Relationship between Na/K-ATPase in thawed sperm and fertility of Angus bulls.

Since bull fertility prediction remains challenging, the identification of potential fertility markers is important considering the economic benefits to the livestock industry. The main goal of this study was to determine the Na/K-ATPase activity and expression in thawed sperm of high (HF)- and low-fertility (LF) Angus bulls. Samples from three different batches/bulls with HF (n = 4) and LF (n = 4) were used. The Na/K-ATPase activity was determined after thawing, whereas sperm kinematics, membrane integrity, and expression of Na/K-ATPase on sperm surface were evaluated immediately post-thaw and after 120 minutes of incubation. Within the same incubation time, there was no difference on sperm membrane integrity, kinematics, and the expression of Na/K-ATPase on the sperm surface between HF and LF bulls. Kinematic parameters of LIN and VCL were not influenced by incubation time in samples from HF and LF, respectively. A tendency (P = 0.06) of higher Na/K-ATPase enzymatic activity for sperm of HF bulls compared to LF bulls was observed (0.49 ± 0.07 and 0.32 ± 0.06, respectively). In conclusion, Na/K-ATPase activity and expression in thawed sperm from Angus bulls are not related to the fertility index after fixed-time artificial insemination. However, sperm kinematics related to hyperactivation might indicate higher sperm cryotolerance for HF bulls.

Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing.

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

Effect of antifreeze protein type III on frozen/thawed of spermatozoa recover from goat epididymis.

The objective of this study was to evaluate the effect of antifreeze protein type III (AFP III) on the freezing of epididymal spermatozoa of goats. A total of 16 pairs of testicles were collected in a slaughterhouse and transported at approximately 5 °C in a thermal box. Epididymal spermatozoa were recovered by retrograde lavage and evaluated using a phase contrast microscope. Then, they were cryopreserved in extender based on Tris-egg yolk, supplemented with AFP III (0, 1, 10, 100 µg/mL), using an automated system. After thawing (37 °C/30 s), the spermatozoa kinetics were evaluated using the CASA automated system; and plasma and acrosome membrane integrity, mitochondrial membrane potential, and intracellular ROS production, by flow cytometry. There was no difference (P ≥ 0.05) between the experimental groups for the parameters of spermatozoa kinetics, mitochondrial membrane potential, and ROS production. However, the integrity of plasma and acrosome membranes of frozen spermatozoa with 100 µg/mL of AFP III was lower (P < 0.05) than the control group. It was concluded that the addition of AFP III to the Tris-egg yolk extender, used in the freezing of sperm obtained from the epididymis of goats, did not improve the preservation of these cells.

Use of Equine Sperm Cryopreservation Techniques as a Conservation Method of Donkey Germplasm.

The aim of this study was to test equine semen cryopreservation techniques for the conservation of donkey germplasm. Ejaculates of three male donkeys were used (n = 18; six ejaculates per donkey; six repetitions), collected by the artificial vagina method. To remove the seminal plasma, the ejaculates were split and submitted to filtration or centrifugation methods. To assess the freezing method, each fraction was submitted to the automated system or the conventional system, and groups were formed: automated centrifuge, automated filtrate, conventional centrifuge and conventional filtrate. After thawing (37°C/30 seconds), were analyzed the sperm kinetic parameters, integrity and functionality of the plasma membrane and mitochondrial membrane potential. Highest sperm concentration (P < .05) was observed in the filtrate groups; the conventional filtrate group presented lower (P < .05) progressive motility and curvilinear velocity compared to the other groups; no difference was observed (P > .05) among the groups for the membrane integrity and functionality, and mitochondrial membrane potential. Thus, centrifugation is the most indicated technique to remove donkey seminal plasma and the automated and conventional freezing methods can be used in donkey semen conservation.

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation.

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 µg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 µg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 µg did not differ from the control or from 8 µg, but was higher than 12 µg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 µg and 12 µg compared to the control and 6 µg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

Effects of L-Carnitine on Equine Semen Quality During Liquid Storage.

l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through ß-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In the second experiment, ejaculates (n = 16) from four Quarter Horses were used. Each ejaculate was split into four treatment groups with different LC concentrations: 0 (control), 0.5, 1.0, and 2.0 mM. Sperm motility, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species content, and plasma membrane stability were evaluated immediately after samples reached 5°C (0 hour) and after 24, 48, and 72 hours. There was a positive correlation (p < 0.05) between LC levels in seminal plasma with both sperm concentration and plasma and acrosomal membrane integrity. Furthermore, the addition of LC (1 and 2 mM) preserved the motility of equine sperm stored at 5°C. It was concluded that the concentrations of LC with seminal plasma present correlate to semen parameters and the addition of LC to skimmed milk-based extender preserves the motility of equine sperm stored at 5°C for up to 48 hours.

Effect of green tea extract (Camellia sinensis) on the spermatic parameters of Wistar rats submitted or not to testicular heat shock.

The aim of this study was to evaluate the effect of green tea extract (GTE) on the spermatic parameters of Wistar rats, submitted or not to testicular heat shock (HS). For this, 48 animals were treated according to the experimental groups (G1: not exposed to HS and untreated; G2: exposed to HS and untreated; G3: not exposed to HS and treated with GTE; G4: exposed to HS and treated with GTE). Subgroups of rats were euthanized on days 15, 30, and 60 to recover the spermatozoa. The total motility (TM), vigor, spermatic morphology and concentration, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity (ACi) were analyzed. The TM was higher in G1 and G3 than in G2 and G4 on day 30, and higher in G4 on day 60. The overall means of TM and vigor were higher in G1 and G3 than in G2 and G4, as well as TM on day 60. For the morphology, G2 and G4 were lower than G1 and G3 on day 15, and G4 was lower than G1 and G3 on day 30. Moreover, in G1 and G3 morphology was higher on days 15 and 30, and in G4 it was lower on day 30, with the overall means being higher in G1 and G3 than in G2 and G4, as well as on days 15 and 60 compared to day 30. The overall mean of ACi, on day 30, was lower than on days 15 and 60 for all the groups. Therefore, HS is shown to be widely deleterious to the gametes, and the daily administration of 100 mg/kg green tea extract does not improve the spermatic parameters of Wistar rats, submitted or not to testicular HS, although it leads to better recovery of spermatic motility and morphology at 60 days.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Concentration of soybean lecithin affects short-term storage success of goat semen related with seminal plasma removal.

The objective of this study was to investigate the need of seminal plasma removal for short-term cooling of buck semen in soybean lecithin (SL) based extender. Each pool was divided equally, and one half was subjected to centrifugation to remove seminal plasma (SP-), while the other half remained with seminal plasma (SP+). Then, both SP+ and SP- samples were diluted in two SL extenders (extender A = 1% SL; extender B = 2% SL), cooled to 5ºC and stored for 48 hours. The sperm kinetics, evaluated by CASA, and plasma membrane integrity (PMI), acrosomal integrity (ACI) and high mitochondrial membrane potential (HMMP), evaluated by epifluorescence microscopy, were determined within five minutes after reaching 5°C (T0), as well as after 24 (T24) and 48 (T48) hours of storage. Interactions (seminal plasma vs. extender vs. time;) were observed for all variables assessed. Total and progressive motility and other variables of sperm kinetics decreased after 24 hours of cooling in the SP+ group, and after 48 hours of storage, these same variables were lower in SP+/B compared to SP-/B groups. Furthermore, SP+ reduced PMI (extender B, T48), HMMP (A and B extenders, T48) and ACI (extender A, T0) compared to SP- samples. The interactions between seminal plasma and soybean lecithin phospholipids seemed to occur in a time-dependent manner. It was concluded that the removal of seminal plasma improves the quality of goat semen that was cooled in a soybean lecithin-based extender, especially when using 2% soybean lecithin.

The Effect of Canthaxanthin on the Quality of Frozen Ram Spermatozoa.

The addition of antioxidants to semen cryopreservation extenders has been employed for combating oxidative damage. This work aimed to evaluate the addition of carotenoid canthaxanthin to a cryopreservation extender of ram semen. Three breeder rams were used and, after semen collection, with 48-hour intervals between collection, the samples were included in the pool formation (n = 6). The experimental groups comprised 0 (control), 0.1, 1, 10, and 25 µM of canthaxanthin. After thawing (37°C/30 s) and incubation at 37°C for 2 hours, semen aliquots from each group were evaluated for sperm kinetics (CASA), the integrity of the plasma and acrosomal membranes (iPAM), intracellular reactive oxygen species (ROS) production, and lipid peroxidation (LPO) by flow cytometry associated with the image. The control group and canthaxanthin 1 µM after incubation at 37°C for 2 hours showed increases of curvilinear velocity and amplitude of lateral head displacement with decreases of linearity, straightness, and wobble (p < 0.05), which were not observed for the canthaxanthin 10 and 25 µM. The supplementation of a Tris-egg yolk extender with canthaxanthin had no effect on the iPAM, intracellular ROS production in viable spermatozoa, or LPO. In conclusion, supplementation with 10 and 25 µM of canthaxanthin in a Tris-egg yolk extender used for ram semen cryopreservation is able to protect ovine sperm from kinetic changes after incubation at 37°C for 2 hours post-thawing.

Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation.

Several studies reveal that vitamin E acts as a cellular stabilizer of unsaturated lipids against oxidative deterioration, thus maintaining structural and functional integrity at the subcellular level. The objective of this study was to evaluate Vitamin E (Trolox) addition to freezing extender for ram spermatozoa. Semen samples were diluted in Tris-yolk egg medium without antioxidant (control group) and with Trolox in different concentrations (30, 60 and 120µM). After thawing (37°C/30s), samples were subjected to analysis for plasma membrane integrity (PMi), acrosome integrity (Aci), mitochondrial membrane potential (MMP), sperm kinematics, and ultrastructural integrity. The Trolox 60 and 120µM groups showed higher percentages of iPMs (P<0.05) when compared to the control group. Differences were observed among groups in sperm kinematic indicators (progressive motility, linearity, straightness, oscillation index, straight-line velocity and average path velocity), with higher values (P<0.05) for the Trolox 60 and 120µM groups. On ultrastructural assessment, Trolox addition at the three concentrations preserved spermatozoon head plasma membranes, while for the spermatozoon tail, plasma membrane preservation at 60µM was higher (P<0.05) than the other groups. The Trolox 60 and 120µM groups presented more mitochondrial ultrastructural preservation than the other groups (P<0.05). These results indicate that Trolox addition to Tris-egg yolk at 60 and 120µM provides greater structural integrity (plasma membrane and mitochondria) and kinematics for ram spermatozoa after cryopreservation.

Espermatozoides caprinos criopreservados em meio à base de leite desnatado acrescido de glutationa reduzida / Goat spermatozoa after freezing in skimmed-milk extender supplemented with reduced glutathione

Visando avaliar o efeito da adição de glutationa reduzida (GSH) ao diluente de congelação de sêmen caprino à base de leite desnatado, utilizou-se sêmen de cinco reprodutores Boer. Após colheita e avaliação, procedeu-se à formação do pool dos ejaculados e diluição em leite desnatado e glicerol 7 por cento, acrescido de antioxidantes: G1) Controle; G2) GSH 2mM mL-1; G3) GSH 5mM mL-1 e G4) GSH 7mM mL-1. As amostras foram congeladas em palhetas (0,25mL) e armazenadas a -196°C. Após descongelação, avaliou-se a integridade de membrana plasmática (iMP) e acrossomal (iAc), potencial de membrana mitocondrial (PMM), cinética e ultraestrutura. Os grupos Controle e GSH (2, 5 e 7mM mL-1) não diferiram (P>0,05) em iMP, iAc, PMM e cinética. Na análise ultraestrutural, os porcentuais de membrana plasmática (cabeça e cauda) e acrossoma íntegros não diferiram (P>0,05) entre grupos. Todavia, o grupo Controle apresentou maior porcentual (P<0,05) de gametas com axonema íntegros do que os de GSH (2, 5 e 7mM mL-1). Maior porcentagem (P<0,05) de espermatozoides com mitocôndrias íntegras foi observada no grupo Controle do que nos de GSH (5 e 7 mM mL-1). Conclui-se que a adição de GSH (2, 5 e 7mM mL-1) em diluente de congelação de sêmen caprino, à base de leite desnatado, não preserva a integridade dos espermatozoides.

Aiming to evaluate in vitro effect of different concentrations of glutathione reduced (GSH) in skimmed-milk and glycerol 7 percent it was used semen from five Boer bucks. After collect and evaluation, a pool of samples was diluted in skimmed-milk and glycerol 7 percent plus antioxidant: G1) Control; G2) GSH 2mM mL-1; G3) GSH 5mM mL-1 and G4) GSH 7mM mL-1. Samples were frozen in straws (0.25mL) and stored at -196°C. After thawing, samples were subjected to integrity of the plasma membrane (iMP) and acrosomal (iAc), mitochondrial membrane potential (MMP), kinematic and ultrastructure analysis. Control and GSH (2, 5 and 7mM mL-1) groups did no differ (P>0.05) in iMP, iAc, PMM and kinematic parameters. In the ultrastructural analysis, percentages of acrosome and plasma membrane (tail and head region) intact did not differ (P>0.05) between groups. However, Control group had higher percentage (P<0.05) of gametes with intact axonemes than those of GSH (2, 5 and 7mM mL-1) groups. Higher percentage (P<0.05) of sperms with intact mitochondrias were observed on Control group than those of GSH (5 and 7mM mL-1). It can be concluded that the GSH (2, 5 and 7mM mL-1) addition in skimmed-milk diluent to freeze goat semen did not preserve sperm integrity.

Integridade de DNA e morfologia espermática de ratos submetidos a campos eletromagnéticos de baixa frequência durante diferentes períodos do desenvolvimento / DNA integrity and sperm morphology of Wistar rats exposed to low frequency electromagnetic fields during different periods of development

A exposição da sociedade a Campos Eletromagnéticos (CEM) vem aumentando vertiginosamente em virtude da ampla expansão tecnológica observada nos últimos anos. Tanto a geração, como a distribuição e a utilização de energia elétrica podem gerar Campos Eletromagnéticos de baixa freqüência (50 e 60 Hz). Pesquisas vêm demonstrando que a exposição a estes CEM podem proporcionar alterações fisiológicas significativas, apesar disto, ainda não estão totalmente esclarecidos a extensão destes efeitos, nem os mecanismos de ação que envolve a interação dos CEM com os organismos biológicos. O presente trabalho teve como principal objetivo verificar os efeitos dos CEM (60 Hz e 1 mT) sobre a integridade de DNA e morfologia espermática de ratos sexualmente maduros, que foram expostos ao CEM durante diferentes períodos do seu desenvolvimento. Os resultados obtidos neste trabalho não encontraram indícios de alterações no DNA dos espermatozóides, porém, foram observadas alterações significativas na morfologia dos espermatozoides após a exposição ao CEM. Estas alterações na morfologia espermática podem reduzir o potencial reprodutivo. Portanto, devemos considerar o CEM como um potencial risco a saúde pública, recomendando- se a realização de mais pesquisas buscando estabelecer níveis seguros de exposição aos CEM.

The society's exposure to electromagnetic fields (EMF) has been growing considerable due to the great technological expansion observed in the last few years. Generation as well as distribution and use of electric energy can generate low frequency electromagnetic fields (50 and 60 Hz). Issues have been demonstrating that EMF exposure could provoke significantly physiological changes, however, the extension of EMF effects weren't totally clarified. The major objective of this issue was to evaluate the EMF (60 Hz and 1 mT) effects on DNA integrity and sperm morphology in Wistar rats with mature sexuality that were exposed during different stages of testicular development. According to our results, EMF did not change DNA integrity, but we could observe morphological changes in sperm after exposure to EMF. These changes in sperm morphology may reduce the reproductive potential. Therefore, we should consider the EMF as a potential risk to public health, recommending the implementation of further research seeking to establish safe levels of exposure to EMF.

Proteínas do plasma seminal de caprinos relacionadas com o índice pluviométrico e a qualidade do sêmen / Proteins of goat seminal plasma related with precipitation index and semen quality

O objetivo deste estudo foi identificar as proteínas do plasma seminal de caprinos da raça Alpina Americana criados na região Nordeste do Brasil que estão relacionadas ao índice pluviométrico e à qualidade do sêmen. O sêmen foi obtido pelo método de vagina artificial a partir de três reprodutores e foi avaliado quanto aos parâmetros macroscópicos e microscópicos. O perfil de proteínas do plasma seminal foi realizado por eletroforese bidimensional. Os parâmetros volume do sêmen, integridade do acrossoma e proteínas totais evidenciaram diferença significativa (P<0,05) entre os períodos de alto (1,7mL, 90,3% e 372μg mL-1,respectivamente) e baixo (1,2mL, 80,3% e 494μg mL-1, respectivamente) índice pluviométrico. Foram detectados, nos períodos de alto e baixo índice pluviométrico, 47 e 49 spots de proteínas com massa molecular relativa de 4 a 106kDa e de 15 a 97kDa e ponto isoelétrico de 3,00 a 8,96 e de 4,48 a 9,83, respectivamente. Apenas no período de alto índice pluviométrico foram observados os grupo de proteínas de 13kDa e 45kDa. Conclui-se que o sêmen de caprinos da raça Alpina Americana criados na região Nordeste do Brasil apresenta-se com melhor qualidade quando colhido no período de alto índice pluviométrico, o que pode ser atribuído à presença das proteínas de 13kDa e 45kDa.

The aim of this study was to identify proteins in seminal plasma of goats raised in the Northeast of Brazil relatedwith precipitation index and semen quality. Semen was obtained from three bucks and evaluated to the microscopic and macroscopic parameters. The profile of seminal plasma proteins was performed by analysis of two-dimensional electrophoresis. Volume, acrosome integrity and total proteins had significant difference (P<0.05) between the periods of high (1.7mL, 90.3% and 372g mL-1, respectively) and low (1.2mL, 80.3% and 494μgmL-1, respectively) precipitation index. It was detected during high and low precipitation index, 47 and 49 spots of proteins with molecular weight of 4 to 106kDa and 15 to 97kDa, and isoelectric point of 3.00 to 8.96, and 4.48 to 9.83, respectively. Only in the period of high precipitation index were observed groups of proteins with 13kDa and 45kDa. It can be concluded that semen of Alpine American goats raised in the Northeast of Brazil has best quality when obtained in the period of high precipitation index, which can be attributed to the presence of protein with 13kDa and 45kDa.

Adição de piruvato de sódio e trolox ao diluidor utilizado para congelação de sêmen de garanhões férteis e subférteis / Effect of pyruvate and trolox added to the extender used for freezing fertile and subfertile stallion semen

O objetivo deste trabalho foi avaliar o efeito do piruvato e trolox (forma solúvel da vitamina E) sobre a qualidade espermática pós-descongelamento. Assim, com o intuito de proteger as células espermáticas dos efeitos deletérios da criopreservação,foram considerados os seguintes tratamentos: T1 (Controle)= INRA82-HEPES sem antioxidantes; T2= INRA82-HEPES + 2mM de piruvato e T3= INRA82-HEPES + 120mM de trolox. As amostras de sêmen descongeladas foram avaliadas quanto à motilidade total (MT) e progressiva (MP), a integridades de membrana plasmática e acrossômica, integridade do DNA, à estabilidade de membrana e ao potencial de membrana mitocondrial (Δψm). A adiηγo de piruvato proporcionou resultados superiores (P<0,05) àqueles obtidos com trolox na motilidade espermática total (9,17 e 14,5 por cento, respectivamente). A adição de piruvato incrementa a motilidade espermática (18,92 e 19,0 por cento, respectivamente) em garanhões férteis e subférteis submetidos à congelação.

The objective of this research was to evaluate the effect of pyruvate and trolox on the thawed sperm quality. For freezing, antioxidants were added to INRA 82-HEPES extender to protect sperm from the deleterious effects of oxidative stress, according to the treatments: T1= INRA82-HEPES without antioxidants; T2= INRA82-HEPES + 2mM of pyruvate and T3= INRA82-HEPES + 120 mM de trolox. The thawed semen samples were evaluated according to the total (MT) and progressive (MP) motility, integrity of plasma and acrossomal membrane, DNA integrity, membrane stability and mitochondrial membrane potential (Δψm). It was observed that the addition of pyruvate resulted in a significantly higher total sperm motility (P<0.05) to those obtained with trolox (9.17 and 14.5 percent, respectively). It can be concluded that the addition of pyruvate improves sperm motility (18.92 and 19.0 percent, respectively) in samples from fertile and sub-fertile stallions submitted to freezing.

Avaliação microbiológica do sêmen fresco e congelado de reprodutores caprinos / Microbiological evaluation of buck fresh and frozen semen

Objetivou-se avaliar a flora microbiana no sêmen fresco e congelado de reprodutores caprinos, assim como a eficácia dos antibióticos estreptomicina, penicilina e gentamicina, na viabilidade de doses de sêmen congeladas. Foram utilizados 25 reprodutores de diferentes raças, submetidos a duas colheitas de sêmen através do método da vagina artificial, após higiene da região prepucial. A primeira colheita do sêmen foi realizada visando o exame microbiológico e a segunda teve como objetivo proceder a congelação, após diluição em leite desnatado, utilizando penicilina + estreptomicina (A1), gentamicina(A2) ou sem antibiótico (A3). Ao proceder a avaliação microscópica no sêmen fresco, evidenciou-se média de 87,92 ± 7,76 % de motilidade individual progressiva (MIP) e 4,96 ± 0,20 de vigor espermático. Em relação à avaliação bacteriana, constatou-se principalmente bactérias do gênero Staphylococcus spp e Bacillus sp. Após a congelação do sêmen, não foram evidenciadas diferenças (P>0,05) entre os grupos quantoa MIP e vigor espermático. Entretanto, na avaliação microbiológicapós-descongelação, a bactéria do gênero Staphylococcus spp esteve presente na maioria das amostras. Observou-se também que a gentamicina (13,3mg/mL) apresentou melhor atividade antimicrobianano processo de congelação do sêmen, concluindo-se que pode ser o antibiótico usado na congelação do sêmen de reprodutores caprinos.

The aim of this research was to evaluate the microbial flora in the fresh and frozen semen of goat reproducers, as well as the effectiveness of the antibiotics estreptomicin, penicillin and gentamicin in cryopreservation of semen. It were used 25 males of different breeds, submitted to two semen collect through the artificial vagina method after cleanliness of prepucial region. The first collection of semen aimed the microbiological exam. The second collection had as goal accomplish freezing, after dilution in skimmed milk, with penicillin+ estreptomicin (A1), gentamicin (A2) or control (A3). After microscopic evaluation, it was evidenced average of 87.92 ± 7.76% of MIP and 4.96 ± 0.20 of spermatic vigor in fresh semen. Regarding bacterin evaluation, it was verified, mostly, bacteria of the gender Sthaphylococcus spp and Bacillus sp. After semen cryopreservation, it was observed that there wasn’t difference (P>0.05) among groups in MIP and spermatic vigor. However, in the microbiological evaluationof frozen-thawed semen, bacteria of Sthaphylococcus spp gender was present in great part of samples. Gentamicin (13.3mg/mL) promoted larger inhibition of the bacterial growth in the semen post-freezing, concluding that gentamicin can be the antibiotic used for freezing of goat semen.

Cocultura de embriões Mus musculus com monocamadas de células de linhagens primária e contínua em um sistema sem fluxo externo de CO2 / Coculture of Mus musculus embryos with monolayers of primary and continuous lineages in a system without external CO2 flow

A eficiência de três monocamadas celulares (linhagem contínua - células Madin and Darby Bovine Kidney/ MDBK;linhagem primária - células de útero e de oviduto bovino) foi testada para verificar a existência de especificidade celular através do desenvolvimento de embriões, desde o estádio de duas células até o de mórula compacta, em um sistema de cocultura sem fluxo externo de CO2. Depois da seleçâo, os embriões (n = 343) foram aleatoriamente distribuídos em diferentes tubos de ensaio, os quais foram colocados a 37°C durante 72 horas. Após o período de cocultura, as porcentagens de mórulas compactas obtidas foram de 87,7 por cento em células de oviduto, 86,2 por cento na monocamada de células uterinas e 88,3 por cento na de células MDBK. Não foi observada diferen- ça significativa entre esses valores e, por isso mesmo, conclui-se que a especificidade celular não é importante para o desenvolvimento in vitro de embriões Mus musculus.

The efficiency of three monolayers (continuous lineage - Madin and Darby Bovine Kidney/ MDBK; primary lineage - bovine celisfrom uterus and oviduct) hás been tested to verify the celular specificity through development of two cells embryos until compact morulaes stage in a coculture system without continuous CO2 flow. The selected mouse embryos (n=343) were randomiy divided into three experimental groups andplaced in closed culture tubes mantained aí 37°C during 72 hours. After the co-culture period, the porcentages of compact morulaes were 87.7 percent in oviduct cells, 86.2 percent in uterus monolayer and 88.3 percent in MDBK cells. It was not observed significative difference between these results and it is possible to condude that celular specificity is not importam to enhance the In vitro development of Mus musculus embryos.

Renovação parcial do meio de cultura como perspectiva de aumentar a eficiência de um sistema de cocultoura de embriões sem fluxo contínuo de CO2 / Partial replacement of the culture medium in order to enhance a embryos coculture system without continuous CO2 flow

Substituindo-se parcialmente (30%) os meios de cultura TC M 199/NaHCO3 (199/NaHCO3) e TC M 199/25mM HEPES (199/HEPES) a intervalos de 24, 48 ou 72 horas, testou-se a possibilidade de incrementar a obtenção de blastocistos expandidos (BLE) a partir de embriões Mus musculus no estádio de duas células incubados sem fluxo contínuo de CO2. Após distribuição aleatória dos embriões em tubos de ensaio contendo 2ml de meio de cultura e monocamada celular de oviduto bovino, os referidos tubos foram hermeticamente fechados e colocados em estufa bacteriológica a 37°C durante 96 horas. A renovação do meio de cultura em qualquer período não incrementou a porcentagem de embriões que alcançou o estádio de BLE, todavia, o número total de BLE obtido com o 199/HEPES (79,0%) foi superior (P £ 0,01) ao verificado com o 199/NaHCO3 (65,0%).

It has been evaluated, in a coculture system without continuous CO2 flow, the possibility to enhance the percentage of blastocists obtained from two cells mouse embryos by partial (30%) replacement (each 24, 48 and 72 hours of intervals) of two culture media (TCM 199/NaHCO3 and TCM 199/25 mM HEPES). The embryos, randomiy allocated in closed cultures tubes with 2 ml of medium and bovine oviduct monolayer, were maintained at 37°C during 96 hours. No diferences between spreaded blastocists were verifica when the media were replaced at ali intervals, however, the medium TCM 199/25mM HEPES was significantiy more effective (P £ 0.01) then TCM 199/NaHCO3.

Preparação de rufiões caprinos pela fixação da curvatura caudal da flexura sigmóide do pênis / Preparation of teaser goats by fixation of the caudal curvature of penis sigmoid flexure

Foram utilizados 15 caprinos sem raça definida com idade variando entre 06 e 36 meses objetivando testar uma técnica cirúrgica para preparo de rufiões por fixação da curvatura caudal da flexwa sigmóide do pênis. Para avaliação dos resultados, os animais eram colocados na presença de fêmeas em estro, a partir do décimo dia da intervenção cirúrgica, durante um período mínimo de seis meses. Os rufiões, quando testados, não apresentaram alteração da libido e mostraram-se incapazes de exteriorizar o pênis, permitindo concluir que a técnica descrita, além de ser rápida e de fácil execução, pode ser utilizada com eficiência na prática de preparo de rufiões caprinos.

In the present study fifteen. undefined breed type bucks, with age varing between six and thirty-six months, were used to test a surgery technique by fixation of the caudal curvature of penis sigmoid flexure for preparation of teaser. To evaluate the results, the teasers buck were placed with goats in oestrus for a minimum period of six months after 10 (ten) days of surgery. During evaluations, the teasers did not present any alteration of the sexual behavior and they were unable to project the penis for outside of the prepuce. There fore, it was concluded that the surgery technique used in these investigation can be easily applied and utilized with effïciency in the preparation of teasers buck.