Comparison of day 2 embryo quality after conventional ICSI versus intracytoplasmic morphologically selected sperm injection (IMSI) using sibling oocytes

Objective: To evaluate whether intracytoplasmic morphologically selected sperm injection (IMSI) could influence early paternal effects by observing embryo quality at day 2.Study design: The study included 30 couples with at least one of the following criteria: male factor infertility, at least 2 previous failures of implantation or previous miscarriages after IVF/ICSI. Sibling oocytes of each patient were randomly assigned to either the ICSI group or the IMSI group. For IMSI, spermatozoa were selected at 8400x magnification through an inverted microscope equipped with Nomarski differential interference contrast optics, Uplan Apo 100x oil/1.35 objective lens and variable zoom lens. For conventional ICSI, spermatozoa were selected at 400x magnification. An embryo was defined as top quality if there were four identical blastomeres on day 2 with no fragments or multinucleation of blastomeres. Data were analysed using the Wilcoxon and chi-squared tests. The significance level was set at P < 0.05. The variables were analysed in relation to the general population and the subpopulations with or without male factor.Results: A total of 331 MII oocytes (30 oocyte retrievals) were selected and injected by the ICSI (n: 172) or IMSI (n: 159) procedure. For IMSI, only spermatozoa classified as morphologically normal at high magnification were used. No differences (P > 0.05) in fertilisation rate (ICSI: 70.9%; IMSI: 70.4%), early embryo cleavage rate (ICSI: 66.9%; IMSI: 60.4%) or cleavage rate (ICSI: 99.2%; IMSI: 99.1%) were observed. on day 2, as compared to ICSI, IMSI provided a similar proportion of top quality embryos (ICSI: 57.8%; IMSI: 52.2%; P > 0.05). These results were not influenced by the presence or absence of male factor.Conclusion: In terms of embryo quality at day 2, IMSI had the same performance as conventional ICSI. However, we cannot exclude the possibility that IMSI effects occur only as a positive later paternal effect. (C) 2010 Elsevier B.V. All rights reserved.

Testicular thermoregulation in Bos indicus, crossbred and Bos taurus bulls: relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production

Mechanisms of testicular thermoregulation, the relationship of scrotal, testicular vascular cone (TVC), and testicular morphology with thermoregulatory capability, and their effects on semen quality and sperm production were studied in 20 Bos indicus, 28 crossbred, and 26 Bos taurus bulls. The ratio of testicular artery length and volume to testicular volume were larger (P < 0.05) in B. indicus and crossbred bulls than in B. taurus bulls (1.03 and 0.94 cm/cm(2). versus 0.48 cm/cm(3); 0.034 and 0.047 ml/cm(3) versus 0.017 ml/cm(3), respectively). Testicular artery wall thickness (average 192.5, 229.0, and 290.0 mum, respectively) and arterial-venous blood distance in the TVC (average 330.5, 373.7, and 609.4 pm, respectively) were smallest in B. indicus, intermediary in crossbred, and greatest in B. taurus bulls (P < 0.05); the proximity between arterial and venous blood was consistent with the estimated decrease in arterial blood temperature after passage through the TVC (5.9, 5.0, and 2.9 degreesC, in B. indicus, crossbred, and B. taurus bulls, respectively). In crossbred and B. taurus bulls, there was a positive top-to-bottom scrotal temperature gradient and a negative testicular subtunic temperature gradient. However, in B. indicus bulls, both scrotal and testicular subtunic temperatures gradients were positive. Differences in the vascular arrangement, characteristics of the artery (e.g. wall thickness) or thickness of the tunica albuginea may have affected the testicular arterial blood and subtunic temperatures in B. indicus bulls. Better testicular thermoregulatory capability was associated with increased scrotal shape (pendulosity), testicular artery length and volume, and top-to-bottom gradient of the distance between the artery wall and the veins in the TVC. Increased semen quality was associated with increased testicular volume and scrotal subcutaneous (SQT) temperature gradient, and with decreased scrotal surface and testicular temperatures. Increased sperm production was associated with increased testicular artery volume, testicular volume, and SQT temperature gradient, and with decreased testicular artery wall thickness, scrotal circumference (SC), and scrotal surface, testicular subtunic, and epididymal temperatures. In conclusion, morphology of the TVC may contribute to the greater resistance of B. indicus bulls to high ambient temperatures by conferring a better testicular blood supply and by facilitating heat transfer between the testicular artery and veins. Testicular thermoregulation was associated with opposing scrotal and testicular subtunic temperatures gradients only in crossbred and B. taurus bulls. Scrotal, TVC, and testicular morphology influence testicular thermoregulatory capability and were associated with differences in semen quality and sperm production. (C) 2003 Elsevier B.V. All rights reserved.

Effect of age and genetic group on characteristics of the scrotum, testes and testicular vascular cones, and on sperm production and semen quality in AI bulls in Brazil

The objectives were to determine the effects of age and genetic group on characteristics of the scrotum, testes and testicular vascular cones (TVC), and on sperm production and semen quality in 107 Bos indicus, B, taurus and cross-bred bulls at three artificial insemination (AI) centers in Brazil. In addition, predictors of sperm production and semen quality were identified. In general, scrotal circumference (SC), scrotal shape score, scrotal neck perimeter, and testicular size (length, width and volume) increased (P < 0.05) with age. Although there were no significant differences among genetic groups for SC or testicular size, B. indicus bulls had the least pendulous scrotal shape, the shortest scrotal neck length, and the greatest scrotal neck perimeter (P < 0.05). Fat covering the TVC was thinner (P < 0.05) in bulls <= 36 months of age and in B. taunts bulls than in older bulls and B. indicus bulls, respectively. Age and genetic group did not affect testicular ultrasonic echotexture. B. indicus bulls tended (P < 0.1) to have the lowest average scrotal surface temperature (SST). In general, ejaculate volume, total number of spermatozoa and number of viable spermatozoa increased (P < 0.05) with age. However, there was no significant effect of age on sperm concentration, motility, major and total defects. The proportion of spermatozoa with minor defects was highest (P < 0.05) in bulls 37-60 months of age. B. indicus bulls had higher (P < 0.01) sperm concentration, total number of spermatozoa and number of viable spermatozoa than B. taunts bulls, with intermediate values for cross-bred bulls. Increased sperm production was associated with increased testicular volume, SC, TVC fat cover, and SST top-to-bottom gradient. Decreased semen quality was associated with increased SC and bottom SST, and decreased scrotal shape, scrotal neck perimeter and vascular cone diameter. In summary, age and genetic group affected the characteristics of the scrotum, testes, and TVC, sperm production and semen quality. In addition, characteristics of the scrotum, testes and TVC were associated with sperm production and semen quality in bulls and could be assessed for breeding soundness evaluation. (c) 2002 Elsevier B.V. All rights reserved.

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P < 0.10) and increased minor sperm defects (P < 0.001) in Year 1. B. indicus bulls had greater (P < 0.005) sperm concentration than B. taurus bulls in both years (1.7 x 10(9)/ml versus 1.2 x 10(9)/ml in Year 1 and 1.6 x 10(9)/ml versus 1.2 x 10(9)/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P < 0.05) total (11.4 x 10(9) versus 8.2 x 10(9)) and viable (6.7 x 10(9) versus 4.9 x 10(9)) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P < 0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3 x 10(9) versus 9.1 x 10(9) and 6.1 x 10(9) versus 5.4 x 10(9) in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P < 0.10) to have more major sperm defects and had more (P < 0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P < 0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Rat epididymal sperm quantity, quality, and transit time after guanethidine-induced sympathectomy

Guanethidine, a chemical that selectively abolishes peripheral noradrenergic nerves, was used to investigate the role of sympathetic innervation in the maintenance of epididymal sperm quantity and quality. Four groups of 10 adult male rats each were treated daily for 21 days, by i.p. injections, with either 0 (saline vehicle), 6.25, 12.5, or 25 mg/kg guanethidine. Norepinephrine content was reduced to undetectable levels in the cauda epididymidis in all guanethidine groups after 3 wk of treatment and was reduced to 7.4% of the control values after 1 wk of 6.25 mg/kg treatment. While body weight gain was significantly decreased at 12.5 and 25 mg/kg compared to that in controls, there was a significant increase in the weights of the seminal vesicles/coagulating glands in all treated groups. The number of homogenization-resistant spermatids per testis and the daily sperm production per testis remained unchanged. The weight of the epididymis was significantly increased at 6.25 and 12.5 mg/kg. Moreover, the number of cauda epididymal sperm and the transit time were increased significantly at 6.25 mg/kg (10.2 days) compared to values in the control cauda (6.3 days). Neither serum testosterone levels nor LH was affected in a dosage-related manner. There were no effects of guanethidine treatment on cauda epididymal sperm motility or morphology. A quantitative analysis of detergent-extracted cauda epididymal sperm proteins by SDS-PAGE revealed no differences, but there were diminutions in seven proteins in homogenates of caput/ corpus tissue. Histologic analysis of testis and epididymis sections revealed no differences between control and denervated animals. In a subsequent experiment the lowest effective dosage (6.25 mg/kg) was given to rats for 1 wk, and an increased number of cauda epididymal sperm and a delay in sperm transit were observed. Our results indicate that low-dosage guanethidine exposure denervates the epididymis within 1 wk, thereby delaying epididymal transit; however, neither 1- nor 3-wk exposure produces qualitative changes in the sperm.

Sexual behaviour, sperm quantity and quality after short-term streptozotocin-induced hyperglycaemia in rats

Studies of diabetes mellitus in the streptozotocin rat model suggest that sexual dysfunctions may result from diabetes-induced alterations of the neuroendocrine-reproductive tract axis. Our investigation was performed to better define the effects of short-term hyperglycaemia on rat epididymal sperm quantity, quality and transit time, using both natural mating and artificial in utero insemination protocols. Male rats were made diabetic with streptozotocin (sc, 40 mg/kg), whereas controls received vehicle. Sexual behaviour was tested after 15 days and sperm fertilizing ability was checked 22 days after the injection through natural mating and artificial in utero insemination. Other parameters such as daily sperm production, testosterone levels, as well as sperm morphology and motility were also investigated. Fifty per cent of the diabetic animals showed no copulatory behaviour during tests and the number of animals reaching ejaculation was smaller in the diabetic group when compared with the control group (33% vs. 83%). Diabetes resulted in decreased body and reproductive organ weights, as well as diminished sperm counts in the testis and epididymis, that were associated with diminution of plasmatic testosterone levels. After natural mating, there was a decrease in the fertility in the diabetic adult male rats (25.5%) compared with control animals (81.5%). However, distal cauda epididymal sperm from diabetic rats displayed normal fertilization ability (91.5%) using in utero insemination. There were no effects of hyperglycaemia on sperm transit time in the epididymis and on spermatogenesis. Our results indicate that diabetes mellitus produces reproductive dysfunction, but does not compromise sperm fertilizing ability in the cauda epididymis in this experimental model.

Effect of ß-Mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.

2016

Components of fractionated stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the accessory glands before and during ejaculation. The stallion s ejaculate is a series of jets that differ in sperm concentration, semen volume and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the total number of sperm. The semen volume and sperm concentration in each of the jets decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with a low sperm concentration. The aims of these studies were to examine the effects of the different SP fractions, and the presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five experiments. The levels of selected enzymes, electrolytes and proteins in different SP fractions were determined. These studies also aimed at assessing the individual variation in the levels of the selected SP components and in the effects of SP on spermatozoa. The association between the components of SP and semen quality, sperm longevity, and fertility was examined with a stepwise linear regression analysis. Compared to samples containing SP during storage, centrifugation and the subsequent removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw motility parameters were also higher in samples containing SP compared to samples stored without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the samples stored with SP than those without SP, and this effect was seen in both sperm-rich and sperm-poor fractions. There were no differences in DNA integrity between fractions stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor fraction after SP removal. The differences between fractions in sperm motility after cooled storage were non-significant. The levels of alkaline phosphatase, acid phosphatase and β-glucuronidase were higher in the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions. The concentrations of sodium and chloride were highest in pre-sperm fluid. In the sperm-poor fraction, the level of potassium was associated with the maintenance of sperm motility during storage. The levels of alkaline and acid phosphatase were associated with sperm concentration and the total number of spermatozoa in the ejaculates. None of the measured SP components were correlated to the first cycle pregnancy rate. In summary, the removal of SP improved DNA integrity after cooled storage compared with samples containing SP. There were no differences in the maintenance of sperm motility between the sperm-rich and sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was found in the sperm-rich fractions stored without SP. In practice, the results presented in this thesis support the use of individual modifications of semen processing techniques for cooled transported semen from subfertile stallions.

Milt quality determination of a critically endangered fish, olive barb (Puntius sarana, Ham.) in Bangladesh

The present study was aimed to evaluate the characteristics of the olive barb sperm. Milt was collected fortnightly from 49 male fish (mean weight 90.8 g and length 18.64 cm) from April to July in 2008. In the olive barb ejaculated milt, volume (µl/g), motility (%), duration of motility (s), concentration (x 10 super(10)/ml) and pH values were found to be 6.06±0.32, 88.27±0.71, 171.41±7.41, 5.16±0.05 and 7.75±0.04, respectively. Milt volume was significantly (P<0.05) correlated with sperm concentration. Milt volume, sperm concentration, motility and duration of motility significantly varied (P<0.05) during spawning season.

Sildenafil citrate improves sperm motility but causes a premature acrosome reaction in vitro

Objective: to determine if sildenafil citrate; a cyclic monophosphate specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction. Design: laboratory analysis of sperm motility after exposure to sildenafil citrate using computer assisted semen analysis (CASA) and acrosome reaction by fluoroscein isothiocyanate labelled peanut agglutinin (FITC-PNA) staining. Setting: An ART unit Patients: 57 male patients Interventions: Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with sildenafil citrate ( 0.67uM) at 37ï?°C for up to 180 minutes. Main outcome measures: both the numbers and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Further, samples revealed that these effects were consistent in the 90% and 45% subpopulations of sperm. In both subpopulations, sildenafil also caused a significant increase in the proportion of acrosome reacted sperm - 22.1% compared with 11.8% in the control group of the good quality fraction (p

Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success

Objective: To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF.
Design: Prospective study.
Setting: Academic research laboratory.
Patient(s): Seventy-five couples undergoing IVF and 28 fertile donors.
Intervention(s): Sperm DNA fragmentation was measured by the alkaline Comet assay in semen and sperm after density gradient centrifugation (DGC). Binary logistic regression was used to analyze odds ratios (OR) and relative risks (RR) for IVF outcomes.
Main Outcome Measure(s): Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy.
Result(s): Men with sperm DNA fragmentation at more than a diagnostic threshold of 25% had a high risk of infertility (OR: 117.33, 95% confidence interval [CI]: 12.72–2,731.84, RR: 8.75). Fertilization rates and embryo quality decreased as sperm DNA fragmentation increased in semen and DGC sperm. The risk of failure to achieve a pregnancy increased when sperm DNA fragmentation exceeded a prognostic threshold value of 52% for semen (OR: 76.00, CI: 8.69–1,714.44, RR: 4.75) and 42% for DGC sperm (OR: 24.18, CI: 2.89–522.34, RR: 2.16).
Conclusion(s): Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome.

Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1 + P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1 + P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates. During late spermatogenesis, around 85% of the histones in the sperm nucleus are replaced with protamines. This process results in sperm chromatin compaction and also transcription silencing. In the human, protamines are comprised of two types: protamine-1 (P1) and protamine-2 (P2). Variations in sperm protamine expression are associated with male infertility. Similarly, sperm DNA integrity is important for male fertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates.