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During mammalian meiosis, Polo-like kinase 1 (PLK1) is essential during cell cycle progression. In oocyte maturation, PLK1 expression is well characterized but timing of posttranslational modifications regulating its activity and subcellular localization are less clear. Small ubiquitin-related modifier (SUMO) posttranslational modifier proteins have been detected in mammalian gametes but their precise function during gametogenesis is largely unknown. In the present paper we report for mouse oocytes that both PLK1 and phosphorylated PLK1 undergo SUMOylation in meiosis II (MII) oocytes using immunocytochemistry, immunoprecipitation and in vitro SUMOylation assays. At MII, PLK1 is phosphorylated at threonine-210 and serine-137. MII oocyte PLK1 and phosphorylated PLK1 undergo SUMOylation by SUMO-1, -2 and -3 as shown by individual in vitro assays. Using these assays, forms of phosphorylated PLK1 normalized to PLK1 increased significantly and correlated with SUMOylated PLK1 levels. During meiotic progression and maturation, SUMO-1-SUMOylation of PLK1 is involved in spindle formation whereas SUMO-2/3-SUMOylation may regulate PLK1 activity at kinetochore-spindle attachment sites. Microtubule integrity is required for PLK1 localization with SUMO-1 but not with SUMO-2/3. Inhibition of SUMOylation disrupts proper meiotic bipolar spindle organization and spindle-kinetochore attachment. The data show that both temporal and SUMO-specific-SUMOylation play important roles in orchestrating functional dynamics of PLK1 during mouse oocyte meiosis, including subcellular compartmentalization.

SummaryThe aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 × 106/ml with or without plasmid pEYFP–NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC–PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.9% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.

Activation of in vitro-matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 microM CA, 5 min; n = 88), CA + BSA (5 microM CA, 5 min; BSA, 5 min; n = 90), IO (5 microM IO, 5 min; n = 91), IO + BSA (5 microM IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 micros; n = 120). Blastocyst rates were higher (p 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.

Research on different cryoprotectants and their associations is important for successful vitrification, since greater cryoprotectant concentration of vitrification solution may be toxic to oocytes. The aim of the present research was to compare the efficiency of immature bovine oocyte vitrification in different associations of ethylene glycol (EG), glycerol and dimethylsulfoxide (Me2SO). In the first experiment, oocytes were exposed to the cryoprotectant for either 30 or 60 s in final solutions of EG + DMSO1 (20% EG + 20% Me2SO) or EG + DMSO2 (25% EG + 25% Me2SO) or EG + GLY (25% EG + 25% glycerol). In the second experiment, the oocytes were vitrified in open pulled straws (OPS) using 30 s exposure of final solutions of EG + DMSO1 or EG + DMSO2 or EG + GLY. Maturation rates of 30 s exposure groups were not different from the control, but 60 s cryoprotectant exposure was toxic, decreasing maturation rates. The vitrification with EG + DMSO2 resulted in enhanced maturation rate (29.2%) as compared with EG + DMSO1 (11.7%) and EG + GLY (4.3%) treatments. These data demonstrate that concentration and type of cryoprotectant have important effects on the developmental competence of vitrified oocytes.

A transferência gênica mediada por espermatozóide (TGME) por ser um método teoricamente simples e barato tem sido usada na produção de animais transgênicos. Entretanto, os resultados são variáveis e inconstantes. Para otimizar a TGME é necessário estabelecer o tempo, a quantidade e o tipo de DNA exógeno que será incubado com os espermatozóides. O estudo dos efeitos destes parâmetros na TGME foi o objetivo deste trabalho. Para avaliar o efeito da adição do DNA exógeno durante 0, 1, 2, 3 e 4 horas de incubação na viabilidade espermática foram utilizadas as sondas fluorescentes Hoechst 33342, Iodeto de propídeo, JC-1 e FITC-PSA. Na avaliação do efeito do tempo de incubação (60, 90 e 120 minutos) nos índices de apoptose e necrose, os espermatozóides foram corados com as sondas fluorescentes Yo-pro e Iodetoto de propídeo e avaliados pelo citômetro de fluxo, e os índices de transfecção avaliados pela PCR em tempo real. Para avaliar o efeito da quantidade e da seqüência de DNA exógeno foram construídas seqüências de tamanhos e estruturas diferentes. Para as seqüências de tamanhos diferentes, foram utilizadas seqüências de 2,2; 5,5 e 8,5kb nas concentrações de 500ng ou 130ng da seqüência de 2,2kb, 323ng da seqüência 5,5kb e 500ng da seqüência de 8,5kb. Para as seqüências de diferentes estruturas foram utilizadas seqüências ricas em oligonucleotideos AT, GC ou intermediária (IN). Foram avaliados o efeito das seqüências de diferentes tamanhos, estruturas e concentrações na indução de apoptose dos espermatozóides pelo citômetro de fluxo e os índices de transfecção por PCR em tempo real. Os resultados mostraram que não houve efeito da adição do DNA exógeno sobre a viabilidade espermática. Entretanto, houve efeito do tempo de incubação onde foi observado maior viabilidade espermática com 1 hora de incubação (35,7%). O tempo de incubação não teve efeito na indução de apoptose, mas foi observado efeito no índice de transfecção apresentando maior quantidade de DNA exógeno associado aos espermatozóides às 2 horas de incubação. A quantidade, o tamanho e a estrutura de DNA exógeno não influenciaram o índice de apoptose, assim como a quantidade de DNA não teve efeito nos índice de transfecção. Entretanto, foi observado efeito do tamanho e da estrutura do DNA, no qual a seqüência de 5,5kb apresentou melhores resultados de transfecção tanto na quantidade de 500ng e 323ng. A seqüência rica em AT também apresentou melhor resultado de transfecção em relação à seqüência rica em GC e a intermediária. Em conclusão, o tempo de incubação reduziu a viabilidade espermática, porém, aumentou os índices de transfecção. A transfecção dos espermatozóides não foi influenciada pela quantidade da seqüência, mas foi influenciada pelo tamanho e estrutura da seqüência. A apoptose e necrose das células espermáticas não foram influenciadas pela quantidade, tamanho e estrutura do DNA exógeno Sperm-mediated gene transfer (SMGT) has been used for transgenic animal production because it is a theoretically simple and low cost method. However, results are various and without repetibility. In order to optimize SMGT it is necessary to determine the time, amount and sequence of exogenous DNA for incubation with spermatozoa. The objective of this study was to verify the effect of these three parameters on SMGT. To assess the effect of exogenous DNA addition on sperm viability after 0, 1, 2, 3 and 4 hours of incubation, fluorescent probes Hoechst 33342, propidium iodide, JC-1 and FITC-PSA were used. To evaluate effects of incubation time (60, 90 and 120 minutes) on apoptosis and necrosis rates, spermatozoa were stained with fluorescent probes Yo-pro and propidium iodide and analyzed by flow cytometry; transfection rates were evaluated by real-time PCR. To assess the effect of exogenous DNA amount and sequence, different sized and structured sequences were made. Different sized sequences of 2.2, 5.5 and 8.5kb were used, with concentration of 500ng or 130ng of 2.2 sequence, 500 or 323ng of 5.5 sequence and 500ng of 8.5 sequence. Different structured sequences were rich in AT, GC or intermediate (IN). The effect of different size, structure and concentration on spermatozoa apoptosis induction were analyzed by flow cytometer and transfection rates by real-time PCR. Results show that no effects occurred on spermatozoa viability after exogenous DNA addition. However, time effect occurred: higher spermatozoa viability was observed after 1 hour of incubation (35.7%). Incubation time had no effect on apoptosis induction; otherwise, transfection rates presented higher amounts of exogenous DNA associated to spermatozoa after 2 hours of incubation. Amount, size and structure of DNA did not influence apoptosis and necrosis rates and DNA amount had no effect on transfection rates. However, size and structure effect were observed: 5.5kb sequence presented better transfection results using 500ng or 323ng. AT- rich sequence also presented better transfection rates than GC-rich and IN sequences. In conclusion, incubation time reduced spermatozoa viability, although enhanced the transfection levels. Spermatozoa transfection was not influenced by DNA sequence amount, but was influenced by the size and structure of the sequence. Apoptosis and necrosis of sperm cells were not influenced by amount, size and structure of exogenous DNA

Apesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal proposito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência genica, na qual a célula espermática tem habilidade espontânea de se ligar a molécula de DNA e internaliza-la. Dado o potencial da transferência genica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência genica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões positivos para o DNA exógeno foram avaliados por PCR. Nenhum efeito de tratamento foi observado nos índices de clivagem, blastocisto e eclosão. Além disso, a porcentagem de blastocistos positivos para o DNA exógeno não diferiu entre os grupos experimentais. Apesar do baixo número de embriões positivos para DNA exógeno, os resultados obtidos mostram que todos os tratamentos apresentaram eficiências similares. A conclusão obtida foi que, apesar de os índices de desenvolvimento embrionário terem sido similares e constante em todos os grupos experimentais, outros fatores como a sequência, o tamanho e a concentração do DNA exógeno devem ser avaliados para melhorar a transferência genica mediada por espermatozoides.

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

A suggested alternative to improve post-thawed ram semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryocapacitation and helping sperm survival. The aim of this study was to evaluate the effect of frozen-thawed ram semen incubation with SP on mitochondrial activity, acrosomal membrane integrity, necrosis and apoptosis. Frozen/thawed semen was divided into two groups: the SP Group and the control group. After 0, 30 and 60 min, fluorescent probes were added to aliquots from each treatment group and evaluated using flow cytometry. There was no difference between treatment groups in almost all viability parameters evaluated, with exception of the apoptosis, which was found increased in SP group. The increase in incubation period resulted in a decreased percentage of sperm with high mitochondrial membrane potential and acrosomal membrane integrity and an increased percentage of necrotic and apoptotic sperm cells. In conclusion, the present study showed that addition of seminal plasma after thawing cryopreserved ram sperm had no identifiable beneficial effect on sperm quality.

A fecundação resulta no aumento intracelular de cálcio que é necessário para a transição do oócito até o estádio de zigoto. Os eventos que ocorrem durante esta transição são caracterizados como ativação, sendo estes dependentes de cálcio. Entretanto, os eventos bioquímicos que ocorrem durante a ativação ainda não estão completamente elucidados. A proteína quinase C (PKC) e a proteína quinase dependente de cálcio/calmodulina (CaMKII), por apresentarem atividade durante a fecundação e por serem ativadas por cálcio são implicadas na regulação dos eventos da ativação. Entretanto, existem muitas dúvidas sobre o real papel destas proteínas na ativação do oócito. Deste modo, o objetivo do presente trabalho foi avaliar o papel da PKC e da CaMKII na ativação de oócitos bovinos. Para tal, oócitos bovinos maturados in vitro foram ativados partenogeneticamente (AP) com cálcio ionóforo A23187 (5μM) por 5 minutos, sendo a retomada da meiose, a organização do citoesqueleto e do retículo endoplasmático (RE) avaliada 1 hora após a ativação. No experimento 1 foi avaliado o papel da CaMKII nestes eventos. Os oócitos foram AP na presença ou ausência de 100M do inibidor de CaMKII (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). A inibição da CaMKII não afetou a retomada da meiose e nem a distribuição dos RE, após a AP. Entretanto, não ocorreu a rotação do fuso meiótico no estádio de telófase II quando a CaMKII foi inibidada. Estes resultados demonstram que embora a CaMKII não tenha efeito na retomada da meiose, esta proteína participa na progressão do ciclo celular de oócitos bovinos, após a AP. No experimento 2 foi avaliado o papel da PKC em oócitos bovinos AP. Os oócitos foram ativados partenogeneticamente na presença ou ausência de 10μM do inibidor de PKC (Bisindolymaleimide I). A inibição da PKC não afetou a retomada da meiose e nem a progressão pelo ciclo celular até o estádio de telófase II. Entretanto, a organização do RE foi afetada pela inibição da PKC. Resultado semelhante foi obtido quando os oócitos foram ativados na presença de citocalasina C, um despolimerizador de filamentos de actina. O presente experimento demonstra a participação da via PKC-actina na organização do RE na ativação de oócitos bovinos. The intracellular calcium increase resulting from fertilization is necessary for oocyte transition to zygote. The events that occur during this transition are characterized as activation, which are dependent on calcium. However the biochemical events that occur during this activation are still not fully elucidated. The protein kinase C (PKC) and the calcium/calmodulin-dependent protein kinase II (CaMKII), are involved in regulating the events of activation, since these proteins have activity during fertilization and are activated by calcium. However there are many doubts about the real role of these proteins in the oocyte activation. Thus, the objective of this study was to evaluate the role of PKC and CaMKII in bovine oocyte activation. For this purpose, in vitro matured bovines oocytes were parthenogenetically activated (PA) by using calcium ionophore A23187 (5μM) for five minutes, and the resumption of meiosis, the cytoskeleton organization and the endoplasmic reticulum (ER) organization were evaluated 1 hour post-activation. In experiment 1, were evaluated the role of CaMKII in these events. The oocytes were PA in the presence or absence of 100M of CaMKII inhibitor (Autocamtide-2 Related Inhibitory Peptide, Myristoylated). The inhibition of CaMKII did not affect the meiosis resumption and the ER after the PA. However, there was no spindle rotation at telophase II stage when the CaMKII was inhibited. These results showed that although the CamKII has no effect on resumption of meiosis, it participates in the regulation of cell cycle progression after PA of bovine oocytes. In experiment 2, was evaluated the role of PKC on PA bovine oocytes. The oocytes were parthenogenetically activated in the presence or absence of 10μM of PKC inhibitor (Bisindolymaleimide I). The PKC inhibition did not affected the resumption of meiosis and the progression through the cell cycle until the stage of telophase II. However, the ER organization was affected by PKC inhibition. A similar result was obtained when the oocytes were activated in the presence of cytochalasin C, which promotes the depolymerization of the actin filaments. The current experiment showed the participation of the PKC-actin pathway at the ER organization in the bovine oocytes activation.

SummaryMyostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A3 (CMA3) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA3 staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA3-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA3-positive sperm cells compared to the others. CMA3 is a simple and useful tool for detecting sperm protamine deficiency in bulls.

O tempo de incubação causa danos nas células espermáticas relacionados a necrose e/ou apoptose. O objetivo deste estudo foi avaliar as mudanças na membrana plasmática relacionadas a apoptose e necrose em espermatozóides bovinos durante 2 horas de incubação. Os espermatozóides foram incubados a 5% (v/v) CO2 em ar por 0, 30, 60, 90 e 120 minutes. Depois de cada periodo, as células espermáticas foram incubadas com as sondas fluorescentes Yo-pro e iodito de propideo (PI) para detectar mudanças na membrana plasmática relacionadas a apoptose e a necrose, respectivamente. Usando Yo-pro/PI assay, três subpopulações diferentes de células espermáticas são detectadas pelo citômetro de fluxo: a) células espermáticas em necrose (PI+ and Yo-pro-/+); b) células espermáticas em apoptose (Yo-pro+ and PI-) e c) células espermáticas vivas (Yo-pro- and PI-). A porcentagem de células vivas (membrana plasmática integra) significativamente diminui durante 2 horas de incubação, por outro lado, a porcentagem de espermatozóides em necrose e apoptose aumentaram durante a incubação. As mudanças na integridade da membrana plasmática foram correlacionadas com o tempo de incubação. Enquanto as células vivas foram correlacionadas negativamente com o aumento do tempo de incubação, necrose e apoptose foram correlacionadas positivamente. Também foi observado que necrose foi o principal dano causado pelo tempo de incubação nas células espermáticas. Conclui-se que o tempo de incubação causa alteração na integridade da membrana plasmática relacionadas a necrose e apoptose nas células espermáticas, sendo que necrose foi observada em maior quantidade em todos os tempos de incubação.

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha -lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position - 1689 (g. AG) and + 15 (g. AG) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide - 46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A - 1689, A - 46, and A + 15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5'-flanking and the 5'-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 +/- 0.65) when compared with non-starved cells (71.44 +/- 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.

O presente trabalho foi realizado com o objetivo de avaliar o desenvolvimento in vitro de embriões bovinos co-cultivados em células da granulosa, do oviduto, BRL e VERO, suplementados com 5% ou 10% de Soro Fetal Bovino (SFB). Os complexos cummulus oócitos foram aspirados, maturados e fecundados in vitro. As estruturas embrionárias foram divididas em oito tratamentos: co-cultivo em TCM 199 contendo células da granulosa, do oviduto, BRL ou VERO adicionadas com 5% ou 10% de SFB. As condições de cultivo foram 38.5 ºC, 5% CO2 em ar e alta humidade por dez dias consecutivos. Os índices de clivagem, blastocisto e eclosão não diferiram (p > 0,05) no co-cultivo com células primárias (granulosa e oviduto) quando a concentração de SFB aumentou de 5 para 10%. Entretanto, no co-cultivo com células de linhagens contínuas (BRL e VERO), quando a concentração de SFB aumentou de 5% para 10%, os índices de blastocistos diminuíram significativamente (p < 0,05) de 33,6 para 16,3 % e de 40 para 16,5% nos embriões bovinos co-cultivados com células VERO e BRL, respectivamente.

The spermatozoa is an ideal vehicle for genetic modification, production of transgenic animals, as well as a biotechnological tool for sperm-mediated gene transfer. However, in order to achieve successful sperm fertilization and exogenous DNA integration, it is necessary for viable cells to remain intact, allowing the sperm to penetrate the oocyte. Fluorescent probes allow evaluation of morphological and functional characteristics of cells, which can be evaluated separately or simultaneously. Therefore, the aim of this study was to validate the simultaneous evaluation of the integrity of plasma and acrosomal membranes of murine sperm using the probes carboxyfluorescein diacetate (CF) and propidium iodide (PI). In order to validate, a standard curve was performed. Sperm were obtained from epididymis and vas deferens from CD-1 mice (8 to 16 weeks of age). Recovered samples were diluted in PBS and then divided into 2 aliquots: one prepared with fresh semen (FS) and the other submitted to Percoll gradient (45%/90%) followed by flash-freezing in liquid nitrogen and thawing (FTP) to induce acrosome damage. Samples were prepared with the following average of FS:FTP: 100:0 (T100), 50 : 50 (T50), and 0 : 100 (T0). Samples were stained using 2 μL Hoescht 33342 (40 μLmL-1 in Dulbecco’s phosphate buffered saline), 3 μL of PI (0.5 mg mL-1 in PBS), 3 μL of CF (0.46 mg mL-1 in DMSO), and were incubated for 8 min at room temperature. After staining, the samples were placed on a slide, coverslipped, and evaluated immediately by epifluorescent microscopy. The Hoescht, PI, and CF fluorescence was detected using a filter with excitation at 352, 538, and 495 nm and emission at 455, 617, and 517 nm, respectively. Approximately 200 sperm cells per slide were examined and classified based on the fluorescence emitted from each probe. Spermatozoa CF+/IP- were considered as intact membranes, CF+/PI+ as acrosome membrane intact and plasma damaged, CF-/PI+ as damaged membranes, and CF-/PI- as acrosome membrane damaged and plasma intact. Hoeschst was used as positive dye. This experiment was replicated 6 times per group, and for statistical analyses, the data of plasma and acrosomal membrane integrity (dependent variables) in the treatments T0, T50, and T100 (independent variables) were submitted to simple linear regression analysis by STATVIEW software (SAS Institute Inc., Cary, NC, USA). The CFDA/PI probes were suitable for the analysis of acrosomal and membrane status of murine sperm and showed a high determination coefficient to plasma membrane integrity (R2 = 0.81; Y = 0.5412x + 6.375) and acrosome integrity (R2 = 0.85; Y = 0.5653x + 11.653). The described protocol was efficient for the simultaneous evaluation of plasma and acrosomal membrane integrity of murine spermatozoa, proving that CFDA can be employed to access acrosomal integrity as an alternative to FITC-PSA. Financial support: FAPESP.

Heat-stress induced maternal hyperthermia has been shown to compromise the series of events associated with oocyte growth and maturation reducing oocyte competence. Such events are regulated by a variety of growth factors and dynamic communication between the oocyte and its surrounding cumulus cells. The objective of the current study was to evaluate the modulatory effects of COCs quality and IGF-I on mitochondrial membrane potential (MMP) and apoptosis in cumulus cells induced by heat shock. In this study high (≥3 layers of compact cumulus cells and homogeneous cytoplasm) and low-grade COCs (

During oocyte growth, transcription activity results in the production of RNA and proteins, which are immediately used or stored. At this time, gene expression is, in part, controlled at the post-transcriptional level through deadenylation and polyadenylation processes. After fertilization, early embryonic cell cycles are mainly supported by maternal mRNA and proteins until the maternal embryonic transition. The extent of the poly A tail of mRNA is known as an important element to determine their stability and can be considered a key marker in embryonic development. Because cell cycle-related genes are implicated in the maturation process and early embryonic cell cycle, study of the poly A tail in oocytes during maturation and early embryonic development may be an useful marker of differential developmental competence. Thus, the aim of the present work was to determine the polyadenylation status of the cell cycle-related genes cdc2, CDK2, CDK4, and Cyclin A, B, D, and E during bovine oocyte in vitro maturation (IVM). A PCR-based technique previously developed by Salles and Strickland (1995 PCR Meth. Appl. 4, 317–321) was used, with some modifications. Cumulus–oocyte complexes obtained from 2- to 8-mm follicles were immediately frozen in liquid nitrogen or IVM for 24 h in TCM-199 supplemented with fetal bovine serum and hormones (LH, FSH, and estradiol). In both cases, cumulus cells were removed by pipetting after a 10-min 0.2% hyaluronidase incubation. The RNA was extracted from pools of 10 immature or IVM oocytes and incubated with phosphorylated oligo-dT and DNA ligase. Thereafter, an anchor adapter [poly(dT) anchor] was targeted to the 5 end of the oligo-dT and incubated with Superscript II (Invitrogen) for reverse transcription. Specific primers were designed at the end of a full known sequence of the genes described above, and PCR was conducted for each gene using a specific primer and the anchor primer. The PCR products were digested with restriction enzymes to verify their specificity and were submitted to polyacrylamide gel electrophoresis. All sequences were amplified in immature and matured oocytes from 2 replicates by using a very small sample. After the restriction enzyme treatment, the amplified products were digested as expected. Although differences in amplicon sizes could be observed between the immature and matured oocytes, these are preliminary results and a higher number of replicates are necessary to determine the changes in the polyadenylation status of these cell cycle-related genes and their biological function. In conclusion, the polyadenylation status of the cell cycle-related genes cdc2, CDK2, CDK4, and Cyclin A, B, D, and E could be effectively performed in small samples, and this may be a powerful tool for studying the differential developmental competence of bovine oocytes and early cleavage embryos. FAPESP.

The aim of this study was to evaluate the efficiency of a low oxygen tension atmosphere (5% CO2, 5% O2, and 90% N2) on swine oocyte maturation in chemically defined media or when supplemented with porcine follicular fluid (PFF). Briefly, oocytes were in vitro matured for 44 h in TCM-199 with 10% PFF or 0.1% PVA added, under a low oxygen tension atmosphere or a normal oxygen tension atmosphere (5% CO2 in air, approximate 20% O2). At 0 and 44 h of maturation, cumulus oophorus cells were removed. To evaluate the migration of cortical granules, oocytes were fixed, permeabilized, and incubated in 100 μg of FITC-PNA mL–1 for 30 min. Oocytes were then incubated in 10 μg mL–1 of Rnase for 30 min and in 10 μg mL–1 of propidium iodide for 10 min to verify nuclear maturation by confocal microscopy (Zeiss LSM 510 Meta). Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify oxidative stress. Data were analyzed by the SAS System for Windows (2000). The nonparametric ANOVA NPAR1WAY procedure was applied to evaluate nuclear maturation rate by comparing groups in pairs. Migration of cortical granules and HSP70 content were analyzed using PROC GLM (LSD test of means). The effects of treatment and manipulation were verified in all analyses. The significance level was 5%, and data were presented as means ± SEM. Results indicated that the percentage of metaphase II oocytes did not differ among groups after 44 h of maturation [PFF 5% O2 (89.16 ± 3.73a), PFF 20% O2 (86.59 ± 6a), PVA 5% O2 (79.62 ± 8.22a), and PVA 20% O2 (93 ± 5.17a)]. However, these groups were different from the 0-h group (0 ± 0b). Results for the percentage of cortical granule migration showed that 0-h oocytes (38.92 ± 2.75a) had lower migration rates compared with other groups. After 44 h of maturation, migration of the cortical granule rate of the PFF-supplemented group under a 5% O2 atmosphere (61.66 ± 3.21b) was different when compared with the PVA 20% O2 group (50.97 ± 3.48c). The other groups showed intermediate results, but without statistical differences [PFF 20% O2 (58.51 ± 2.5bc) and PVA 5% O2 (53.75 ± 3.14bc)] for the migration of cortical granules. Moreover, no difference at pixel quantification of HSP70 was observed among groups after 44 h of maturation [PFF 5% O2 (116.45 ± 40.94a), PFF 20% O2 (44.44 ± 12.66a), PVA 5% O2 (29.95 ± 7.95a), and PVA 20% O2 (58.49 ± 22.2a)], although these groups were different from the 0-h group (247.41 ± 38.59b). Although the HSP content decreased throughout in vitro maturation of swine oocytes under the low and high oxygen tension atmospheres, it can be concluded that a low oxygen tension atmosphere did not affect nuclear maturation and rates of cortical granule migration regardless of maturation media supplementation. Financial support: FAPESP (grant no. 05/01420-7).

The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed. This work was supported financially by FAPESP 03/0156-9.

A recently suggested alternative to improve post-thaw ovine semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryo-capacitation and helping sperm survival in the female tract. The aim of this study was to evaluate the effect of thawed ram semen incubation with seminal plasma for 15 or 30 min as assessed by the fluorescent probes, Hoechst 33342 (40 µg mL−1), propidium iodide (0.5 mg mL−1), JC-1 (76.5 µM in DMSO), and FITC-PSA (100 µg mL−1 in Dulbecco's PBS (DPBS)), for cellular viability, plasmatic damage, mitochondrial activity, and acrosomal damage, respectively. Five ejaculates were collected from 4 different animals via artificial vagina and were pooled to eliminate individual differences. After thawing, semen was divided into 2 groups, one diluted with seminal plasma (1 : 1, 30% in DPBS) and the other in DPBS (1 : 1). After 15 and 30 min, fluorescent probes were added to 25 µL of each group and 100 cells were counted under a epifluorescence microscope (Olympus IX81, motorized inverted research microscope). Spermatozoa were classified under 8 categories: alive, damaged acrosome, and high mitochondrial activity (ADH); dead, damaged acrosome, and high mitochondrial activity (DDH); alive, damaged acrosome, and poor mitochondrial activity (ADP); dead, damaged acrosome, and poor mitochondrial activity (DDP); alive, intact acrosome, and high mitochondrial activity (AIH); dead, intact acrosome, and high mitochondrial activity (DIH); alive, intact acrosome, and poor mitochondrial activity (AIP); or dead, intact acrosome, and poor mitochondrial activity (DIP). Statistical analysis was performed by Fisher test at a 5% level. The results are presented in Table 1. There was no significant difference between groups except for the AIH category which was reduced during incubation in either DPBS or SP for 15 or 30 min. These last data allow the conclusion that ram sperm is highly susceptible to cryogenic damage. Nevertheless, the high percentage of the AIH category suggests that the spermatozoa that can resist the freezing protocol remain alive and intact. Hence, we can infer that ram frozen semen has approximately half the fertilizing potential of fresh semen. In addition, we observed a negative effect of the incubation period as the decreased percentage of the AIH category was accompained by an increase of the DDP and DIP ones. We conclude that seminal plasma incubation after thawing does not benefit sperm quality. It is necessary to study the seminal plasma constitution to conclude why this experiment differed from other published data. Table 1.Spermatozoa percentages in the categories during incubation periods with or without seminal plasma This work was supported financially by FAPESP 05/55256-3

Current data about the kinetics of exogenous DNA interaction with spermatozoa are controversial. The amount of DNA associated with the spermatozoa is proportional to the incubation period. However, a great amount of exogenous DNA, associated or internalized, can activate endonucleasis, which cleaves the exogenous and/or endogenous DNA similarly to apoptosis. The aim of this work was to evaluate the effect of the incubation period of bovine spermatozoa with exogenous DNA on transfection rate and induction of apoptosis, oncosis, or initial oncosis and/or late apoptosis of sperm cells. For that, 5 × 106 spermatozoa/mL were incubated with 500 ng of plasmid pEYFP-Nuc (Clontech, Mountain View, CA, USA) for 60, 90, and 120 min. Sperm cells were then subjected to electrophoresis to remove non-associated exogenous DNA. DNA (endogenous and exogenous) was extracted and real-time PCR was performed to quantify exogenous DNA/spermatozoa association. Each reaction was performed with 12.5 µL of Platinum SYBR Green; 0.5 µL of ROX; 5.6 µL of H2O; 5 µL of sample (1 µg); 0.7 µL of primer sense (5′-ATGGCCGACAAGCAGAAGAAC-3′) and 0.7 µL of primer anti-sense (5′-TGCCGTCCTCGATGTTGTG-3′); 500 nM each. Real-time PCR was conducted for 40 cycles of 95°C for 15 s and 64°C for 1 min. Apoptosis and oncosis analyses were evaluated by flow cytometry with 106 sperm/mL stained with 2 µL of Yo-pro (100 µM) for 20 min and with 10 µL of propidium iodide (6 µM) for 10 min at room temperature. Data were analyzed by MINITAB Realease 14 Statistical Software (Minitab, Inc., State College, PA, USA), subjected to ANOVA, and compared by Fisher's or Tukey's test (P ≤ 0.05) for transfection rate and apoptosis/oncosis, respectively. Linear regression obtained by the amplification of pEYFP-Nuc at different concentrations showed efficiency of R2 = 0.9987. The results summarized in Table 1 show that increase in length of the incubation period increases the amount of exogenous DNA associated with spermatozoa and does not affect the average of live spermatozoa in apoptosis, oncosis, and initial oncosis and/or late apoptosis groups. Table 1.Quantification of exogenous DNA associated with bovine sperm cells and percentages of live, apoptotic, and oncotic spermatozoa This work was supported financially by FAPESP 03/07456-8 and 03/10234-7.

There are many methods to produce transgenic animals, although when considering bovine species, those methods offer low repeatability, high costs, and low transgene integration efficiency. To overcome these difficulties, sperm mediated gene transfer (SMGT) could be used as an alternative to produce transgenic embryos. This technology allows carrying exogenous DNA into the oocyte during the fertilization period, once spontaneous binding exists between spermatozoa and exogenous DNA. The aim of this study was to compare 4 methods for incorporating DNA into sperm cells—sperm incubation, capacitation, electroporation, and lipofection—to verify the efficiency of embryo production with exogenous DNA, employing spermatozoa as a vector. Cumulus–oocyte complexes (COCs) from abattoir-derived bovine ovaries were randomly divided into 5 groups (4 experimental groups: sperm incubation, sperm capacitation with calcium ionophore, electroporation, and lipofection; and 1 control group). In vitro maturation was performed in TCM-199 medium supplemented with 10% FCS, sodium pyruvate, gentamycin, FSH, hCG, and estradiol in an incubator at 39�C, in an atmosphere of 5% CO2 in air and high humidity for 24 h. After Percoll gradient (45/90%) separation at 600g for 30 min, spermatozoa were washed in Talp Semen medium (200g for 5 min). For in vitro fertilization (IVF), 5 � 106 spermatozoa were used to inseminate microdroplets with 20 matured oocytes from all groups: incubation with exogenous DNA for 1 h, sperm capacitation (250 nM of calcium ionophore) for 5 min followed by sperm incubation for 1 h, electroporation (500V), and lipofection (Effectene�; Qiagen, Mississauga, Ontario, Canada). The EYFP-Nuc plasmid (500 ng mL-1; Clontech, Mountain View, CA, USA) was used as exogenous DNA. Oocytes inseminated with non-treated sperm were considered as the control group. The presumptive zygotes were co-cultured with a granulosa cell monolayer in SOFaa medium in an incubator at 39�C, with an atmosphere of 5% CO2 in air and high humidity. The blastocyst rate was analyzed by ANOVA. Embryos from all experimental and control groups were subjected to PCR to detect internalized EYFP, using primers specific to this exogenous DNA, and considering positive embryos those that showed a fragment of 440 bp after electrophoresis. The fragment of 440 bp from positive embryos was sequenced to check correspondence to the EYFP. Electroporation (17.95%) and sperm capacitation (15.12%) were more efficient for EYFP-positive embryo production when compared to the lipofection protocol (6.25%). Sperm incubation (12.5%) did not show a significant difference from the other groups. Sequenced PCR-positive products for EYFP showed 100% homology with nucleotides of EYFP at GenBank. In conclusion, it is possible to use SMGT to deliver exogenous DNA into an oocyte at the time of IVF. This work was supported financially FAPESP 03/08542-5 and 03/07456-8.

Mammalian sperm cells have a spontaneous ability to take up exogenous DNA in a process regulated by specific protein. However, little is known about the effect of exogenous DNA on sperm cell association. The aim of this work was to evaluate the effect of size, concentration, and structure of exogenous DNA sequence in apoptosis and oncosis induction and the association with spermatozoa. To evaluate the effect of size and concentration of exogenous DNA, sequences of 2.2, 5.5, and 8.5 kb were used at the concentrations of 500 ng (Experiment 1) or 5 × 1011 molecules (Experiment 2). To assess the effect of structure (Experiment 3), 3 sequences of approximately 500 bp were used, containing 65.7% of AT (AT sequence), 46.4% of AT (IN sequence), and 38.6% of AT (GC sequence). To evaluate apoptosis and oncosis in all treatments, sperm cells were incubated with exogenous DNA for 1 h, stained with 2 µL of Yo-Pro (100 µM) for 20 min, and 10 µL of propidium iodide (6 µM) for 10 min, and then analyzed by flow cytometry. To evaluate the transfection rates, sperm cells, after incubation, were separated from non-associated exogenous DNA by 2.5% gel electrophoresis. DNA (genomic and exogenous) was extracted and association rates obtained by real-time PCR. Data were submitted to ANOVA and compared by Tukey's test (P ≤ 0.05). The number of viable, apoptotic, oncosis, or in initial apoptosis/late oncosis spermatozoa incubated with different concentrations or sequences of exogenous DNA did not differ among groups. In Experiment 1, a higher DNA association with sperm cells was observed in the 5.5 kb sequence in comparison with the 2.2 and 8.5 kb. In Experiment 2, the 8.5 kb sequence had the lower association with the spermatozoa in comparison with the 2.2 kb and 5.5 kb. In Experiment 3, there was a higher interaction of the AT sequence when compared to IN and GC sequences. Reference data suggest that big sequences of exogenous DNA have advantages in the interaction with spermatozoa. The present work is the first one to show that the advantage of the big sequences on interaction became a disadvantage in relation to the ability of these cells to internalize this DNA. Table 1. Quantification of different exogenous DNA associated to sperm cells Financial support was provided by FAPESP 03/10234-7 and 03/07456-8.

Several studies have been performed over the years to promote the understanding and improvement of the sperm-mediated gene transfer technique. However, little is known about the effect of exogenous DNA in the sperm cells. The aim of this study was to evaluate the effect of the incubation period and exogenous DNA addition on mitochondrial activity and acrosomal status of bovine spermatozoa. Frozen-thawed semen was separated by Percoll gradient (45/90%) at 600g for 30 min, and the pellet was resuspended and washed by centrifugation in sperm-TALP at 200g for 5 min. The spermatozoa were resuspended in fertilization medium (without heparin) at a concentration of 5 × 106 spermatozoa/mL and incubated at 39°C, 5% CO2 in air with 500 ng pEYFP-Nuc/mL (Clontech, Mountain View, CA, USA) (DNA group) for 1 and 2 h or without DNA (control group) for 0, 1 and 2 h. JC-1 (Molecular Probes; Invitrogen Brasil, Ltd., Sao Paulo, Brazil) was used to determine mitochondrial potential and fluorsecein isothiogyanate (FITC-PSA; Sigma-Aldrich, Brazil) was used to assess acrosomal status. Two microliters of JC-1 (76.5 μM in DMSO) and 50 μL of FITC-PSA (100 μg/mL in DPBS) were added to 150 μL of IVF medium + 5 × 106 spermatozoa/mL; the mixture was incubated at 25°C for 10 min. DNA incorporation was evaluated by p-EYFP-Nuc PCR amplification. All treatments were repeated ten times and data were analyzed by ANOVA and Tukey test (P < 0.05). The results are described in Table 1. The sperm were classified as: Class 1 (intact acrosome and high mitochondria potential), Class 2 (intact acrosome and low mitochondria potential), Class 3 (reacted acrosome and high mitochondria potential) and Class 4 (reacted acrosome and low mitochondria potential). In both groups, the Class 1 sperm decreased over time, whereas an increase in number was verified for Class 2 sperm. There was no significant difference in numbers among the incubation periods in both groups for Class 3 sperm. However, there was an increase in Class 4 sperm number over time, indicating that the main effect of the incubation period was the loss of mitochondria potential. There was no effect of the exogenous DNA addition on sperm viability in relation to the control group, indicating that the exogenous DNA had no effect on mitochondrial activity and acrosomal status of bovine semen. Table 1. Effect of incubation period and exogenous DNA addition on sperm viability This work was supported by FAPESP 03/10234–7; 03/07456–8.

Recently, progress has been achieved in reproduction biotechnology with coconut water as semen extender for domestic animals and in bovine oocyte in vitro maturation and embryo culture. The aim of this study was to evaluate the dehydrated coconut water (ACP250) as semen extender and its effect on in vitro sperm capacitation in swine. Eighteen ejaculates were collected from three crossbred boars of Landrace and Duroc. Semen samples were extended in ACP250 solution or Beltsville Thawing Solution (BTS) and submitted to sperm capacitation after extension (EXT) or after cooling (COO) at 16°C for 24 h. The semen was centrifuged at 400g for 8 min. After centrifugation, the samples were standardized at 2 × 107 spermatozoa/mL, subjected to sperm capacitation in TALP medium with 5 mM of caffeine, and incubated at 38.5°C and 5% of CO2 in high humidity for three h. Four treatments were tested, T1 (ACP250 + EXT), T2 (ACP250 + COO), T3 (BTS + EXT), and T4 (BTS + COO). Sperm capacitation was evaluated by Coomassie Blue G stain, as follows: capacitated spermatozoa were stained in tenuous blue and not capacitated in intense blue. The Coomassie Blue G staining technique was described by Larson and Miller (1999 Mol. Reprod. Dev. 52, 445–449), who demonstrated that sperm from a variety of mammalian species can be stained by this technique, and an adequate observation of the acrosomal status was possible as well. This procedure was described as simple, fast, inexpensive and reliable, and it requires no fluorescence or DIC optics. For statistical analysis, SAS (System for Windows; SAS Institute, Inc., Cary, NC, USA) was utilized (P < 0.05). Comparing the sperm capacitation rates in relation to fresh vs. cooled semen, there were significant differences between T1 (22.3%) and T2 (43.7%) and between T3 (23.4%) and T4 (48.1%) (P < 0.05). In relation to BTS or ACP250 extenders, there were no significant differences between T1 and T3 and between T2 and T4 (P > 0.05). In conclusion, the ACP250 solution is an option for in vitro sperm capacitation in swine, specially with chilled extended semen. This work was supported by FAPESP 01/11931-8 and ACP Biotechnology – CE.