oocytes without a visible polar body to undergo cleavage and in vitro development. More in vivo matured than in vitro matured oocytes underwent cleavage at Day 1 (46.6% v. 12.6%) and developed to the morula stage by Day 4 (66.7% v. 46.1%), but no blastocysts were obtained at Day 7 in either group. In Experiment 3, we evaluated the in vivo viability of domestic cat embryos derived from ICSI of in vitro matured oocytes. Morula stage embryos were transferred to 18 domestic cat recipients either on Day 4 or 5 after oocyte recovery. A total of 3 domestic cat recipients were pregnant after transfer to recipients on Day 5. Two pregnant cats delivered two normal and healthy live male kittens on Day 68 of gestation and the remaining cat delivered a male kitten on Day 62 that died during the last two days of gestation. These results demonstrate that: (1) inadequate cytoplasmic maturation of in vitro matured domestic cat oocytes is the main cause of deficient oocyte activation; (2) the injection of oocytes without a visible polar body is a useful technique to evaluate oocyte cytoplasmic maturation; and (3) blastocysts obtained after ICSI of in vitro matured oocytes are viable and not a result of parthenogenesis.