Cytotoxic screening of plants from the Brazilian Atlantic Forest has led to the identification of Casearia arborea and Sorocea hilarii as sources of antitumor compounds.

In the present study, we have evaluated the cytotoxic activity of 282 extracts from 72 native plant species of the Brazilian Atlantic Forest biome. As a result, Casearia arborea and Sorocea hilarii leaves extracts showed cytotoxic activity against three tumour cell lines tested (B16F10, SW480 and Jurkat). After bioassay-guided fractionation, the bioactive fractions were submitted to the dereplication study via High-performance Liquid Chromatography, connected to High-resolution Mass Spectrometry (HPLC-ESI-QTOF/MS) analysis, combined with a Global Natural Products Social Molecular Networking (GNPS) tool. A combination of bioactivity-guided and dereplication approaches resulted in the putative annotation of 27 clerodane diterpenes and 9 flavonoids as main compounds present in the cytotoxic fractions of C. arborea. Regarding the active fraction of S. hilarii, 10 megastigmans, 17 spirostane steroids derivatives and 2 lignans were putatively identified. In conclusion, Casearia arborea and Sorocea hilarii are potential sources of antitumor compounds.

The effects of increasing amounts of milk replacer powder added to whole milk on mammary gland measurements using ultrasound in dairy heifers.

The aim of this study was to determine the effect of increasing total solids (TS) in the liquid diet during the preweaning phase on mammary gland development in dairy heifers. The increase in TS was obtained with the addition of milk replacer powder to whole milk. Crossbreed Holstein-Gyr heifers (n = 60) were distributed in 4 treatments with different TS concentrations: 13.5% (n = 15), 16.1% (n = 15), 18.2% (n = 15), and 20.4% (n = 15). The liquid diets were provided from 5 to 55 d of age. From 56 to 59 d of age, the total amount of liquid feed was reduced by half. Heifers were weaned at 60 d and monitored until 90 d of age. Ultrasound mammary gland evaluations were performed weekly between 5 to 11 wk of age, using a B-mode ultrasound equipped with microconvex transducer at a frequency of 6 MHz. In those same weeks, the manual palpation of mammary parenchyma (PAR) was performed. Increased TS concentration of liquid diet during the preweaning period increased body weight of heifers, but did not alter PAR growth and the deposition of adipose tissue in the mammary gland evaluated by ultrasonography. The oval-shaped structure of PAR was altered after 2 mo of age. In the evaluated period, PAR growth was isometric with respect to the body growth rate. Palpation scoring of PAR had a strong correlation with the ultrasound evaluation of the PAR.

Teofilina como agente capacitante do sêmen bovino / Theophylline as a capacitating agent of bovine semen

Objetivou-se avaliar a teofilina como agente capacitante substituto ou associado à heparina sobre a reação acrossômica dos espermatozoides e o desenvolvimento de embriões produzidos in vitro. O experimento foi realizado com quatro touros e três tratamentos, totalizando 12 grupos experimentais. O sêmen dos touros foi avaliado nos tratamentos descritos a seguir: tratamento 1 (HEP): heparina - 10µg/mL; tratamento 2 (TEO): teofilina - 5mM; tratamento 3 (HEP + TEO): heparina (10µg/mL) + teofilina (5mM), por zero, seis, 12 e 18 horas, corados com trypan blue/Giemsa para avaliação da reação acrossômica. Para a produção dos embriões, os agentes capacitantes foram adicionados aos meios de fertilização. Na análise espermática, a taxa de reação acrossômica verdadeira foi maior (P<0,05) no tempo zero hora, enquanto para espermatozoides mortos, as maiores taxas (P<0,05) foram nos tempos de 12h (84,46±5,82) e 18h (86,75±4,19). A taxa de embriões produzidos (37,97±13) e a taxa de eclosão (33,50±14) foram maiores (P<0,05) para o tratamento HEP. Não foi observada diferença (P>0,05) entre touros na análise de reação acrossômica nem na PIVE. A utilização da teofilina foi tão eficiente quanto a da heparina na indução da reação acrossômica, no entanto resultou em menores taxas de produção embrionária.(AU)

The sperm capacitating process should take special attention during in vitro embryo production (IVEP) once that affects the success of embryo production. The study aimed to evaluate theophylline as substitute capacitating agent or in combination with heparin on the sperm acrosome reaction and development of embryos produced in vitro. The experiment was carried out using 4 bulls and 3 treatments, establishing 12 experimental groups. Each bull was evaluated in the following treatments: Treatment 1 (HEP): Heparin - 10mg/mL; Treatment 2 (THEO): Theophylline - 5mM; Treatment 3 (HEP + THEO), Heparin (10mg/mL) + Theophylline (5mM). The semen of bulls was incubated in each treatment for 0, 6, 12 and 18h, stained with Trypan blue / Giemsa and analyzed by electron microscopy for assessment of acrosome reaction. Using sperm of same bulls, capacitating agents were added to the fertilization media, for IVEP. In sperm analysis, the true acrosome reaction rate was higher (P<0.05) in time 0h, while sperm dead rates were highest (P<0.05) at 12h (84.46±5, 82), and 18h (86.75±4.19). The produced embryos rate (37.97±13) and hatching rate (33.50±14) were larger (P<0.05) for HEP treatment. There was no difference (P>0.05) between bulls in acrosome reaction analysis neither for IVEP. The use of theophylline was as effective as heparin in the induction of the acrosome reaction, although it resulted in lower embryo production rates.(AU)

Effect of increasing amounts of milk replacer powder added to whole milk on postweaning performance, reproduction, glucose metabolism, and mammary fat pad in dairy heifers.

The aim of this study was to evaluate the effects of increasing the total solids (TS) content of liquid feed, by adding increasing amounts of milk replacer powder to whole milk, on age at puberty and postweaning performance, glucose metabolism, and mammary fat pad of dairy heifers. Crossbreed Holstein × Gyr heifers (n = 58) were distributed into 4 different treatments during the preweaning period. Treatments consisted of liquid feeds with TS content of 12.5 (actual TS = 13.5 ± 0.53%; n = 15), 15.0 (actual TS = 16.1 ± 0.03%; n = 15), 17.5 (actual TS = 18.2 ± 0.14%; n = 13), or 20.0% (actual TS = 20.4 ± 0.24%; n = 15), which were provided up to 59 d old. From 60 to 89 d old, the animals received the same starter offered during the preweaning period in addition to corn silage ad libitum starting at 70 d old. From 90 to 104 d old, the animals were adapted to a total mixed ration. At 105 d old, the animals were distributed in 4 paddocks equipped with electronic feed and water bins and were fed the same total mixed ration ad libitum. A period of adaptation to the electronic feed bins occurred from 105 to 119 d old. Feed and water intake and body weight and body frame development were assessed until puberty. Mammary gland evaluations were performed monthly by ultrasonography from 120 d of age until puberty onset. Puberty onset was determined as plasma progesterone concentration greater than 1 ng/mL in 2 consecutive samples collected 7 d apart. The date of puberty onset was recorded as the collection day of the first of these samples. A glucose tolerance test was performed at 280 d of age. The increased TS content of the liquid feed fed during the preweaning period did not affect dry matter intake, performance, age at puberty, glucose metabolism, or mammary gland fat pad at later stages of rearing process. Conversely, hip width and heart girth increased linearly up to 150 and 240 d of age, respectively, as a result of the increased TS concentration of the liquid feed during the preweaning period.

Intestinal development of bovine foetuses during gestation is affected by foetal sex and maternal nutrition.

We aimed to evaluate the effects of maternal nutrition (MN) and foetal sex on the intestinal development of bovine foetuses throughout different days of gestation (DG). Forty-four multiparous, dry Holstein × Gyr cows with average initial body weight of 480 ± 10 kg were fed the same diet of either restricted feeding at 1.15% of body weight (CO, n = 24) or fed ad libitum (overnourished, ON, n = 20). Six cows from CO group and five cows from ON group were slaughtered at 139, 199, 241 and 268 DG, and foetuses were necropsied to evaluate the intestinal development. The mass, length and density of foetal intestines were not affected by MN (p ≥ 0.260). An interaction between MN and DG was observed for the villi length of jejunum (p = 0.006) and ileum (p < 0.001). Villi length of jejunum and ileum was higher (p < 0.10) in foetuses from ON-fed cows than in foetuses from CO-fed cows at 139 DG. However, at 199 DG, the villi length of jejunum and ileum of foetuses from CO-fed cows was higher than in foetuses from ON-fed cows. Despite these differences, MN did not affect the villi length of jejunum and ileum at 268 DG (p > 0.10). Female foetuses had greater small intestine mass (p = 0.093), large intestine mass (p = 0.022), small intestine mass in proportion to body mass (p = 0.017) and large intestine mass in proportion to body mass (p < 0.001) than male foetuses. Female foetuses had also longer small intestine (p = 0.077) and greater small intestine density (p = 0.021) and villi length of jejunum (p = 0.001) and ileum (p = 0.010) than males. We conclude that MN affects the pathway for the development of foetal villi length throughout the gestation in bovine foetuses without changing the final villi length. Female foetuses had higher intestinal mass, density and villi length than males during the foetal phase in bovines.

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.

Effects of day of gestation and feeding regimen in Holstein × Gyr cows: III. Placental adaptations and placentome gene expression.

This study investigated the influence of day of gestation (DG) and feeding regimens (FR) on the expression of genes responsible for placenta development, nutrient transfer, and angiogenic factors in Holstein × Gyr cows. Forty pregnant multiparous Holstein × Gyr cows with an average initial body weight of 482±10.8kg and an initial age of 5±0.8 yr were allocated to 1 of 2 FR: ad libitum (AL; n=20) or maintenance level (ML; n=20). Maintenance level was considered to be 1.15% of body weight (dry matter basis) and met 100% of the net energy requirements and AL provided 190% of the total net energy requirements. Cows were slaughtered at 4 DG: 139, 199, 241, and 268d. After the cows were slaughtered, the placenta and uterus were separated and weighed. Caruncles and cotyledons were individually separated, counted, and weighed. Placenta expressed as kilograms and grams per kilogram of empty body weight (EBW) was heavier in ML- than in AL-fed cows at 268d of gestation. Placenta expressed as kilograms and grams per kilogram of EBW was the lightest at 139d of gestation, and the greatest mass was observed at 268d in ML-fed cows. However, in AL-fed cows, the heaviest placenta expressed as grams per kilogram of EBW was observed from 199d of gestation. Placentomes expressed as grams per kilogram of EBW were heavier in ML-fed cows during gestation, and the number of placentomes was greater in ML-fed cows at 268d of gestation. We observed that IGFR1 and IGFR2 were involved in placenta adaptations when ML was provided, as their expression in placentome cells was greater in ML-fed cows at 268d of gestation. The genes responsible for angiogenesis were also greater in ML-fed cows: VEGFA, GUCY1B3, HIFA, FGF2, and NOS3 were altered by FR and DG interaction and they were greater in ML-fed cows at 268d of gestation. In addition, VEGFB and ANGPT2 did not show interactions between FR and DG, but they were greater in ML-fed cows. Thus, we suggest that the placenta from an ML-fed cow develops adaptations to the reduced nutrient supply by altering its structure and gene expression, thereby developing mechanisms for potential increased nutrient transfer efficiency to the fetus.

Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos.

The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p < 0.05). The number of dead cells in vitrified embryos of Gr. 2 and Gr. 3 was higher (42.6 ± 26.2 and 63.2 ± 34.65, respectively) in comparison with Gr. 1 (conventional freezing, 10.1 ± 8.5, p < 0.05). Embryos vitrified with dimethylformamide included the same quality of apoptotic cells that Gr. 1 (conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification.

Efeito da base genética materna e da estação de parição sobre variáveis produtivas de fêmeas primíparas Holandês x Zebu / Effect of the maternal genetic basis and of the calving season on productive variables of primiparous Holstein x Zebu cows

Avaliaram-se variáveis produtivas de 78 fêmeas primíparas mestiças de base genética materna Gir ou Guzerá, paridas na estação seca ou chuvosa, utilizando-se o procedimento GLM do SAS. A estação de parição interferiu (P<0,05) no peso ao parto (PP), no escore da condição corporal ao parto (ECCP), no ganho de peso durante a gestação, no período de serviço (PS) e no intervalo de partos (IDP). O PP e ECCP foram maiores (P<0,05) nas primíparas que pariram na estação seca em relação às que pariram na estação chuvosa. Os valores foram de 475,19±39,81kg, 4,07±0,44 e 420,67±37,80kg e 3,62±0,37, respectivamente, para a estação seca e chuvosa. O PS e o IDP foram menores (P<0,05) entre as vacas que pariram na estação seca, 132,02±91,94 dias e 13,90±3,06 meses, em relação às paridas na estação chuvosa, 190,07±77,27 dias e 15,84±2,58 meses. As fêmeas de base genética Gir apresentaram melhor desempenho (P<0,05) que as de base genética Guzerá para as características produção de leite, duração da lactação e produção de leite por dia de intervalo de partos, cujos valores, nessa ordem, foram de 2276,15±656,47kg, 306,08±46,55 dias, 5,06±1,38kg/dia para as fêmeas de base Gir e de 1733,74±678,41kg, 265,62±67,11 dias, 4,37±1,68kg/dia para as de base Guzerá.

Productive variables of 78 crossbred Holstein x Zebu primiparous cows of Gir or Guzera maternal genetic basis, calved in the dry or rainy season were evaluated using the GLM procedure of the SAS. The calving season affected the weight at calving (WC), body condition score at calving (BCS), service period (SP) and calving interval (CI). The WC and BCS were higher (P<0.05) por cows calved in the dry season, 475.19±39.81kg and 4.07±0.44, than in the rainy season, 420.67±37.80kg and 3.62±0.37, respectively. The SP and CI were shorter (P<0.05) for cows calved in the dry season, 132.02±91.94 days and 13.90±3.06 months, than cows calved in the rainy season, 190.07±77.27 days e 15.84±2.58 months, respectively. The Gir based cows had higher milk yield, longer lactation period and higher milk yield per day of calving interval than the Guzera based cows, respectively, 2276.15±656.47kg, 306.08±46.55 days, 5.06±1.38kg/day for the Gir and 1733.74±678.41kg, 265.62±67.11 days, 4.37±1.68kg/day for the Guzera based cows.

Influência da presença do bezerro no momento da ordenha sobre o desempenho reprodutivo de vacas mestiças Holandês-Zebu / Effect of calf presence during milking on reproductive performance of Holstein x Zebu crossbred cows

Avaliou-se a influência da presença do bezerro no momento da ordenha sobre os parâmetros reprodutivos de 75 vacas primíparas e multíparas mestiças Holandês-Zebu, divididas em três grupos experimentais: GI - vacas ordenhadas sem a presença do bezerro; GII - vacas ordenhadas com a presença momentânea dos bezerros, sendo estes retirados da sala de ordenha logo após a descida do leite; e GIII - vacas ordenhadas com a presença constante dos bezerros, sendo estes amarrados ao lado da vaca durante toda a ordenha. O escore médio da condição corporal ao parto foi de 3,1. O período de serviço, dias para o retorno ao cio e as taxas de manifestação de cio e gestação até os 120 dias pós-parto não foram influenciados (P>0,05) pelo tipo de manejo de ordenha.

This study was carried out to evaluate the effect of calf presence in the milking parlour on the reproductive performance of seventy five crossbred (Holstein Î Zebu) cows randomly allocated into three groups: group I - cows milked without the calf presence; group II - cows mantained with their calves during short period of time before milking to promote milk ejection and thereafter the calves were put out the milking room until the cows had finished milking; and group III - cows mantained with the calf during milking. The average body condition score at calving was 3.1. The service period, number of days to return to estrous and estrous and pregnant rates until 120 day postpartum were not influenced (P>0.05) by milking management of cows. The calf presence in the milking parlour did not influence the reproductive performance of Holandês-Zebu crossbred cows.

Indução do estro no pós-parto em vacas primíparas Holandês-Zebu / Induction of estrus in the postpartum of Holstein-Zebu heifers through norgestomet

Avaliou-se o efeito do peso corporal no início do tratamento com progestágeno sobre as características reprodutivas de vacas mestiças Holandês-Zebu no pós-parto. Foram utilizadas 64 vacas, divididas em quatro grupos: GI - vacas com peso corporal entre 390-458kg e submetidas a tratamento hormonal com norgestomet, GII - vacas com peso corporal entre 464-562kg e submetidas a tratamento hormonal com norgestomet, GIII - vacas com peso corporal entre 374-451kg (controle) e GIV - vacas com peso corporal entre 452-545kg (controle). Os animais do grupo II manifestaram o primeiro estro no pós-parto mais cedo que os demais (64,4 dias - GII vs. 109,4-GI; 143,2-GIII e 105,1-GIV dias), e apresentaram menor período de serviço (94,6 dias vs. 125,5; 160,9 e 131,0 dias, na mesma ordem de citação anterior). Quanto às taxas de manifestação de estro e de gestação final, não se verificaram diferenças (P>0,05) entre os tratamentos. Os animais do GII apresentaram o menor período de serviço e os do GIII, o maior (94,6 vs. 160,9). Não houve influência do tratamento hormonal nem do peso corporal sobre a produção de leite e duração da lactação. O uso do implante de progestágeno nos animais que apresentaram maiores peso e condição corporal no início do tratamento respondeu por menor intervalo entre o parto e o primeiro estro. O uso do progestágeno em animais mais leves esteve associado ao retorno mais rápido à atividade ovariana cíclica no pós-parto.

Parameters determining the quality of charqui, an intermediate moisture meat product.

Charqui is a typical Brazilian meat product obtained by salting and sun-drying beef samples. The chemical, physical and microbiological characteristics of the charqui were evaluated throughout processing and storage. The results confirm charqui is an intermediate moisture meat product (A(w) = 0·70-0·75). A close relationship between moisture, pretein and ash vaiues was found, suggesting the possibility of using the resulting charqui A(w) value as a parameter to define the product instead of the official moisture and mineral residue contents. The TBA determination, which expresses the state of lipid oxidation, rapidly reached the maximum value, corroborating the previous observations on the salt pro-oxidant role, and then decreased gradually. A gradual decrease in microorganism count during processing and storage of charqui was also observed. These results indicate the feasibility of obtaining a final product with a low level of microbial count when raw materials of good quality, and adequate handling conditions, are used for charqui production.