Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine.

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 µM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 µM, with no embryo formation occurring at 24 µM. Further analysis showed that embryos treated with 12 µM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.

Effects of EGF and melatonin on gene expression of cumulus cells and further in vitro embryo development in bovines.

Despite previous research demonstrating the benefits of including growth factors and antioxidants to maturation medium to support embryo production, to date the effect of epidermal growth factor (EGF) and melatonin (Mel) on oocyte competency has not been studied. This study supplemented in vitro maturation (IVM) medium with EGF (10 ng/ml) and Mel (50 ng/ml) alone, or in combination, and evaluated cumulus cell (CC) gene expression and the development and quality of parthenogenetic blastocysts. No differences in CC gene expression levels indicative of developmental potential were found among the treatment groups. Antioxidant gene CuZnSOD was significantly (P < 0.05) decreased in CCs from the Mel group. Moreover, blastocyst rates on day 7 were significantly increased in EGF or Mel (P < 0.05), but not EGF+Mel. Significant decrease (P < 0.05) in GPX1, CuZnSOD, SLC2A1 and HSPA1A (P = 0.07) mRNA levels was observed in blastocysts from the Mel group. OCT4 gene expression was significantly increased (P < 0.05) in EGF+Mel and confirmed using immunofluorescence. Our results indicate that, despite the lack of changes of competence-related genes in CCs, IVM medium supplemented with Mel improved the culture environment sufficiently, resulting in improved blastocysts. Moreover, EGF and Mel combined during maturation increased OCT4 gene and protein expression in blastocysts, indicating its potential for stem cells.

Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro / Atividade antioxidante de óleo essencial obtido de Lippia origanoides melhora a qualidade de embriões bovinos produzidos in vitro

The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50µM/mL Cysteamine; T3)2.5µg/mL; T4)5.0µg/mL and T5)10.0µg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.(AU)

O objetivo desse estudo foi avaliar a suplementação de meio de cultura de embriões com antioxidante obtido do extrato oleoso da Lippia origanoides no desenvolvimento e na qualidade de blastocistos produzidos in vitro. Oócitos coletados de ovários de matadouros foram maturados e fertilizados in vitro segundo procedimento laboratorial padrão. Zigotos foram cultivados em meio SOF suplementado de acordo com os seguintes tratamentos: T1) meio de cultivo embrionário sem suplementação antioxidantes; T2) 50µM/mL Cisteamina; T3) 2,5µg/mL; T4) 5,0µg/mL e T5) 10,0µg/mL do antioxidante obtido do extrato oleoso de Lippia origanoides. No sétimo dia de cultivo, os blastocistos foram fixados e avaliados para taxa de apoptose, número total de células e massa celular interna através do teste TUNEL. O uso de antioxidantes durante cultivo não aumentou (P>0,05) a taxa de produção final de blastócitos. Os tratamentos T2, T3, T4 e T5 tiverem menor índice apoptótico (p>0,05 - 4,5±1,1%, 8,4±2,5%, 3,4±1,1% e 5,5±0,9%, respectivamente) quando comparados a T2 (10,0±1,4%). O valor de massa celular interna não diferenciou (p>0,05) entre embriões de diferentes tratamentos. A adição de antioxidante obtido do extrato oleoso de Lippia origanoides reduziu a taxa de apoptose e melhorou a qualidade sem aumentar a produção in vitro de embriões bovinos.(AU)

Bone marrow mononuclear cells versus mesenchymal stem cells from adipose tissue on bone healing in an Old World primate: can this be extrapolated to humans? / Células mononucleares de medula óssea versus células-tronco mesenquimais de tecido adiposo em cicatrização de lesão óssea em primata: pode ser extrapolado para humanos?

In veterinary medicine, the cell therapy is still unexplored and there are many unanswered questions that researchers tend to extrapolate to humans in an attempt to treat certain injuries. Investigating this subject in nonhuman primates turns out to be an unparalleled opportunity to better understand the dynamics of stem cells against some diseases. Thus, we aimed to compare the efficiency of bone marrow mononuclear cells (BMMCs) and mesenchymal stem cells (MSCs) from adipose tissue of Chlorocebus aethiops in induced bone injury. Ten animals were used, male adults subjected, to bone injury the iliac crests. The MSCs were isolated by and cultured. In an autologous manner, the BMMCs were infused in the right iliac crest, and MSCs from adipose tissue in the left iliac crest. After 4.8 months, the right iliac crests fully reconstructed, while left iliac crest continued to have obvious bone defects for up to 5.8 months after cell infusion. The best option for treatment of injuries with bone tissue loss in old world primates is to use autologous MSCs from adipose tissue, suggesting we can extrapolate the results to humans, since there is phylogenetic proximity between species.(AU)

Na medicina veterinária, a terapia celular ainda é inexplorada e há muitas perguntas não respondidas, o que leva os pesquisadores a uma tendência a estender a terapia para os seres humanos, na tentativa de tratar certas lesões. Investigar esse assunto em primatas não humanos revela-se uma oportunidade sem precedentes para compreender melhor a dinâmica das células-tronco contra algumas doenças. Assim, objetivou-se comparar a eficiência das células mononucleares de medula óssea (BMMCs) e das células-tronco mesenquimais (MSCs) do tecido adiposo de Chlorocebus aetiops na lesão óssea induzida. Foram utilizados 10 animais, adultos do sexo masculino, submetidos à lesão óssea nas cristas ilíacas. As MSCs foram isoladas e cultivadas; de forma autóloga, as BMMCs foram infundidas na crista ilíaca direita e as MSCs de tecido adiposo na crista ilíaca esquerda. Após 4,8 meses, a crista ilíaca direita foi totalmente reconstruída, enquanto a crista ilíaca esquerda continuou apresentando defeito ósseo evidente por até 5,8 meses após a infusão. A melhor opção para o tratamento de lesões com perda de tecido ósseo em primatas do Velho Mundo é a utilização de MSCs autólogas de tecido adiposo, sugerindo que se podem estender os resultados para seres humanos, uma vez que há proximidade filogenética entre as espécies.(AU)

Effects of partial replacement of soybean meal with other protein sources in diets of lactating cows.

The protein nutrition of dairy cows is of great importance because of its direct influence on milk production, reproductive efficiency, and feeding cost. Eight first-lactation Holstein cows were randomly assigned to two contemporary 4 × 4 Latin squares in a 2 × 2 factorial design to evaluate the effects of replacing soybean meal with yeast-derived microbial protein (YMP) as a protein source (0% or 1.5% of dry matter (DM)) and its combination with slow-release urea (SRU; 0% or 0.75% of DM) on DM intake and milk production and composition, as well as blood parameters and nitrogen balance. Each experimental period lasted 28 days, with 21 days of adaptation and 7 days of data collection. The diets were formulated to attend the nutritional recommendations of the National Research Council and consisted of 49% forage (47% corn silage and 2% Tifton hay) and 51% concentrate, with 16.8% CP and 1.6 Mcal net energy for lactation/kg DM. For diets without YMP, the inclusion of SRU decreased DM intake, milk production as well as N intake and balance, but did not affect efficiency of production, milk composition or most of blood parameters. On the contrary, for diets with YMP, DM intake and milk production were increased by inclusion of SRU, while minor effects were observed for milk efficiency and composition, blood parameters as well as N intake, excretion and balance. When diets with SRU were compared, the inclusion of YMP increased DM intake, 4% fat-corrected milk, and N intake and balance (P<0.05), with no differences in milk production (kg/day), milk energy, efficiency of milk production or most of the blood parameters. For diets without SRU, YMP inclusion decreased DM intake, milk production, milk energy, N intake, fecal N and N balance (P<0.05), with no effects on milk efficiency and composition, or most of blood parameters. In conclusion, the use of YMP, SRU or both as partial substitutes of soybean meal in the diet of lactating cows has no negative effects on productivity parameters.

The effects of Trolox on the quality of sperm from captive squirrel monkey during liquefaction in the extender ACP-118™.

SummaryThe aim of this study was to evaluate the effect of incubating semen for different periods (90, 270 or 450 min) with or without Trolox® (100 or 150 µM) on the quality of sperm from Saimiri collinsi. Sperm motility, vigour, and plasma membrane integrity (PMI) were evaluated in both fresh semen and semen incubated for different time periods, i.e. 90, 270 or 450 min of incubation. Supplementation of semen extender with Trolox® 100 µM improved sperm motility, vigour and PMI for up to 270 min of incubation.

Imunidade celular em vacas Holandesas soropositivas para o Vírus da Leucose Bovina (BLV) durante o período de transição / Cellular immunity in Holstein cows seropositive for Bovine Leukosis Virus (BLV) during the transition period

Esta pesquisa avaliou a dinâmica dos leucócitos e das subpopulações de linfócitos em vacas Holandesas soropositivas para o BLV no período de transição. Amostras de sangue (n=72) provenientes de 12 vacas foram coletadas entre as semanas -2 e +3 para a realização do leucograma, imunofenotipagem, dosagem de cortisol e haptoglobina (Hp). O perfil leucocitário foi caracterizado por leucocitose, neutrofilia, monocitose e eosinopenia próximo ao parto. Linfocitose e elevada proporção de linfócitos B CD21+ foram achados constantes entre as semanas -2 e +3; assim, as vacas foram testadas e confirmadas soropositivas para o BLV. Os valores das subpopulações de linfócitos T apresentaram-se baixos durante o período de transição, observando-se dois picos máximos que coincidiram com as elevações nas concentrações de cortisol no parto (2,11µg/dL) e semana +3 (1,97µg/dL). Hp apresentou aumento crescente de -2 (166µg/mL) a +3 (576µg/mL), provavelmente associada à elevada taxa de infecções uterinas observadas nas semanas +2 e +3. As vacas soropositivas para o BLV apresentaram leucograma de estresse próximo ao parto, exceto para linfócitos. A linfocitose e as elevadas proporções de células B CD21+, associadas com as baixas proporções de células T, podem ser indicativo de imunossupressão e predisposição aos processos inflamatórios no período pós-parto.(AU)

This research evaluated the dynamics of leukocytes and lymphocytes subsets in seropositive Holstein cows for BLV during the transition period. Blood samples (n=72) from 12 cows were harvested from week -2 up to week +3 to perform leukogram, immunophenotyping, cortisol and haptoglobin (Hp). Leukocytes pattern was characterized by leukocytosis, neutrophilia, monocytosis and eosinopenia around calving. Lymphocytosis and high proportions of B cells CD21+ were a constant finding between week -2 and +3, thus cows were tested and confirmed seropositive for BLV. The values of T lymphocytes subsets were low during the transition period, observing two peaks that coincided with high levels of cortisol at delivery (2.11µg/dL) and week +3 (1.97µg/dL). Hp had gradual increase from week -2 (166µg/mL) until week +3 (576g/mL) probably due to high rate of uterine infection detected between week +2 and +3. The seropositive cows for BLV presented stress leukogram around delivery, except for lymphocytes. Lymphocytosis and the high proportions of B cells, associated with the low proportions of T lymphocytes, can be indicative of immunosuppression and predisposition to the inflammatory process observed in the post-partum period.(AU)

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation.

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.

Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

Description of bovine major histocompatibility complex class IIa haplotypes using parthenogenetic embryo-derived cells.

In this study, we report an approach to characterize individual BoLA haplotypes using cells from parthenogenetic bovine embryos derived from slaughterhouse ovaries. Eight of the 15 parthenogenetic embryos so obtained had not undergone meiotic recombination on the BoLA region and were suitable to describe BoLA haplotypes. Detailed analysis of the BoLA class IIa region identified seven different class IIa haplotypes, including six not previously described and two new alleles of BoLA-DQA and one BoLA-DQB. Our method provided reliable sources of homozygous DNA to describe BoLA haplotypes.

Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella).

Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.

Maturação in vitro de oócitos bovinos em meios suplementados com quercetina e seu efeito sobre o desenvolvimento embrionário / In vitro maturation of bovine oocytes in medium supplemented with quercetin, and its effect on embryonic development

A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF2HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P<0,05) nos grupos tratados com 0,4, 2, 10 e 50∝M de quercetina (56,9, 59,5, 53,6 e 49,6%, respectivamente) e com 100∝M de cisteamina (50,4%) em relação ao grupo controle (42,3%). Na comparação entre os dois antioxidantes, a quercetina (0,4 e 2µM) foi superior na produção de embriões (56,9 e 59,5%, respectivamente) em comparação com cisteamina (50,4%). As taxas de embriões eclodidos foram similares (P>0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina.

Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF2HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P<0.05) in the groups treated with 0.4, 2, 10 and 50∝M of quercetin (56.9, 59.5, 53.6 and 49.6%, respectively) and 100 ∝M cysteamine (50.4%) compared to the control group (42.3%). Comparing the two antioxidants, quercetin (0.4 to 2µM) was superior in embryo production (56.9 and 59.5% respectively) compared with cysteamine (50.4%). The rates of hatched embryos were similar (P>0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine.

Bovine papillomavirus in Brazil: detection of coinfection of unusual types by a PCR-RFLP method.

Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.

Effect of triiodothyronine on developmental competence of bovine oocytes.

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRß, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development.

Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.

Short-term culture of ovarian cortical strips from capuchin monkeys (Sapajus apella): a morphological, viability, and molecular study of preantral follicular development in vitro.

The aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not ß-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys' preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys.

Characterization of folliculogenesis and the occurrence of apoptosis in the development of the bovine fetal ovary.

The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 µm) to the secondary follicular stages (38.0 ± 14.9 µm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life.

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella).

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Evaluation of apoptosis as a mechanism of follicular cell atresia in the ovaries of cattle (bos indicus) and buffalo (bubalus bubalis) fetuses

The objective of this study was to investigate the occurrence of apoptosis in the ovaries of cattle and buffalo fetuses between 4 and 8 months old by the terminal deoxynucleotidyltransferase - mediated dUTP nick end labeling (TUNEL) assay . Histological analysis of the ovarian t issue showed that apoptosis occurred at all ages evaluated , presenting a similar pattern among different fetal stages in both species . Within species, secondary follicles displayed a higher (P 0.05) of apoptotic follicular cells among the three follicular classes compared . C omparing resul ts between species, secondary follicles had a higher (P < 0.05) mean number of TUN EL positive cells in bovine fetuses; however , this difference was proportional to the larger number of follicular cells present in secondary follicles in this species . In summary, the TUNEL method was effective for the detection of apoptosis in the support ing cells of ovarian follicles from bovine and buffalo fetuses with apoptosis occurring at similar rates in both species between 4 and 8 months of gestational age. Further studies are needed to better understand the dynamics of apoptosis as a regulator of follicular atresia in fetal ovaries from these species, as well as the potential involvement of the oocyte in this process.