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Moringa oleifera seeds are used globally as a treatment for water and contain the lectins cMoL and WSMoL, which display coagulant activity. In this study, we sought to determine the cytotoxicity and genotoxicity of the M. oleifera seed extract (SE), prepared with the same procedure that people use for treating water, as well as cMoL and WSMoL, in human peripheral blood mononuclear cells (PBMCs). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, while genotoxicity was evaluated using the comet assay, with cell nucleoids categorized in classes ranging from 0 (without damage) to 4 (maximum damage). The PBMCs treated with SE, cMoL, and WSMoL displayed viability higher than 60% in treatments with concentrations up to 100 µg/mL. In addition, SE and cMoL displayed low genotoxicity owing to the detection of nucleoids in class 1. However, the number of nucleoids in all classes increased when 50 and 100 µg/mL of WSMoL was administered, reaching a damage frequency of 50.0%. Although M. oleifera SE, cMoL, and WSMoL were not cytotoxic to PBMCs after 24 h of exposure, dose-dependent genotoxic effects were observed, especially with WSMoL. These findings indicate that caution must be exercised when selecting a lectin/extract concentration for water treatment.

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 (P 0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P 0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; 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

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p 0.05) between GLY and DMF, but ACI was higher (p 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 106 sperm/ml) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio®). Subsequently. they were filled in straws (0.5 ml) and subjected to freezing and storage (-196 °C). Immediately after thawing (37 °C, 30 s), semen samples were evaluated for kinetics (CASA), plasma and acrosomal membrane integrity, and mitochondrial membrane potential (flow cytometry). The addition of 50 and 100mM sucrose to the freezing extender increased (P < 0.05) the parameters of TM, PM, VCL, VSL, and VAP, compared to the control group. The WOB parameter of the group supplemented with 100 mM sucrose was higher (P < 0.05) than the control group. Higher values ​​(P < 0.05) of ALH and BCF were observed in groups treated with sucrose (25, 50, and 100 mM), compared to BotuCrio®. The semen frozen in the presence of 100 mM sucrose presented higher percentages (P < 0.05) of sperm with intact plasma and acrosomal membranes, and high mitochondrial membrane potential in relation to the other groups. It is concluded that the addition of sucrose to equine semen freezing extender increase motility (50 and 100 mM), plasma and acrosomal membrane integrity preserve, and high sperm mitochondrial membrane potential (100 mM) after thawing.

This study was conducted to evaluate the effects of lycopene on quality parameters of cryopreserved ram semen. Semen samples were collected from three Santa Inês rams to form a seminal pool (n = 8). Aliquots from each pool were diluted in Tris-egg yolk extender to create experimental groups containing 0 µM (control), 0.1 µM, 1 µM and 5 µM lycopene. The samples were evaluated for sperm kinetics, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species (ROS) production and lipid peroxidation immediately after thawing and after incubation for two hours at 37 °C. The addition of lycopene had no effect on the parameters that were evaluated at the time of thawing when compared with the control. However, after incubation, the groups with added lycopene showed a decrease in progressive motility. All experimental groups showed a significant reduction in linearity and straightness following incubation. Furthermore, the 5 µM lycopene group showed a decrease in wobble and an increase in amplitude of lateral head displacement. In conclusion, the addition of lycopene to the freezing extender of ram semen affected the kinetics parameters of cryopreserved spermatozoa after a two-hour incubation period.Keywords: carotenoid, flow cytometry, kinetic, sheep

The goals of this study were to investigate (a) the localisation of Na+, K+-ATPase in ram sperm; (b) cryopreservation like-changes on median fluorescence intensity (MFI) and the expression of Na+, K+-ATPase on the sperm surface (labeled sperm); and (c) the influence of Na+, K+-ATPase inhibition on sperm kinematics. Exp. 1: pooled semen samples (n = 7) from four rams were diluted and frozen. The following sperm features were evaluated in fresh-diluted and post-thawed samples: MFI and expression of Na+, K+-ATPase (Bodipy® FL-Ouabain), plasma and acrosomal membrane integrity (IP; FITC-PNA), sperm viability and plasma membrane stability (Yo-Pro and Merocyanine), as well as sperm kinematics (CASA). Exp. 2: pooled semen samples (n = 5) from four rams were diluted and incubated in medium treated with or without Na+, K+-ATPase inhibitor (ouabain, 10−4 M). Sperm kinematics (CASA) were determined prior to (0 h) and after incubation (3 h). In both fresh and thawed sperm, Na+, K+-ATPase was localised in the middle piece and cryopreservation did not influence the MFI in sperm with intact plasma membranes. However, cryopreservation reduced (P

Semen cryopreservation is responsible for decrease the gamete fertility, due to structural and functional damages. Among the various causes, oxidative stress, resulting from the higher generation of reactive oxygen species (ROS), has been attributed to affect semen quality. Thus, it was objectified evaluate the effect of Trolox on ram and goat sperm, subjected to freezing. Semen pools of goat (n=5) and ram (n=6) were diluted in skimmed milk (7% glycerol) or Tris-egg yolk (5% glycerol) extender, respectively, added or not of Trolox (0, 20 or 40 μM/ml) and frozen. After thawing (37 °C/30 s), aliquots of semen were evaluated for lipid peroxidation by high performance liquid chromatography, coupled with a photodiode array detector (HPLC-DAD), and flow cytometry (C11-BODIPY581/591), besides of plasma membrane and acrosome integrity by fluorescence microscopy, and sperm kinetics by computerized sperm analysis (CASA). The antioxidant treatment with Trolox did not determine significant effects (p>0.05) on lipid peroxidation, plasma membrane integrity, acrosomal integrity and on the kinetic parameters evaluated. Thus, it is concluded that Trolox (20 or 40 μM) did not have a protective or deleterious effect on goats and ram sperm, submitted to freezing. El proceso de criopreservación del semen provoca una disminución de la fertilidad de los gametos debido a daños estructurales y funcionales. Entre las diversas causas, el estrés oxidativo, resultante de la mayor generación de especies reactivas de oxígeno (ROS), ha sido atribuido por afectar a la calidad seminal. Así, se pretendía evaluar el efecto de Trolox en los espermatozoides de cabra y oveja sometidos a congelación. Se diluyeron pooles de semen de cabras (n=5) y ovejas (n=6) en diluyentes a base de leche desnatada (Glicerol 7%) o de Tris-gema de huevo (Glicerol 5%), respectivamente, añadidos o no de Trolox (0, 20 o 40 µM/mL), y sometido a congelación. Tras la descongelación (37 °C/30 s), se evaluó la peroxidación lipídica de las alícuotas, mediante cromatografía líquida de alto rendimiento acoplada a un detector de matriz de diodos (HPLC-DAD) y también mediante citometría de flujo (C11-BODIPY581/591), la integridad de las membranas plasmáticas y acrosómicas, por médio de microscopía de fluorescencia, y la cinética de los espermatozoides, mediante el sistema computarizado de análisis de esperma (CASA). El tratamiento antioxidante con Trolox no determinó efectos significativos (p>0,05) sobre la peroxidación lipídica, la integridad de las membranas plasmáticas y acrosomales y los parámetros cinéticos evaluados. Así pues, se concluye que Trolox (20 o 40 μM) no presenta un efecto protector o perjudicial sobre los espermatozoides de cabra y oveja sometidos a congelación. O processo de criopreservação do sêmen ocasiona a diminuição da fertilidade dos gametas, devido a danos estruturais e funcionais. Entre as várias causas, o estresse oxidativo, resultante da maior geração de espécies reativas de oxigênio (ROS), tem sido atribuído por afetar a qualidade seminal. Assim, objetivou-se avaliar o efeito do Trolox sobre os espermatozoides caprinos e ovinos submetidos à congelação. Pools de sêmen caprino (n=5) e ovino (n=6) foram diluídos em diluidores a base de leite desnatado (Glicerol 7%) ou Tris-gema de ovo (Glicerol 5%), respectivamente, adicionados ou não de Trolox (0, 20 ou 40 µM/mL), e congelados. Após descongelação (37 °C/30 s), alíquotas foram avaliadas quanto à peroxidação lipídica, por cromatografia líquida de alta performance acoplada a detector de arranjo de diodos (HPLC-DAD) e por citometria de fluxo (C11-BODIPY581/591), à integridade de membrana plasmática e acrossomal, por microscopia fluorescente, e à cinética espermática, pelo sistema computadorizado de análise espermáticas (CASA). O tratamento antioxidante com Trolox não determinou efeitos significativos (p>0,05) sobre a peroxidação lipídica, integridade de membranas plasmática e acrossomal e parâmetros cinéticos avaliados. Assim, conclui-se que o Trolox (20 ou 40 μM) não apresenta efeito protetor ou nocivo sobre os espermatozoides caprinos e ovinos submetidos à congelação.

l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through β-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 (

The aim of this study was to replace fetal bovine serum (FBS) with platelet-rich plasma (PRP) for in vitro production of bovine embryos. The maturation media (TCM-199 medium) for the cumulus-oocyte complexes (COCs) was supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC). After fertilization, the presumed zygotes were randomly distributed in culture medium supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC) for 7 days. Cumulus cell (CC) expansion was greater (P

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.

We investigated whether the recombinant leptin (1, 10, 100 ng/mL) influences the meiotic maturation of goat oocytes, whether the MAPK and JAK2/STAT3 pathways mediate the effects of leptin during in-vitro maturation, and whether leptin differently affects the abundance of mRNAs relevant to leptin signal transduction and apoptosis in oocytes and cumulus cells. The addition of leptin to the maturation medium positively affected the number of oocytes that completed nuclear maturation. Nuclear oocyte maturation stimulated by leptin was significantly impaired when we added the specific inhibitors of MAPK (U0126) and JAK2/STAT3 (AG490) to the maturation medium. The addition of leptin (10 ng/mL) during maturation did not affect the expression of AMPKα1, PPARα, Caspase 3, and BCL2 genes in oocytes or cumulus cells. The PPARγ and BAX mRNA abundances were significantly reduced in cumulus cells in the leptin group compared to the control group. Our results demonstrate that supplementation of the in-vitro maturation medium with leptin significantly improves nuclear maturation and reveal the important role of the MAPK and JAK2/STAT3 signaling pathways in establishing the leptin-mediated nuclear maturation of goat oocytes. Moreover, leptin treatment affects PPARγ and BAX gene expression in cumulus cells.

Adiponectin is an adipokine secreted primarily by adipocytes and is involved in the control of male and female reproductive functions. Circulating levels of adiponectin are inversely correlated with body fat mass, and its biological effects are predominantly mediated through two receptors, AdipoR1 and AdipoR2. The aim of the present study was to verify the expression of the adiponectin system (adiponectin and its receptors, AdipoR1 and AdipoR2) in goat ovary using qPCR and immunohistochemistry analyses and further investigate the in vitro effects of recombinant adiponectin (5 μg/mL and 10 μg/mL) on goat oocyte nuclear maturation. We demonstrated that the mRNA and proteins of the adiponectin system are present in goat ovary. Gene and protein expression of AdipoR1 and AdipoR2 was detected in follicular cells (oocyte, cumulus, granulosa and theca) of small and large antral follicles, while adiponectin mRNA was not detected in oocytes from small and large follicles or in large follicle cumulus cells. Finally, addition of various concentrations of adiponectin in maturation medium affected the number of oocytes that reached metaphase II. In conclusion, in the present study, we detected expression of adiponectin and its receptors AdipoR1 and AdipoR2 in goat ovarian follicles. Furthermore, we demonstrated that recombinant adiponectin increases nuclear maturation of goat oocytes in vitro .

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.

This study evaluated the effect of leptin on the in vitro culture of isolated sheep early antral follicles. Early antral follicles (300–450 μm) were isolated and cultured for 12 days in tissue culture medium 199 (TCM 199) supplemented with glutamine, hypoxanthine, transferrin, insulin, selenium, ascorbic acid, bovine serum albumin (BSA) and recombinant follicle stimulating hormone (rFSH) (TCM 199+: control medium) or TCM 199+ supplemented with 2 or 10 ng/mL leptin. After culture, oocytes were subjected to in vitro maturation (IVM). The parameters analyzed were morphology, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥110 μm) rates. After IVM, reactive oxygen species (ROS) levels, mitochondrial activity, meiotic stages and meiotic resumption rates were also analyzed. After 12 days of culture, the concentration of 2 ng/mL of leptin showed a higher percentage of morphologically normal follicles, fully-grown oocytes (≥110 μm), active mitochondria and meiotic resumption compared to the control medium (TCM 199+; P 0.05). After culturing, no significant differences existed among treatments in terms of the follicle diameter and ROS levels. In conclusion, the addition of 2 ng/mL leptin to the base culture medium is capable of improving follicular survival, oocyte growth, mitochondrial activity and meiotic resumption after the in vitro culture of isolated sheep early antral follicles.

The aims of the study were to characterize leptin and it is receptor (LEPR) proteins immunoexpression in ovine ovaries and to evaluate the effects of leptin on development of secondary follicles cultured in vitro. The ovaries were collected and fixed for immunohistochemical analysis. Additional pairs of ovaries were collected and secondary follicles were isolated and cultured, for 18 days, in α-MEM+ alone or supplemented with 10 or 25 ng/mL of leptin. The antrum formation and fully grown oocytes rates were higher in 25 ng/mL leptin than all treatments. GSH levels and mitochondrial activity were higher in 10 or 25 ng/mL leptin than α-MEM+. 25 ng/mL leptin showed a higher percentage of MII than the α-MEM+. In conclusion, leptin and its receptor are expressed in ovine ovaries and 25 ng/mL leptin promoted higher in vitro maturation rates by improving follicular development, GSH levels and mitochondrial activity of ovine oocytes compared to control medium. Made available in DSpace on 2019-11-14T18:28:56Z (GMT). No. of bitstreams: 1 Immunolocalizationofleptinand2019.pdf: 1686099 bytes, checksum: abf22569096650debedd8a6fa114616a (MD5) Previous issue date: 2019

Este estudo avaliou a influência do estresse térmico na resposta superovulatória e no perfil bioquímico de ovelhas Dorper superovuladas em ambiente tropical semiárido. Foram utilizadas 13 ovelhas da raça Dorper, as quais foram submetidas à sincronização do estro e ao tratamento superovulatório, em dois períodos diferentes [termoneutro (TN) e estresse térmico (ET)]. As ovelhas foram inseminadas por laparoscopia utilizando sêmen congelado 42-43 horas após a remoção do dispositivo intravaginal. Cinco dias após a inseminação artificial, foi realizada laparotomia para avaliação da resposta ovariana e lavagem uterina. Coletou-se sangue para avaliação das variações nas concentrações séricas de glicose, frutosamina, triglicerídeos, β-hidroxibutirato (BHB), creatinina, ureia, proteína total, albumina, AST/TGO, GGT, Fosfatase Alcalina, Cálcio, Fosfato e Magnésio. Os períodos termoneutro (TN) e de estresse térmico (ET) tiveram temperaturas médias de 22,01 °C e 27,61 °C, respectivamente, com umidade relativa de 31,9%. Não houver diferença na resposta ovulatória e recuperação de estruturas entre os períodos TN e ET. As concentrações dos metabólitos séricos sofreram variação entre períodos térmicos e entre momentos de avaliação. Em conclusão, o período térmico não afeta a eficiência do protocolo de superovulação em ovelhas Dorper criadas em ambiente tropical semiárido, mas provoca alterações nos metabólitos séricos.

The aim of this study was to evaluate the fertilization rate of cows that were superovulated and artificially inseminated with sex-sorted semen. Cows were treated with an intravaginal progesterone device plus estradiol benzoate (day 0). Superstimulation treatments began four days after with eight applications of FSH at 12 h intervals. D-Cloprostenol was administered on day 6. Progesterone device was removed on day 7, and LH was administered on day 8. The treatments were divided as follows: NonSx, two AI with non-sorted semen were conducted 12 and 24 h after LH; Sx12&24, two AI with sex-sorted semen were conducted 12 and 24 h after LH; and Sx24&36, two AI with sex-sorted semen were conducted 24 and 36 h after LH. Embryos were recovered on day 16 and were evaluated and classified. Percentage of fertilized embryos tended to be greater for the non-sorted semen than the sex-sorted semen. The number of unfertilized oocytes was smaller when the non-sorted semen was used relative to the sex-sorted semen. There was no difference between the treatments that used sexed semen. In conclusion, the use of sex-sorted semen in superovulated dairy cows results in greater numbers of unfertilized oocytes than non-sorted. However, when only sorted semen is used AI should be performed 24 and 36 h after LH.

Recent studies have shown that adiponectin, an adipokine predominantly produced by adipose tissue, regulates several reproductive processes. However, the mechanisms of action of adiponectin on the maturation of goat oocytes remain to be determined. The aim of this study was to investigate whether (a) adiponectin influences the meiotic maturation of goat oocytes; (b) MAPK MEK 1/2 mediates the effects of adiponectin; and 3) adiponectin differentially affects mRNA relative abundance of genes relevant for adiponectin signal transduction in goat oocytes. The addition of adiponectin (5 μg/ml) during the maturation of goat oocytes resulted in a higher percentage of successful nuclear maturation compared to those of the group without adiponectin (p < 0.05). Adiponectin-stimulated nuclear oocyte maturation was significantly impaired by a mitogen-activated protein kinase MEK 1/2 inhibitor, U0126 (p < 0.05). There was no evidence of any adiponectin-induced difference in the relative transcript abundances of AdipoR1, AdipoR2, AMPKα1, AMPKα2, PPARα and PPARγ genes. In conclusion, these results indicate that adiponectin has a positive effect on the meiotic maturation of goat oocytes through the MAPK MEK 1/2 pathway. Furthermore, the adiponectin does not affect the relative abundance of genes relevant for adiponectin signal transduction in goat oocytes.

The present study is the first attempt to evaluate the use of fluorescent microscopy as a tool for determining the sperm qualitity in penaeid shrimp species. The probes propidium iodide and 6-carboxyfluorescein diacetate (CFDA) were used in combination to assess sperm quality of Litopenaeus vannamei captive broodstock and wild stocks of Farfantepenaeus subtilis and Litopenaeus schmitti. L. vannamei showed a significant higher amount of live cells (80.87%) after 93 days in captivity, when compared to animals entering (day 0) the maturation system (61.03%). The percentages of live sperm cells for wild-caught F. subtilis and L. schmitti were above 50% over the 12-month sample period in northeastern Brazil. These results demonstrate that the fluorescent microscopy can be used as a tool to determine the sperm quality in penaeid, allowing the evaluation of male performance in aquaculture systems, as well as to determine their reproductive cycle in fisheries research.

Summary The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml−1] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post-thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml−1 Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P

Leptin, a hormone that was originally identified in adipocytes, has been implicated in the regulation of ovarian folliculogenesis through endocrine, autocrine and/or paracrine mechanisms. The aim of this study was to investigate the expression patterns of leptin (LEP) and its receptor (LEPRb) in different types of ovarian follicular cells from goats. In small follicles, the expression levels of LEP were higher (P0.001) in granulosa cells than in theca cells, cumulus cells and oocytes. The expression of LEP in granulosa cells was higher (P0.001) in small follicles than in large follicles. In large follicles, the expression of LEPRb was higher (P0.05) in granulosa cells than in theca cells, cumulus cells and oocytes. Higher expression (P0.05) of LEPRb was detected in granulosa cells isolated from large follicles than in granulosa cells isolated from small follicles. Immunohistochemical analyses revealed the presence of the LEP and LEPR proteins in follicles at all stages of development. The most intense staining for LEP and LEPR was observed in the cytoplasm of oocytes and the surrounding granulosa cells. In conclusion, it was demonstrated that leptin and its receptor are expressed at both the mRNA and protein levels in goat ovarian follicles. Furthermore, the presence of a leptin signaling system in the caprine ovary suggests a potential regulatory role for leptin in follicular development and the maturation of goat oocytes.

In the study, we have examined the antitumor and antimicrobial activities of the methanol extract, the fractions, a fraction of total alkaloids and two alkaloids isolated from the stem of Erythroxylum caatingae Plowman. All test fractions, except the hexane fractions, showed antimicrobial activity on gram-positive bacteria and fungi. The acetate: methanol (95:5), acetate, chloroform and hexane fractions show the highest cytotoxicity activity against the NCI-H292, HEp-2 and K562 cell lines using MTT. The absence of hemolysis in the erythrocytes of mice was observed in these fractions and 6β-Benzoyloxy-3α-(3,4,5- trimethoxybenzoyloxy) tropane (catuabine B). Staining with Annexin V-FITC and JC-1 was used to verify the mechanism of action of the compounds of E. caatingae that showed cytotoxicity less than 30 μg/mL in leukemic cells. After 48 h of incubation, we observed that the acetate: methanol (95:5), acetate, and chloroform fractions, as well as the catuabine B, increased in the number of cells in early apoptosis, from 53.0 to 74.8%. An analysis of the potential of the mitochondrial membrane by incorporation of JC-1 showed that most cells during incubation of the acetate: methanol (95:5) and acetate fractions (63.85 and 59.2%) were stained, suggesting the involvement of an intrinsic pathway of apoptosis.

In developing countries, such as Brazil, goat business is in a considerable expansion and the use of biotechnology may be an important tool to increase productivity of livestock in genetic improvement programs. However, more studies are necessary to gain a better understanding of the potential uses of such techniques, including their usefulness to improve reproductive efficiency, which could contribute to the development of less favoured regions. The objective of this review is to describe the main reproductive biotechniques used in Brazil, such as artificial insemination, embryo transfer, in vitro fertilization and transgenesis, as well as the results obtained with their use.

The aim of the present study was to evaluate the in vitro and in vivo effect of the addition of superoxide dismutase (SOD) and reduced glutathione (GSH) to ram semen freezing extender. Significant differences (p 0.05). It can therefore be concluded that the addition of SOD 100 U/ml and GSH 2 and 5 mM preserves the acrosome integrity of frozen ram spermatozoa, while the addition of SOD 100 U/ml to Tris egg-yolk extender offers protection to the membranes of sperm cells after thawing.

This study was conducted to evaluate the effect of the reproductive (multiparous or nulliparous) and climatic (dry or rainy period) conditions on the number and quality of embryos collected from Boer does. Fifty does, being 33 multiparous and 17 nulliparous, were synchronized for oestrus with CIDR® devices for a period of 11 days and superovulated with 250UI of FSH-p. The animals were observed for oestrous behaviour at 12 h intervals, after CIDR® withdrawal. Does were mated and six days later flushed transcervically to recover the embryos. There was no significant difference (P>0.05) among the average number of structures and viable embryos recovered from the multiparous and nulliparous females. The average number of structures and viable embryos recovered from multiparous does had no significant difference (P>0.05) between dry and rainy periods. The average number of structures and viable embryos classified as G1 recovered from nulliparous was lower (P

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii that affects reproductive performance in small ruminants. Although the T. gondii life cycle is well understood since 1960s, several aspects related to its infection remain unclear. In the present study we hypothesized that sheep inseminated with T. gondii -contaminated semen would develop toxoplasmosis. In order to test that hypothesis, 41 sheep were experimentally infected with semen spiked with the organism. Females were divided in three groups (G1–G3): (a) females in G1 group were inseminated with semen containing 6.5 × 10 4 tachyzoites; (b) females in G2 group with semen containing 4 × 10 7 tachyzoites; and (c) females in G3 group with tachyzoite-free semen (control group). To confirm T. gondii infection via semen, serological tests were performed using indirect immunofluorescence reaction and the detection of parasite DNA in the blood stream using the nested PCR test. While in G1 group only 5/15 (33.3%) of the females presented seroconversion, all sheep in G2 15/15 (100%) seroconverted. The nested PCR test showed that 14/15 (93.3%) of the females in the G1 and 14/15 (93.3%) in the G2 group were positive for T. gondii while in the G3 group all samples were negative. In addition, ultra-sound test evidenced that in sheep presented embryonic reabsorption in animals from the infected groups. In conclusion, insemination using fresh semen experimentally contaminated with different infectant doses of T. gondii tachyzoites was able to infect sheep, leading to the possibility of toxoplasmosis transmission via semen.

The effects of deslorelin acetate use in inducing ovulation need to be clarified to improve the results of equine embryo transfer. The mRNA abundance for angiogenic factors and LH receptor (LHR) in corpus luteum (CL) was studied in mares with natural (control group [CG]) and induced ovulation with deslorelin acetate (treatment group [TG]; follicles: ≥ 35 mm). Transrectal ultrasonography was used to verify the ovulation day, and on Days 4, 8, and 12 after ovulation (Day 0), CL samples were obtained through ultrasound-guided biopsy. The messenger RNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and LHR genes were analyzed by real-time polymerase chain reaction. A positive correlation was observed between VEGF and LHR (P < 0.00001, r = 0.78), and it was possible to detect higher LHR expression in the TG than in the CG on Day 4 (P < 0.05). Moreover, this expression was higher on Days 4 and 8 than on Day 12 in the TG. Basic fibroblast growth factor was also expressed in luteal tissue on all days for both groups; however, these differences were not significant. In conclusion, deslorelin acetate was effective for the induction of ovulation in mares, resulting in higher expression of LHR, especially on the fourth day after ovulation. In addition, VEGF expression was influenced by induced ovulation, with a lower level on Day 12, which is expected in nonpregnant mares.

O objetivo deste trabalho foi estudar a eliminação de Toxoplasma gondii no sêmen de carneiros naturalmente infectados. Foram utilizados 65 reprodutores submetidos inicialmente à pesquisa de anticorpos anti-T. gondii por meio da técnica de imunofluorescência indireta (IFI). Os carneiros sorologicamente positivos foram submetidos à colheita de sêmen para detecção do DNA de T. gondii. Na sorologia observaram-se 6/65 (9,2%) carneiros positivos, enquanto no PCR nested de sêmen 4/6 (66,6%) carneiros foram positivos. Conclui-se que a detecção, por meio da técnica da PCR nested, da forma proliferativa de T. gondii no sêmen de carneiros naturalmente infectados, reforça a necessidade de se pesquisar sobre a possibilidade da transmissão horizontal do parasito via sêmen na espécie ovina. The aim of this paper was to study the Toxoplasma gondii shedding in the semen of naturally infected rams. Sixty-five rams were initially submitted to anti-T. gondii antibody detection by indirect immunofluorescence (IIF). Serologically positive rams were then submitted to semen collection for T. gondii DNA detection. In the serology, 6/65 (9.2%) rams were positive, while in the nested PCR of semen there were 4/6 (66.6%) positive rams. It can be concluded that detection of the proliferative form of T. gondii in semen of naturally infected rams by the nested PCR technique reinforces the need to investigate possible horizontal transmission of this parasite via semen in sheep.

Contents The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle-stimulating hormone (FSH) i.m. in six descending doses at 12-h intervals. The goats received 4.8 μg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate-buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick-end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E2) and progesterone (P4) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p

Summary This work aimed to evaluate the effect of stabilisation times, glycerol concentration, and the catalase and superoxide dismutase supplementation of diluent on parameters of frozen-thawed spermatozoa from epididymis of Nelore bulls: Experiment 1: spermatozoa diluted in Tris-egg yolk with glycerol (3%, 5% or 7%) and stabilisation times (0, 2 or 4 hr at 5°C); Experiment 2: Tris-egg yolk only, Tris-egg yolk with catalase (CAT, 50 or 100 U ml−1) or superoxide dismutase (SOD, 50 or 100 U ml−1). Frozen-thawed spermatozoa were evaluated for kinetic parameters, plasma membrane and acrosome integrity, mitochondrial activity and IVF capacity. ALH and BCF were affected (p

a b s t r a c t 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 subcell- ular 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 with- out antioxidant (control group) and with Trolox in different concentrations (30, 60 and 120 M). After thawing (37 ◦ C/30 s), samples were subjected to analysis for plasma mem- brane integrity (PMi), acrosome integrity (Aci), mitochondrial membrane potential (MMP), sperm kinematics, and ultrastructural integrity. The Trolox 60 and 120 M 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, linear- ity, straightness, oscillation index, straight-line velocity and average path velocity), with higher values (P < 0.05) for the Trolox 60 and 120 M groups. On ultrastructural assessment, Trolox addition at the three concentrations preserved spermatozoon head plasma mem- branes, while for the spermatozoon tail, plasma membrane preservation at 60 M was higher (P < 0.05) than the other groups. The Trolox 60 and 120 M 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 120 M provides greater structural integrity (plasma membrane and mitochondria) and kinematics for ram spermatozoa after cryopreservation.

Neoplastic transformation is the abnormal proliferation of cells. These transformations are often related to changes in cell surface glycoconjugates which can be detected by lectins. We evaluated the anti-tumor potential of BlL, a galactoside-binding lectin isolated from Bothrops leucurus venom as well as its cytotoxicity and hemolysis activity. The phosphatidylserine externalization and mitochondrial membrane potential were also determined. BlL exhibited cytotoxic activity against all tumor cell lines tested by induced phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by apoptosis.

Avaliou-se a integridade de espermatozoides ovinos colhidos e criopreservados em diferentes situações climáticas na região semiárida do estado da Paraíba. As colheitas de sêmen foram realizadas em duas épocas do ano, período seco e período chuvoso, utilizando cinco machos adultos da raça Santa Inês, com histórico favorável de fertilidade. Os ejaculados foram avaliados quanto à motilidade, vigor, concentração e morfologia espermática, após a formação do pool e diluição em Tris-gema, na concentração final de 240x10(6) espermatozoides/mL. Motilidade total e vigor espermático não diferiram entre as épocas seca e chuvosa. Valores de integridade do acrossoma e da membrana plasmática, e o potencial de membrana mitocondrial foram mais baixos (P0.05) at the time with less rainfall. We concluded that sheep reproduction raised in semi-arid Paraiba region suffers climatic condition influence and the lowest rainfall period is deleterious to the sperm cells integrity subjected to cryopreservation process. It is suggested that ram spermatozoa cryopreservation be done in period of greatest rainfall in this region.

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n=4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1=0.04%, SL G2=0.08% SL and G3=0.16%) for a final concentration of 240×106spermatozoa/mL. The semen samples were packed in straws (0.25mL), frozen using an automated system and stored in liquid nitrogen (−196°C). After thawing (37°C/30s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P>0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.