Region-specific expression of aquaporin subtypes in equine testis, epididymis, and ductus deferens.

The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, -3, -7, and -9). Overall, we inferred that fluid transport in the stallion testicle involved a collaboration of AQP subtypes (primarily AQP2, -5, -7, and -8). Based on immunohistochemistry, expression of AQP subtypes analyzed (i.e., AQP0, -2, -5, and -9) was localized to Leydig cells and elongated and round spermatids. Functional significance of AQP expression by Leydig cells remained uncertain. In elongated and round spermatids, AQP s likely contributed to the volume reduction observed during spermatogenesis. Subtypes AQP2 and AQP9 were the predominant forms expressed in epididymal tissue. Regulation of AQP2 expression, especially in the epididymal head, seemed to occur at the post-transcriptional level, as protein expression upon immunohistochemistry was pronounced, despite low transcript abundance. In epididymal tissue, AQPs likely contributed to fluid resorbtion, given their localization on the apical membrane of principal cells.

Expression of aquaporin water channels in equine endometrium is differentially regulated during the oestrous cycle and early pregnancy.

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.

Ultrastructural and rheological properties of bovine vaginal fluid and its relation to sperm motility and fertilization: a review.

The tubular genital organs of the mammalian female reproductive tract provide different luminal microenvironments that can act as an aid or a barrier to gamete transport and the first steps of embryonic development. The mechanism of sperm transport through the genital tract has not been fully established but, first of all, sperm cells encounter and need to overcome mucous-like secretions to reach the site where fertilization occurs. One of these secretions, vaginal fluid, is a complex biological product, which is mainly derived from cervical mucus. In this review, we focus on recent studies that describe the relationships between the ultrastructural and rheological properties of bovine vaginal fluid and sperm motion. In the light of this, we propose bovine vaginal fluid as a morphofunctional model for the study of sperm transport in mammals.

Rheological and ultrastructural properties of bovine vaginal fluid obtained at oestrus.

The properties of cervical-vaginal fluid are under strict hormonal control: and in mammals in which semen is deposited in the anterior vagina, changes produced in these properties can result in a lower or higher resistance to sperm motion. The aim of this study was to determine whether the structural organization of bovine vaginal fluid is related to its rheological properties. Vaginal fluid samples were collected from 41 cows at oestrus: 20 at the middle of oestrus (between 8 and 12 h after starting) and 21 at the end of oestrus (between 18 and 22 h). Flow behaviour was determined using a viscosimeter, and the ultrastructural analysis was performed by scanning electron microscopy. Six samples showed newtonian behaviour: three collected at the middle and three collected at the end of oestrus. Newtonian samples had dense and compact matrices arranged as membranes with rough, irregular surfaces, and sparse, thin filaments (< 150 nm). Non-newtonian samples collected at the end of oestrus (n = 18) had a higher (P = 0.016) consistency index (K = 944 +/- 229 mPa.s(n)) than those collected at the middle of oestrus (n = 17; K = 237 +/- 84 mPa.s(n)). Thick filaments (> 700 nm) that were either randomly arranged with thinner filaments forming a mesh or heavily cross-linked by thin filaments (50-150 nm) were observed in all non-newtonian samples collected at the end of oestrus, while medium-diameter filaments (between 200 and 500 nm) forming loose networks were observed in non-newtonian samples collected at the middle of oestrus. These findings indicate a close relationship between the molecular organization of the structural elements of bovine vaginal fluid and its rheological behaviour. Vaginal fluid dramatically reduces its mechanical barrier effect during the course of oestrus but always appears to maintain its three-dimensional filamentous structure. The images of vaginal fluid showing newtonian behaviour would appear to support previous results, suggesting that this property may be related to bovine infertility.

Factors affecting pregnancy loss from gestation Day 38 to 90 in lactating dairy cows from a single herd.

The present study was designed to establish whether factors such as previous estrus synchronization, corpus luteum and embryo number at the time of pregnancy diagnosis, changes in body condition score, milk production, clinical disease (mastitis or lameness) and the inseminating bull affect pregnancy loss from 38 to 90 days of gestation. We derived data from 601 pregnant lactating dairy cows from a single herd. Pregnancy diagnosis was performed by ultrasonography between Day 38 and 44 following insemination. We also recorded corpus luteum and embryo number at this time. Pregnancy loss was defined as a negative pregnancy diagnosis on the second palpation per rectum undertaken between 90 and 96 days after insemination. Data were analyzed using multiple logistic regression methods. Cows that had an additional corpus luteum were eight times less likely to miscarry. The risk of pregnancy loss was 3.1 times higher in cows bearing twins. A one unit reduction in body condition score from previous partum to 30 days postpartum resulted in a 2.4-fold increase in pregnancy loss. We noted a higher incidence of pregnancy loss in cows inseminated using semen from one of the six bulls used. This particular bull led to a 3.4-fold increase in the rate of pregnancy loss. Logistic regression analysis showed no significant effects of previous estrus synchronization, milk production, clinical disease, body condition at previous partum or at pregnancy diagnosis, or body condition change between previous partum and pregnancy diagnosis. Our findings indicate a positive relationship between the presence of an additional corpus luteum and the maintenance of gestation. Risk factors for pregnancy loss were twin pregnancy, reduced body condition after previous parturition and the inseminating bull.

Intraperitoneal insemination in mammals: a review.

This review focuses on factors associated with the development of intraperitoneal insemination in mammals. Findings to date indicate that fertility improves as the sperm cell concentration rises, but that the optimal sperm number differs in each species. Sperm washing before intraperitoneal insemination favours fertility. Peritoneal fluid shows a variable effect on spermatozoa, depending on the hormonal status of the female. The optimal time for insemination appears to be just prior to ovulation. The technique may be performed either through the abdominal or the vaginal wall. Verification of sperm deposition in the proximity of the ovaries improves fertility rates. Although associated with some risk of infection and an immune reaction against spermatozoa, the intraperitoneal technique rarely gives rise to severe anaphylactic shock, peritonitis, adhesion formation and the production of anti-sperm antibodies and these complications may be prevented by adequate sperm pretreatment and antibiotic therapy. The success of intraperitoneal insemination in humans, with results comparable with those of intrauterine insemination in the treatment of infertility, suggest the potential use of this technique in domestic mammals, especially in those in which intrauterine insemination poses practical difficulties. Some of the methods applied in human intraperitoneal insemination, such as confirming the position of the needle in the peritoneal cavity, and sperm pre-treatments might also improve results in domestic species. Conversely, the use of the animal model should help to develop some aspects of this technique in humans.

Anatomicohistological characteristics of the genital tubular organs of the female brush-tailed porcupine (Atherurus africanus, Gray 1842) from Gabon.

The present study examines the anatomic and histological characteristics of the female genital tract of the wild brush-tailed porcupine (Atherurus africanus). A description of the macroscopic and microscopic features of the tubular genital organs based on observations made in 18 wild adult females is presented. The histological stains used were haematoxylin and eosin, 1% toluidine blue, PAS-haematoxylin and Masson's trichrome. Observations indicate that the fimbriated infundibulum of the oviduct completely surrounds and is attached to the entire ovarian surface. The uterus is bicornuate, being composed of two uterine horns separated by a velum uteri, a small uterine body and a cervix. In both pregnant and non-pregnant females, the uterine horns and body of the uterus present a pseudostratified, columnar ciliated epithelium containing sparse simple tubular glands. In pregnant females, the endocervical canal is filled by a viscous cervical secretion. The different patterns observed in the endometrium and myometrium of the uterine horns and in the vaginal epithelium suggest a relationship with the reproductive state of the brush-tailed porcupine female.

Posttranslational processing of PH-20 during epididymal sperm maturation in the horse.

It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.

Persistent ovarian follicles in dairy cows: a therapeutic approach.

Anestrus is common during the postpartum period in high-producing dairy cows. In a previous investigation, we were able to diagnose persistent follicles of 8 to 12 mm in anestrous cows. This report describes 2 consecutive studies. The objectives of the first were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate 2 therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with the Ovsynch protocol. For Study 1, anestrous cows were considered to have a persistent follicle if it was possible to observe a single follicular structure > 8 mm in the absence of a corpus luteum or a cyst in 2 ultrasonographic examinations performed at an interval of 7 d. At diagnosis (Day 0), cows were assigned to 1 of 3 treatment groups. Cows in Group GnRH/PGF (n=17) were treated with 100 microg GnRH i.m., and 25 mg PGF2alpha i.m. on Day 14. Cows in Group PRID (n=18) were fitted with a progesterone releasing intravaginal device (PRID, containing 1.55 g of progesterone) for 9 d and were given 100 microg GnRH i.m. at the time of PRID insertion, and 25 mg PGF2alpha i.m. on Day 7. Cows in Group Control (n=18) received no treatment. The animals were inseminated at observed estrus and were monitored weekly by ultrasonography until AI or 5 weeks from diagnosis. Blood samples were also collected on a weekly basis for progesterone determination. The mean size of persistent follicles on Day 0 was 9.4 +/- 0.04 mm. Progesterone levels were < 0.2 ng/mL during the first 35 d in 16 of 18 Control cows. Cows in the PRID group showed a lower persistent follicle rate (16.7% < 70.6% < 88.9%; P < 0.0001; PRID vs GnRH/PGF vs Control, respectively); a higher estrus detection rate (83.3% > 29.4% > 11.1%; P < 0.0001) and a higher pregnancy rate (27.8% > 5.9% > 0%; P = 0.02). For the second study, 145 cows with persistent follicles were randomly assigned to 1 of 2 treatment groups: cows in Group Ovsynch (n=73) were treated with 100 microg GnRH i.m. on Day 0, 25 mg PGF2alpha i.m. on Day 7, and 100 microm GnRH i.m. 32 h later. Cows in this group were inseminated 16 to 20 h after the second GnRH dose (Ovsynch protocol). Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1, and were inseminated 56 h after PRID removal. Cows in the PRID group showed a higher ovulation rate (84.8% > 8.2%: P < 0.0001); a higher pregnancy rate (34.2% > 4.1%; P < 0.0001) and lower follicular persistence rate (22.2% < 63%; P < 0.0001) than those in Ovsynch. Our results indicate that persistent follicles affect cyclic ovarian function in lactating dairy cows. Cows with persistent follicles can be successfully synchronized and time inseminated using progesterone, GnRH and PGF2alpha but show a limited response to treatment with GnRH plus PGF2alpha.

Confocal scanning laser microscopy examination of bovine vaginal fluid at oestrus.

This article reports the study of the structural elements of bovine vaginal fluid at oestrus using a confocal scanning laser microscope (CSLM) to examine samples collected from 10 lactating cows at the time of insemination. The filamentous glycoproteins, which form the structural component of vaginal fluid, were stained with fluorophore acridine orange for 10 min. This microscopy technique permits visualization of a network of non-oriented filaments of different thickness in a three-dimensional array. This arrangement was repeated throughout the sample and was common to all samples. Interfilament spaces varied in width between cows and ranged from 1 to 20 microns. The present results provide a new, non-aggressive technique for the study of biological samples that show hydrogel-like behaviour. Due to its simplicity, the CSLM technique proposed offers the advantages of (1) providing realistic images of vaginal fluid structure, free from artifacts arising from sample preparation, and (2) the possibility of observing specimens shortly after collection. CSLM proved to be a useful tool for the examination of vaginal fluid and may also serve as a good control for artifacts associated with scanning electron microscopy procedures.

A scanning electron microscopic study of the peritoneal mesothelium covering the genital tract and its ligaments in the cow.

This study was undertaken to describe the surface features of the peritoneal mesothelium covering the genital tract and adjacent ligaments of the cow during the oestrous cycle. The relationship between mesothelial surface and spermatozoa was also evaluated after intra-uterine and intraperitoneal insemination. Surface features of mesothelial cells from 25 cyclic cows were examined by scanning electron microscopy and by image analysis. Presence of spermatozoa was evaluated by scanning electron microscopy in seven additional cows. In the external side of the infundibulum, the oviductal mucosa exceeds the free margin, forming a continuous band measuring 2.5-10 mm in width. This oviductal epithelium shows cyclical variations with a predominance of ciliated cells during the follicular phase. In respect of the mesothelium, no clear morphological differences were observed associated with the side of ovarian bursa (internal versus external), or with the phase of the oestrous cycle. Mesothelial cells covering the uterus and mesometrium have a higher microvilli density and length and a smaller cell surface area than in the oviduct and adjacent structures. The presence of solitary cilia in the mesosalpinx and mesotubarium superius (infundibulo-cornual ligament) of some specimens was also observed. When samples were processed without postfixation in osmium tetroxide, a layer of amorphous material covered all surfaces. After intra-uterine insemination of five cows, no spermatozoa were found on their peritoneal mesothelium. Numerous spermatozoa were found after intraperitoneal insemination being attached throughout mesothelial surfaces. These results indicate that there are morphological differences between regions, but no cyclic changes, in the surface features of mesothelial cells covering the genital tract and adjacent ligaments of the cow, and that spermatozoa can bind to mesothelial surfaces after intraperitoneal insemination.

Evidence for a functional glycogen metabolism in mature mammalian spermatozoa.

The glycogen content in fresh raw dog spermatozoa was 0.22+/-0.03 micromol/mg protein. This matched with the presence of a glycogen-like staining in the head and midpiece. Glycogen levels lowered to 0.05 micromol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 micromol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35+/-0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25+/-0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo.

A structural study of the bovine vaginal fluid at estrus.

The present study describes the structural components of the bovine vaginal fluid at estrus by scanning electron microscopy (SEM) following critical point- and freeze-drying preparation procedures. Confocal scanning laser microscopy (CLSM) was also used to evaluate the structural integrity of samples, and a control sample was assessed by adding sperm to the vaginal fluid. Samples were collected from 10 cows at the time of artificial insemination, prepared for SEM by using critical point- and freeze-drying procedures, gold coated, and observed by SEM. Mesh size and filament thickness were measured with an image analyzer. Of the 10 samples processed, 4 were considered altered following critical point drying. Compaction and lack of filaments were observed in these samples. A small area of one sample showed a honey comb-like structure when freeze drying was used. Nonoriented filaments with different thicknesses and with a network-like structure were observed throughout the remainder of the samples. Filaments throughout all samples were also observed by CSLM. After critical point drying, the mesh area ranged from 0.8 to 101.4 microns 2; the minor axis from 0.7 to 10.8 microns; and filament thickness from 40 to 442 nm. Using freeze drying, the mesh area ranged from 0.9 to 493.8 microns 2; the minor axis from 0.7 to 27.5 microns; and filament thickness from 40 to 800 nm. When samples were freeze dried, mesh values were similar to the interstrand channels observed by CSLM. In sperm-vaginal fluid samples, following critical point- or freeze-drying procedures, spermatozoa were oriented randomly in the vaginal fluid and did not seem to alter filamentous structure. Our data suggest that the freeze-drying procedure better preserves the true structural dimensions of the vaginal fluid. Furthermore, the filamentous structure of the vaginal fluid does not appear to impede sperm transport.

A scanning electron microscope study of the structural component of the bovine vaginal fluid at oestrus.

The ultrastructure of the vaginal fluid of cows at oestrus was analysed by scanning electron microscopy. Samples of vaginal fluid were collected from 7 normally cycling and healthy cows, 3 to 4 years of age, in oestrus. Evaluation of the patterns of crystallization was used as a previous assessment of the quality of the samples. SEM images revealed a structure made up of non-oriented filaments constituting a network with a three-dimensional arrangement. These filaments were of different diameters and ranged between 50 and 400 nm. Mesh size, defined as the minimum distance measurement between strands, ranged between 2 to 8 microns.

The relationship of rheological behavior of the vaginal fluid at the time of insemination to the pregnancy rate in dairy cows.

The objective of this study was to determine whether rheological properties of the vaginal fluid at the time of insemination could be used as an indicator of potential fertility in dairy cows. Thixotropy and flow behavior were evaluated. Vaginal samples were collected from 347 cows at the time of insemination, within a 12- to 24-h interval after the onset of a natural estrus. Logistic regression analysis showed no significant effects of thixotropy measurements on the pregnancy rate, while flow behavior and the index of consistency (K) had a significant effect. The decrease in the consistency index (K) was related to the increase in the pregnancy rate (odds ratio = 0.99; P = 0.014), and the risk of nonpregnancy was higher (odds ratio = 0.46; P = 0.024) in cows in which vaginal fluid had a Newtonian behavior. Non-Newtonian behavior was recorded in 294 samples (85%) while 53 behaved as Newtonian fluids. For samples from all cows, the mean value of the consistency index was 310 +/- 28.2 milliPascals.second(n) (x +/- SEM) and ranged from 0 to 3881 milliPascals.second(n). In Newtonian samples, the consistency index mean was 2.6 +/- 0.6 milliPascals.second(n) and 367.6 +/- 30.4 milliPascals.second(n) for non-Newtonian samples. Of the 347 cows, 131 (38%) became pregnant. For flow behavior classification, 15 (28.3%) cows in which samples had Newtonian behavior and 116 (39.5%) cows with non-Newtonian samples became pregnant. Therefore, the highest probability of pregnancy was shown by cows in which samples had non-Newtonian behavior and the lowest consistency index values. Our data suggest that flow behavior measurements of the vaginal fluid at the time of insemination may be an indicator of potential fertility in cattle.

Effect of reproductive disorders previous to conception on pregnancy attrition in dairy cows.

This study was undertaken to determine whether reproductive disorders previous to conception influence pregnancy attrition in dairy cows. Reproductive disorders were registered and pregnancy diagnoses were performed as a part of a reproductive health program at 9 commercial dairy herds in northeastern Spain. Data from 3022 diagnosed pregnant lactating cows were used. Pregnancy diagnosis by palpation per rectum was performed from 33 to 70 d post insemination. Pregnancy attrition was registered when the pregnancy diagnosis was negative at the second palpation carried out between 120 and 150 d following insemination. Data analysis was performed by multiple logistic regression methods. Pregnancy attrition rates were 2.6 and 1.8 times higher in cows with previous pyometra and retained placenta, respectively, than in cows without these disorders. No effect of endometritis, ovarian cysts and repeat breeding was shown on pregnancy attrition. Our results suggest that additional efforts to reduce the risk of retained placenta and pyometra should decrease the incidence of pregnancy attrition in dairy cows.

Influence of management factors on pregnancy attrition in dairy cattle.

The present study was conducted to determine the influence of management factors on pregnancy attrition in dairy cattle. Data from 3162 diagnosed pregnancies in parous cows and 1050 in heifers at 9 commercial dairy herds in northeastern Spain were used. Pregnancy diagnosis by palpation per rectum was performed from 30 to 70 d post insemination. Pregnancy attrition was registered when pregnancy diagnosis resulted negative in a second palpation carried out between 120 and 150 d following insemination. Overall proportion of pregnancy losses was 7.9% (9.6% in parous cows and 2.8% in heifers). Data analysis was performed by multiple logistic regression methods. For all animals, effect of time of pregnancy diagnosis was shown (Odds ratio = 0.97 for 1 d increase; P = 0.0042). Conceptus loss in heifers was lower than in parous cows (Odds ratio = 0.28; P = 0.0001), and a higher proportion of pregnancy attritions was detected in animals inseminated in spring, summer and winter, compared to those inseminated in autumn (P < 0.04). Herd effect on pregnancy attrition was also significant. Similar results were observed in the subanalysis for parous cows and, furthermore, no effect of lactation number and of interval from previous calving to pregnancy was shown in this group. In heifers, no effect of time of pregnancy diagnosis and of insemination season on pregnancy attrition was shown, and only a herd effect was observed. Our data suggest that the influence of parity status (heifer vs cow) could affect the proportion of pregnancy attrition rather than early diagnosis, and, in pregnant cows, adaptation to seasonal changes associated with temperature decreases seem more efficient.

Persistent truncus arteriosus in a diprosopic newborn calf.

A newborn diprosopic female calf had a partially duplicated head with two faces each exhibiting a mouth, a snout, an anomalous incomplete mandible, two eyes and a lateral ear. A single ear with two small auditory canals was present on the midline between the two medial eyes. A type 1 persistent truncus arteriosus and hypoplasia of the thoracic portion of thymus were the most outstanding extracranial defects. In the heart, a persistent foramen secundum and a large patent foramen ovale allowed communication between the right and left atria. In the right ventricle, the small conus arteriosus was separated in part from the inflow tract by an anomalous 'septomarginal muscular septum'. An interventricular septal defect was also present. A large undivided truncus arteriosus, exhibiting a tricuspid truncal valve at its origin, arose for the most part from the conus arteriosus of the right ventricle. The truncus gave rise to the brachiocephalic trunk, the aortic arch, a small pulmonary trunk, from which the left and right pulmonary arteries emerged, and two coronary arteries. The etiology and pathogenetic mechanisms implicated in the appearance of persistent truncus arteriosus are reviewed. It is suggested that a deficit or insufficiency in the cranial neural crest may play a role in the pathogenetic mechanisms leading to the production of persistent truncus arteriosus and related defects in cephalic duplications.

Meteorological air pollution potential for Santiago, Chile: Towards an objective episode forecasting.

The geography and climate of the Santiago basin are, in general, unfavorable for the diffusion of air pollutants. Consequently, extreme events occur frequently during the high pollution season extending from April to August. The meteorological conditions concurrent with those extreme events are mainly associated with the leading edges of coastal lows that bring down the base of the semipermanent temperature inversion reducing the dirunal growth of the surface mixed layer. In order to produce an objective 12 to 24-hour episode forecast, a two-way multivariate discriminant analysis has been used in the definition of a meteorological air-pollution potential index (MAPPI), separating high and low meteorological air-pollution potential days. The same procedure has been applied in the selection of the most efficient predictors for the MAPPI objective forecast, based on 12 and 24 UTC radiosonde data at Quintero, about 100 km to the NW of Santiago. Results indicate about 70% correctly forecasted days, with satisfactory skill-scores relative to persistency. The strong persistency characterizing the most efficient predictors in the 12-hour objective forecast scheme, makes the prediction of the first and last days of any particular air-pollution potential episode particularly difficult. To overcome this problem, a new set of predictors based on continuous measurements near the level of the top of the temperature inversion layer (900 hPa during air-pollution episodes) is being tested. Preliminary results indicate that the time-integrated zonal wind component at that level is a reliable precursor for both the onset and the end of air-pollution potential episodes.

Development in vitro of rabbit embryos after freezing by two-step or ultra-rapid cooling methods.

The effects of rapid freezing by two-step and ultra-rapid cooling methods on 2-cell, 8 to 16-cell, compacted morula and early blastocyst stages of rabbit embryos were examined. Dimethylsulfoxide (DMSO, 3.5 mol/l) combined with sucrose (0.25 mol/l) was used as the freezing medium. The embryos were loaded into plastic straws with the freezing medium, held during 2.5 min and then were directly plunged into liquid nitrogen (ultra-rapid cooling) or after a time from 30 to 45 min held at -27 degrees C (two-step cooling). After rapid thawing embryo development was evaluated by in vitro developmental capacity shown by the embryos and was compared to control. All studied embryonic stages developed in vitro after two-step and ultra-rapid cooling procedures. However, higher developmental rates were obtained when the two-step cooling method was used. Using the two-step cooling method, best results were obtained at compacted morula and blastocyst stages, and no significant differences were shown when compared with control embryos at blastocyst stage. All embryonic stages to which the ultra-rapid cooling method was applied showed lower developmental rates than control. The results show that a high proportion of rabbit embryos can develop in vitro after freezing by two-step cooling method.