Effects of conjugated linoleic acid or betaine on the growth performance and fatty acid composition in backfat and belly fat of finishing pigs fed dried distillers grains with solubles.

The objectives of this study were to determine the effects of conjugated linoleic acid (CLA) or betaine on the growth performance, carcass characteristics and fatty acid composition in backfat and belly fat of pigs fed distillers dried grains with solubles (DDGS). Thirty-two (60±2 kg) crossbred barrows (Duroc×Landrace×Yorkshine) were assigned to one of four diets randomly: (1) the control diet containing no corn DDGS (control group); (2) the diet containing 30% corn DDGS (DDGS-fed group); (3) the diet containing 30% corn DDGS and 10 g/kg CLA (CLA-fed group); (4) the diet containing 30% corn DDGS and 1 g/kg BET (BET-fed group). The pigs fed DDGS showed that the percentages of C18:2, polyunsaturated fatty acid (PUFA) and iodine value (IV) increased, while C18:1, saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) decreased. Pigs fed the DDGS+CLA or DDGS+betaine diets showed the increased percentage of SFA, and the decreased percentage of C18:2, PUFA and IV. In conclusion, results confirmed that the diets containing 30% DDGS had no detrimental effects on growth performance, but increased the percentage of PUFA and IV and decreased the percentage of SFA and MUFA in the backfat and belly fat. However, supplementation with CLA or BET can part reverse these effects on carcass fat in finishing pigs.

Sperm hyperactivation as quality control for sperm penetration assay.

The sperm penetration assay (SPA) is subject to considerable variation, and controls are needed to verify the accuracy of the results. It is proposed that sperm hyperactivation (HA) can serve as a quality control check for the SPA. The objective was to determine if there was an association between the SPA outcome and sperm HA measured at various times during the SPA procedure. The data showed a significant correlation between percent sperm HA and percent zona-free oocyte penetration by sperm preincubated for three hours prior to sperm-oocyte interaction (short preincubation). Some sperm hyperactivity was observed in liquefied raw semen samples, but this was insignificantly related to SPA results. Low correlation was observed between SPA results and sperm HA determined immediately after centrifuge washing of sperm. The results suggest that it is possible to utilize sperm HA measured immediately after the sperm-oocyte interaction period as a quality control check of SPA results.

Assessment of sperm for cryopreservation using the hypoosmotic viability test.

In summary, the hypoosmotic viability parameter was significantly correlated with the outcome of the thawed sperm motility. The prefreeze supravital staining for sperm viability and the hypoosmotic sperm swelling test were not predictive of the thawed sperm total motility. The hypoosmotic viability parameter was not correlated to the postwarmed sperm motility after refrigeration. The results indicated that the integrity of the sperm membranes at the head were more important than the tail membrane.

Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid.

The present study demonstrated the presence of HPV gene sequences in Percoll-washed sperm cells using polymerase chain reaction primers targeting smaller gene regions. Up to 64% of the sperm specimens were shown to contain gene sequences indicative of the presence of HPV. Human papillomavirus type 16 was detected about twice as often as HPV type 18. The results suggest the possible role of sperm as a vector for HPV.

White blood cells in semen affect hyperactivation but not sperm membrane integrity in the head and tail regions.

The presence of high numbers of peroxidase-positive PML in ejaculated semen significantly reduced sperm HA, an important step leading to sperm capacitation. Sperm membranes at both the head and tail regions, as assessed by the hypo-osmotic viability parameter and the hypo-osmotic sperm swelling test, respectively, were not affected by peroxidase-containing leukocytes. Sperm motility was not affected, but sperm curvilinear and straight line velocity parameters were reduced in the presence of high concentrations of leukocytes in the ejaculate. The results suggested that the effect of leukocytes on sperm was through a reduction in sperm hyperactive motility but not through alterations in the sperm head and tail membranes.

Sperm as a noninvasive gene delivery system for preimplantation embryos.

OBJECTIVE: To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. DESIGN: Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. SETTING: Clinical and academic research environment. MAIN OUTCOME MEASURE: Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. RESULTS: Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. CONCLUSION: The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

Utilization of bioreactance technique as indicator for preload responsiveness during living donor liver donation.

BACKGROUND: Fluid restriction has been used to decrease central venous pressure as a strategy of fluid management during living donor liver donation (LDLD). However, potential risks such as hemodynamic instability are also involved during the procedure. Monitoring of preload or preload responsiveness is therefore crucial during LDLD. The aim of this study was to explore the efficiency of an innovative bioreactance method that introduced the noninvasive cardiac output monitor (NICOM, Cheetah Medical, Vancouver, Wash, United States), as surrogate indicator for preload responsiveness during LDLD. METHODS: Twenty-six patients underwent LDLD and were enrolled in this study. Fluid was restricted until the organ was harvested. The ultrasonic cardiac output monitor (USCOM Ltd, Sydney, Australia) was applied to measure stroke volume (SV) before and after 500 mL of 6% hydroxyethyl starch 130/0.4 (Voluven, Fresenius Kabi, Friedberg, Germany) fluid challenge, and the value of SV variation (SVV) from NICOM was also recorded. Fluid responsiveness was defined if SV increased by more than 15%. Receiver operating characteristic (ROC) curve was performed. RESULTS: The area under the ROC curve was 0.6023. The optimal cutoff value of SVV from NICOM was 12% during LDLD. CONCLUSIONS: Our study has shown that SVV measured from the bioreactance technique is a poor indicator for monitoring preload responsiveness during LDLD.

Corrected flow time is a good indicator for preload responsiveness during living donor liver donation.

BACKGROUND: Corrected flow time (FTc) has been utilized as preload indicator in recent literature. Accurate estimation of preload status during living donor liver donation (LDLD) is important due to fluid restriction. We evaluate the effectiveness of FTc as a surrogate of preload indicator during LDLD. MATERIALS AND METHODS: Twenty-five patients undergoing LDLD were enrolled in the study. Administration of intravenous fluid was restricted before lobectomy was performed. After the organ was harvest, fluid challenge with 500 mL of Voluven (130/0.42, Fresenius, Friedberg, Germany) was performed. Stroke volume (SV) was measured with ultrasonic cardiac output monitor (USCOM; USCOM Pty, Ltd, Sydney, Australia) before and after the fluid challenge. The FTc value obtained with USCOM before fluid challenge was recorded. Fluid responsiveness was defined as an increase in SV of more than 15%. Receiver operating characteristic (ROC) curve was performed. RESULTS: The area under ROC curve was 0.9. The optimal cutoff FTc value was 340 milliseconds during LDLD. CONCLUSIONS: FTc is a noninvasive, easily obtainable, and essentially good preload indicator during LDLD.

Cardiac output derived from arterial pressure waveform analysis in patients undergoing liver transplantation: validity of a third-generation device.

BACKGROUND: Hemodynamic monitoring is essential to a successful liver transplantation procedure. FloTrac, a hemodynamic monitor that uses arterial-waveform-based pulse contour analysis for cardiac output (CO) measurement, has proven useful in many clinical settings. One of the primary foci of FloTrac's recent third-generation software upgrade was improving its accuracy in low systemic vascular resistance status. We evaluated the accuracy of the upgraded FloTrac monitor during liver transplantation. MATERIALS AND METHODS: Twenty-eight patients undergoing liver transplantation were enrolled in the study. Two sets of CO were measured with a radial arterial line connected to a FloTrac monitor (COFT) and a pulmonary artery catheter connected to a continuous cardiac output Vigilence monitor (COPAC). Simultaneous CO measurement was performed and recorded every 5 minutes throughout the surgery. Bland-Altman analysis was used to estimate the accuracy. The comparative method and reference method were considered interchangeable if the limits of agreement did not exceed a threshold set a priori at the greater of ±1 L/min, or a percentage error of lesser than 30%. RESULTS: In all, 3234 paired data were collected. The bias was -0.8 L/min and the limits of agreements were -5.6 to 4.0 L/min. Percentage error was 75%. Regression analysis of the systemic vascular resistance index (SVRI) and the bias between COPAC and COFT showed that the bias was inversely related to the SVRI [r2=0.49; P<.001, y=-32.1983+9.9978 Log(x)]. CONCLUSIONS: Despite a software upgrade, the effectiveness of the FloTrac artery-derived cardiac output monitor for CO measurement during liver transplantation remains limited.

Stroke volume variation derived by arterial pulse contour analysis is a good indicator for preload estimation during liver transplantation.

BACKGROUND: Accurate determination of preload during liver transplantation is essential. Continuous right ventricular end diastolic volume index (RVEDVI) has been shown to be a better preload indicator during liver transplantation than the filling pressures. However, recent evidence has shown that dynamic variables, in this case stroke volume variation (SVV), are also good indicators of preload responsiveness. In this study, we evaluated the correlation between SVV, which we derived from arterial pulse contour analysis and RVEDVI. METHODS: In this study, we looked for possible relationships between SVV obtained through FloTrac/Vigileo monitor, central venous pressure (CVP), pulmonary arterial occlusion pressure (PAOP), and RVEDVI in 30 patients undergoing liver transplantation. Measurements were taken at 11 defined points during different phases across liver transplantation. Each set of measurement was taken during a steady state, which means at least 15 minutes elpased after any changes occured in either the infusion rate of catecholamines or ventilator settings. Pearson's test was used for correlation estimation. RESULTS: There was a statistically significant (P<.01) relationship between SVV and RVEDVI with a correlation coefficient of -0.87. The correlations between CVP (r=0.42), PAOA (r=0.46), and RVEDVI were less strong. CONCLUSION: We conclude that SVV is a good indicator for preload estimation during liver transplantation. A higher SVV value is associated with a more hypovolemic fluid status.

Cardiac output derived from arterial pressure waveform analysis: validation of the third-generation software in patients undergoing orthotopic liver transplantation.

BACKGROUND: The upgraded third-generation software (version 3.02) for the FloTrac/Vigileo system has been developed to particularly improve the accuracy of cardiac output (CO) measurements in hyperdynamic conditions. The aim of our study was to compare the CO values obtained using the FloTrac/Vigileo system during orthotopic liver transplantation (OLT) with those obtained in the same circumstances using a Swan-Ganz catheter (bolus thermodilution method). METHODS: Twenty consecutive recipients scheduled for OLT were studied. Simultaneous CO values measured by both devices were obtained at 10 predefined time points throughout the surgery. A percentage error of not more than 30% was established as the criterion for device interchangeability. RESULTS: A total of 200 paired measurements were obtained. The CO values derived from the FloTrac/Viligeo ranged from 2.8 to 10.9 L/min, with a mean of 5.91±1.81 L/min. The values from bolus thermodilution ranged from 2.2 to 13.2 L/min, with a mean of 6.12±2.07 L/min. The bias was 0.22, and the limits of agreement were -3.13 to 3.56 L/min. The percentage error between the FloTrac/Viligeo and bolus thermodilution measurements was 54.93%. The percentage errors of paired measurements in three surgical phases by subgroup analysis were 43.50% (dissecting phase), 62.9% (anhepatic phase), and 56.05% (reperfusion phase), respectively. CONCLUSION: CO measurements obtained using the less invasive arterial waveform FloTrac/Vigileo system upgraded with the third-generation software had poor intraoperative agreement with pulmonary artery thermodilution CO measurements in patients undergoing OLT.

Diadenosine tetraphosphate (Ap4A) and triphosphate (Ap3A) signaling of human sperm motility.

The ubiquitous dinucleotide polyphosphate, diadenosine tetraphosphate (Ap4A), has been shown to be a signal molecule for DNA replication in mammalian cells. In this study, Ap4A and a related compound, diadenosine triphosphate (Ap3A), were tested for possible signaling functions in human spermatozoa. A computerized automated semen analyzer was used to detect changes in spermatozoa motility parameters. Cryopreserved-thawed donor spermatozoa were washed and incubated in 0.1 mM Ap4A, 0.1 mM Ap3A, or control medium. The data indicated that both Ap4A and Ap3A decreased the percentage of motile spermatozoa after 4 or more hours of incubation in vitro. The two dinucleotide polyphosphates caused an increase in the amplitude of lateral spermatozoa head displacement parameter only at the start of incubation. The other spermatozoa kinematic parameters were unaffected. No opposing ying-yang dual actions of Ap4A to Ap3A were seen. From the results, Ap4A and Ap3A were observed to be potential inhibitory signals of spermatozoa motility after prolonged exposure.

Rapid identification of deoxyribonucleic acid sequence differences in cytochrome P-450 21-hydroxylase (CYP21) genes with denaturing gradient gel blots.

Denaturing gradient gel electrophoresis was used to identify single-base differences in the cytochrome P-450 21-hydroxylase (CYP21) genes of 132 unrelated control individuals and family members of three unrelated patients with 21-hydroxylase deficiency. The salt-wasting variety was caused by gene deletion and gene conversion/deletion mutations in affected members of two families studied. The simple virilizing form, present in the third family, was caused by an apparent point mutation not detectable by routine Southern blots. We have detected many restriction fragment melting polymorphisms in the CYP21 genes of the members of both salt-wasting families and normal individuals with denaturing gradient gel electrophoresis. We also identified a restriction fragment melting polymorphism specific for the simple virilizing patient in the third family. The data demonstrate that the CYP21 genes are highly polymorphic and that denaturing gradient gel electrophoresis is useful for genomic deoxyribonucleic acid analysis of patients with 21-hydroxylase deficiency.

A double method sperm wash for artificial insemination.

Recently, there have been concerns regarding the presence of reactive oxygen species (ROS) produced during sperm processing for insemination. However, the sperm wash methods that yielded low ROS levels also had low sperm recovery after processing. The objective of this study was to compare sperm recovery after swim-up from pellet, overlay, and 2-layer Percoll wash methods with the recovery after the double method wash. The latter method consisted of a combination of 2 sperm wash methods, namely, the overlay and the Percoll method. Motile sperm were first collected through the overlay method. The leftover semen was then processed through the 2-layer Percoll method to scavenge motile sperm and the resultant pellet combined with the pellet from the overlay method. In this manner, the level of ROS was kept to a minimal, sperm recovery was improved, and a mixture of sperm with different surface properties was produced as a result of using different processing methods. The results indicated an improvement in sperm recovery and in total sperm motility in noncryopreserved sperm after using the double method wash when compared with the other wash methods. The study suggests that the double method wash is a feasible method for processing sperm for insemination.

Combined supravital staining and hypoosmotic swelling test.

The parameter of sperm viability in hypoosmotic solution (VHOS) provides information concerning the membrane integrity of the sperm head. The objective of this study was to determine the association between the VHOS parameter and the sperm penetration assay. The VHOS parameter correctly predicted 70.0-71.4% of the failed sperm penetration assay samples in the short duration preincubation groups. A combination of both the VHOS parameter and the hypoosomotic sperm swelling (HOS) test significantly reduced the number of false negative results. In general, a sperm sample with an abnormal VHOS result and an abnormal HOS test result would be associated with a negative sperm penetration assay. Washing by centrifugation appeared to weaken the sperm head membranes while the swim-up method selected for sperm with strong head and tail membranes. After the various processing methods unique changes in the integrity of the sperm head and tail membranes for each sperm sample may help to identify the optimal method of preparation for individual patients undergoing the newer assisted reproductive technologies such as sperm microinjection.

The relationship between human sperm fertilizing capacity and histocompatibility linked antigen (HLA) alleles gene sequences.

Heterodimer membrane glycoproteins expressed from hypervariable genes located in the histocompatibility linked antigen (HLA) class II genes on chromosome 6 have been shown to induce activation of lymphocytes and are involved in human sperm binding processes. The objective was to identify an association between HLA-DQA1, -DRB1 or -DPB1 genes and sperm kinematic parameters and sperm penetration of oocytes. The results showed reduced sperm hyperactivation and decreased sperm penetration of zona-free oocytes when the HLA-DRB1 allele was present. The reduced hyperactive motility was not attributed to alterations in sperm kinematic parameters. In contrast, the HLA-DPB1 gene only affected sperm count, linearity of movement and sperm head dimensions. HLA-DQA1 had no effect on the sperm parameters. The data suggest a link between HLA-DRB1 and HLA-DPB1 genes and sperm concentration, sperm movement characteristics and fertilizing capacity.

Clinical pregnancy rate after the double method wash and intrauterine insemination.

The protocol for intrauterine insemination (IUI) involves sperm processing using different methods that have produced varying results. These sperm wash methods do not take into consideration the problems of the exact timing of ovulation and the requirements of sperm cells at different stages of capacitation. The objectives of this study were 1) to use the double method wash previously reported to produce a mixture of different populations of sperm cells and determine the pregnancy outcome after IUI and 2) to compare the sperm kinematic parameters after the double method wash with those after the centrifuge (or whole-population) wash method. Patients were divided into either the double method group (n = 119) or the centrifuge method group (n = 76). The Hamilton-Thorn HTM-C automated sperm motility analyzer (Hamilton-Thorn Research, Danvers, MA) was used to analyze sperm motility parameters. Pregnancy outcomes were evaluated after controlled ovarian stimulation and IUI. An almost 2-fold increase was seen in the pregnancy rate with the double method wash compared with the centrifuge method wash. Sperm motility and velocity were also enhanced in the double method wash groups. The results support the usefulness of the double method wash for the preparation of sperm for IUI.

Temporal and constitutive expression of homeobox-2 gene (Hu-2), human heat shock gene (hsp-70), and oncogenes C-sis and N-myc in early human trophoblast.

Recent studies of the genetic basis of animal development indicate that homeobox genes, protooncogenes, and some heat shock genes may play a role in early embryogenesis. To investigate the possibility that these genes function in early human embryonic development, we monitored the expression of a human homeobox gene (Hu-2), two human protooncogenes (C-sis and N-myc), and a human heat shock gene (hsp-70) in human trophoblasts at 7 to 13 weeks gestational age. All these genes were found to be expressed in the tissues analyzed. The hsp-70 gene was expressed at nearly constant levels throughout the development period surveyed, whereas N-myc, C-sis, and Hu-2 showed a coordinated pattern of regulated expression. These results are consistent with a functional role of these genes in the early course of human development.

Uptake of exogenous human papilloma virus L1 DNA by oocytes and detection by the polymerase chain reaction.

OBJECTIVE: The purpose of this study was to determine if oocytes were capable of taking up exogenous DNA such as human papillomaviral (HPV) DNA and evaluate the zona pellucida as a barrier to the entry of foreign DNA into the oocyte. METHODS: The experiment consisted of four groups of hamster oocytes exposed to HPV DNA fragments: Group A, zona-free oocytes (n = 5); Group B, oocytes with an intact zona pellucida (n = 5); Group C, oocytes fixed in 4% buffered formalin solution for 20 min (n = 5); and Group D, zona-free oocytes (n = 4). Group C oocytes served as an internal control to ensure adequate washing of the oocytes after incubation. RESULTS: The zona pellucida was not a barrier to foreign DNA molecules. The PCR did not detect L1-HPV and beta-globin gene sequences in the untreated hamster oocyte. Uptake of the smaller DNA fragments such as that amplified from the beta-globin region was independent of active oocyte cell processes. CONCLUSION: Oocytes cultured in vitro can passively take up exogenous DNA fragments. The results suggest a possible role of oocytes as vectors for foreign DNA.

Identification of male-factor semen samples prior to insemination and in vitro fertilization.

Semen analyses carried out as part of the clinical in vitro fertilization or intrauterine insemination protocols provide important information that determine the type of clinical treatment of the male partner and the sperm processing method. It is postulated that the sperm of male-factor patients cannot survive hypoosmotic stress conditions because of defective sperm membrane function. To test this, 0.1 ml of semen from each of 102 patients was placed in 1.0 ml of 150 mosmol/liter eosin citrate fructose solution and incubated for 30 min at 37 degrees C. The percentage viability of the sperm cells was then determined. The results indicated that patients with two or more abnormal semen parameters had a significantly lower percentage viability while in the hypoosmotic solution (40.6 +/- 4.7%), in contrast to non-male-factor patients (69.0 +/- 1.6%). Donor sperm (N = 32) serving as controls (73.3 +/- 2.1%) had a viability in hypoosmotic solution similar to that of non-male-factor patients. The data suggest that sperm of male-factor patients are less able to survive the hypoosmotic stress conditions as shown by the percentage viability in hypoosmotic solution and emphasize the importance of using less stressful sperm processing methods for in vitro fertilization or insemination in these patients.