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.

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)

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.

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.

Artificial oocyte activation using calcium ionophore in ICSI cycles with spermatozoa from different sources

The present study evaluated the effect of artificial oocyte activation (AOA) with calcium ionophore A23187 oil intracytoplasmic sperm injection (ICSI) cycles using spermatozoa from different sources. The 314 cycles evaluated were divided into three groups according to sperm origin, the ejaculated group (n = 92), the epididymal group (n = 82). and the testicular roup (n = 140). Each group was further split into experimental subgroups, depending oil whether or no AOA was performed. In additions the cycles of women younger than 36 years were evaluated separately. For each experimental group, ICSI outcomes were compared between subgroups. No significant difference was observed between subgroups for all sperm origin groups. When evaluating only the cycles of women younger than 36 years of age, AOA increased the percentage of high-quality embryos (74.5 versus 53.0%. P = 0.011) and the implantation rate (19.3 versus 10.5%, P = 0.0025) when it was used with ejaculated spermatozoa, and the percentage of high-quality embryos (64.4 versus 50.3%, P = 0.006) when epididymal spermatozoa were used. These results may suggest that both sperm maturity and oocyte quality play a role in oocyte activation. However. this study is to be continued to confirm these findings.

Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa

Objective: To evaluate the effect of artificial oocyte activation (AOA) on intracytoplasmic sperm injection (ICSI) cycles using surgically retrieved sperm.Design: Laboratory study.Setting: Fertility/assisted fertilization center.Patient(s): Couples undergoing surgical sperm retrieval for ICSI (n = 204).Intervention(s): Application of calcium ionophore A23187 for AOA.Main Outcome Measure(s): Cycles were divided into experimental groups according to the origin of the sperm used for injection and the type of azoospermia: [1] testicular sperm aspiration in nonobstructive-azoospermic patients (TESA-NOA group, n = 58), [2] TESA in obstructive-azoospermic patients (TESA-OA group, n = 48), [3] and percutaneous epididymal sperm aspiration in obstructive-azoospermic patients (PESA-OA, n = 98). For each experimental group, cycles where AOA was applied (subgroup: activation) were compared with cycles in which AOA was not applied (Subgroup: control). The fertilization, high-quality embryo, implantation, and pregnancy rates were compared among the subgroups.Result(s): For patients undergoing TESA, AOA did not improve ICSI outcomes for either type of azoospermia. However, for cases in which the injected sperm were retrieved from the epididymis, a statistically significantly increased rate of high-quality embryos was observed with AOA.Conclusion(s): Artificial oocyte activation may improve ICSI outcomes in azoospermic patients when epididymal, but not testicular spermatozoa, are injected. (Fertil Steril (R) 2009;92:131-6. (C)2009 by American Society for Reproductive Medicine.)

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.

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk-based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration. © 2013 Elsevier Inc.

Cryopreservation of bovine spermatozoa from the epididymal tail using conventional and automated methods

Cryopreservation of sperm is important to preserve the gerrnplasm from animals of genetic value, which can die unexpectedly. This study compares conventional and automated methods of cryopreservation of spermatozoa obtained from the epididymis of bulls post-mortem. Twenty-two epididymides were obtained from a commercial slaughterhouse. Spermatozoa were collected from the tail of the epididymis using the retrograde flow technique. Thus, the samples, which were diluted in 10 ml of extender without glycerol (Botubov (R) I, Botupharma, Botucatu, SP, Brazil), were evaluated on motility, sperm vigor, structural and functional (swelling hypoosmotic test) membrane integrity, mitochondrial activity, sperm viability and ADN fragmentation. The samples were divided into two aliquots and diluted in extender with glycerol (Botubov (R) II, Botupharma, Botucatu, SP, Brazil) at a concentration of 50x10(6) motile sperm/0.5 French straws. One sample was frozen by the conventional method (4 hours at 5 degrees C, in a refrigerator and 20 min in nitrogen vapor) and the other by the automated method (Cryogen (R) Dualflex, Neovet, Uberaba, MG, Brazil). The parameters were higher in all the tests of fresh sperm samples, with the exception of the swelling hypoosmotic test, which showed no significant difference when the results were compared with sperm frozen by the conventional method. The average motility of fresh spermatozoa was 74%, and conventional and automated averages were 29 and 25%, respectively. Therefore, although cryopreservation techniques reduce sperm quality parameters, the viability of the sperm is maintained, and these methods can be used to preserve sperm.

Effect of testicular thermoregulation on the quality of buffalo sperm

The process of spermatic division and differentiation (spermatogenesis) occurs with intratesticular temperature lower that the corporal temperature and for that is essential that the testicular thermoregulation mechanism occurs properly. For evaluation of the scrotal surface temperature can be used the infrared thermography or testicular sensors, besides that, can be evaluated the blood flux in the spermatic cord through the Doppler ultrasonography. Thus, the aim of this study is to analyze the testicular thermoregulation in adult buffaloes through scrotal thermography and Doppler ultrasound of testicular artery and verify its effect on sperm quality. For that were used seven healthy buffaloes, with age of 3 and 4 years, of the Murrah breed. The animals were subjected to 3 semen collections using artificial vagina, with one day of interval. In addiction, the retal temperature measurement (RT) with dry bulb thermometer, the measurement of scrotal surface temperature (SST) and body surface temperature (BST) through infrared thermography and the pulsatility (PI) and resistivity (RI) index of testicular artery by Doppler ultrasonography, were performed using 2 distinct moments: animals previously placed to shade (M1) and animals subjected to 4 hours of sun (M2). All parameters were compared by T test and the correlations were performed by Pearson test using the In Stat Graph Pad 3 (R) program. The significant level considered was 5%. There was an increase (p<0,05) of RT, SST, SNT and RI in M2. increasing trend was observed (0,05>p>0,01) PI and RI between M1 and M2. There was a low correlation between SST and semen quality. The results of this study allow us to conclude that adult buffaloes have low ability to perform body and testicular thermoregulation in situations of enviromental heat stress. However, this low capacity of testicular temperature maintenance demonstrated no correlation with the sperm kinetic parameters and sperm morphological defects in buffalo spermatozoa.

Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)