Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P < 0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P < 0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P > 0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P > 0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P > 0.05) in the presence of heparin and PHE (P < 0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development. © 2013 The Society for In Vitro Biology.

Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development

Lipid peroxidation and vitamin E in serum and follicular fluid of infertile women with peritoneal endometriosis submitted to controlled ovarian hyperstimulation: a pilot study

Objective: To assess the level of lipid peroxidation (LP) and vitamin E in the follicular fluid and serum of infertile patients, with or without endometriosis. who were submitted to ovulation induction for assisted reproduction procedures. Design: Prospective study. Setting: Assisted conception unit, university hospital. Patient(s): Infertile patients 20 to 38 years of age were selected prospectively and consecutively and were divided into the endometriosis group (17 patients with pelvic endometriosis) and the control group (19 patients with previous tubal ligation or male factor and without endometriosis). Intervention(s): Peripheral blood samples were collected on D1 (before the beginning of the use of gonadotropins), D2 (day of hCG administration), and D3 (day of oocyte retrieval). On D3, follicular-fluid samples free from blood contamination also were collected and stored. Main Outcome Measure(S): Lipid peroxidation was assessed by malondialdehyde quantification by spectrophotometry, and measurement of vitamin E was performed by HLPC. Result(s): On D1, no significant difference in LP was observed between groups. However, vitamin E levels were significantly higher in the control group. On D2, LP levels were significantly higher in the endometriosis group compared with in the control group, and vitamin E levels continued to be significantly higher in the control group. On D3, there was no significant difference in serum and follicular-fluid levels of LP and vitamin E between groups. However, on D3, vitamin E levels were found to be significantly higher in serum than in follicular fluid in both groups, whereas malondialdchyde levels were significantly lower in follicular fluid than in serum only in the control group. Conclusion(s): Before the beginning of ovulation induction, a significant decrease in vitamin E was observed in patients with endometriosis, perhaps because antioxidants are consumed during oxidation reactions. After ovulation induction with exogenous gonadotropins, the group of patients with endometriosis not only presented increased lipid peroxidation but also maintained lower vitamin E levels than the control group, a fact that hypothetically could compromise oocyte quality in endometriotic patients. However, on the day of oocyte retrieval, both serum LP potential and vitamin E levels were found to be similar in the two groups. (Fertil Steril(R) 2008; 90:2080-5. (C) 2008 by American Society for Reproductive Medicine.)

Influência do método de separação dos espermatozóides viáveis (swim-up) na eficiência de seleção do sexo de bovinos por gradiente descontínuo de densidade e o impacto no melhoramento genético animal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Quantificação diferencial de transcritos em espermatozóides de suínos suplementados com vitaminas e minerais

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determination of trace elements in bovine semen samples by inductively coupled plasma mass spectrometry and data mining techniques for identification of bovine class

The reproductive performance of cattle may be influenced by several factors, but mineral imbalances are crucial in terms of direct effects on reproduction. Several studies have shown that elements such as calcium, copper, iron, magnesium, selenium, and zinc are essential for reproduction and can prevent oxidative stress. However, toxic elements such as lead, nickel, and arsenic can have adverse effects on reproduction. In this paper, we applied a simple and fast method of multi-element analysis to bovine semen samples from Zebu and European classes used in reproduction programs and artificial insemination. Samples were analyzed by inductively coupled plasma spectrometry (ICP-MS) using aqueous medium calibration and the samples were diluted in a proportion of 1:50 in a solution containing 0.01% (vol/vol) Triton X-100 and 0.5% (vol/vol) nitric acid. Rhodium, iridium, and yttrium were used as the internal standards for ICP-MS analysis. To develop a reliable method of tracing the class of bovine semen, we used data mining techniques that make it possible to classify unknown samples after checking the differentiation of known-class samples. Based on the determination of 15 elements in 41 samples of bovine semen, 3 machine-learning tools for classification were applied to determine cattle class. Our results demonstrate the potential of support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF) chemometric tools to identify cattle class. Moreover, the selection tools made it possible to reduce the number of chemical elements needed from 15 to just 8.

Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin-like Growth Factor I, Insulin and Membrane Integrity of Bull Spermatozoa

Contents This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 mu M beta-mercaptoethanol (beta-ME) and 50 mu M cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.

Predicting the mating system from phenotypic correlations between life-history and sperm quality traits in the Alpine whitefish Coregonus zugensis

The mating behavior and reproductive strategies of Alpine whitefish like Coregonus zugensis (Nusslin) are poorly understood, probably because they spawn in deep water where direct observations are difficult. In this study, we interpret life-history and sperm quality traits of fish that we caught from their spawning place. We found that males invest heavily into gonadal tissue (up to 5.6% of their body weight), which is, in comparison to other fish, consistent with external fertilization, distinct pairing and moderate to high communal spawning, or no pairing and low to moderate communal spawning. Sperm competition theory and recent experimental studies on other salmonids predict that males optimize ejaculate characteristics in relation to the costs of sperm and the level of competition they have to expect: dominant males are predicted to invest less into ejaculate quality and to have slower spermatozoa than subdominant males. We found that spermatozoa of older males are slower than those of younger males. Moreover, older males have larger breeding tubercles, a secondary sexual trait that has, in some previous studies, been found to be linked to good condition and to good genetic quality. Our results suggest that C. zugensis has age-linked reproductive strategies, that multimale spawning is common, i.e., that sperm competition plays a significant role, and that older males are on average dominant over younger males at the spawning place.

Effect of psychological stress on the L-arginine-nitric oxide pathway and semen quality

It has been reported that mental stress causes abnormality of spermiogram parameters. We investigated the effect of psychological stress on the L-arginine-nitric oxide (NO) pathway. Semen samples were collected from 29 healthy fourth semester medical students just before (stress) and 3 months after (non-stress) the final examinations. Psychological stress was measured by the State Anxiety Inventory questionnaire. After standard semen analysis, arginase activity and NO concentration were measured spectrophotometrically in the seminal plasma. Measurements were made in duplicate. During the stress period, sperm concentration (41.28 ± 3.70 vs 77.62 ± 7.13 x 10(6)/mL), rapid progressive motility of spermatozoa (8.79 ± 1.66 vs 20.86 ± 1.63%) and seminal plasma arginase activity (0.12 ± 0.01 vs 0.22 ± 0.01 U/mL) were significantly lower than in the non-stress situation, whereas seminal plasma NO (17.28 ± 0.56 vs 10.02 ± 0.49 µmol/L) was higher compared to the non-stress period (P < 0.001 for all). During stress there was a negative correlation between NO concentration and sperm concentration, the percentage of rapid progressive motility and arginase activity (r = -0.622, P < 0.01; r = -0.425, P < 0.05 and r = -0.445, P < 0.05, respectively). These results indicate that psychological stress causes an increase of NO level and a decrease of arginase activity in the L-arginine-NO pathway. Furthermore, poor sperm quality may be due to excessive production of NO under psychological stress. In the light of these results, we suggest that the arginine-NO pathway, together with arginase and NO synthase, are involved in semen quality under stress conditions.