Canine sperm vitrification with nonpermeable cryoprotectants and coconut water extender

This study was aimed to assess the efficiency of coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants for canine semen vitrification, using a simple method that yields a high survival rate of spermatozoa for clinical use. Twelve ejaculates from 12 adult normozoospermic dogs were collected separately by digital manipulation and only the second semen fraction was used in this study. After evaluation of volume, concentration, viability, total and progressive motility, velocity parameters and morphology, semen was diluted with a coconut water extender (50% (v/v(volume per volume)) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% and 0.25M sucrose until final concentration of 100x106 spermatozoa/ml. After equilibration at 5ºC for 60 minutes, semen was vitrified by "direct dropping method" into liquid nitrogen in spheres with a volume of 30 µl. After a week of storage the spheres were devitrified as three of them were dropped into 0.5 mL of CaniPlus AI medium (Minitüb, Germany), which was previously warmed in a water bath at 42ºC for 2 minutes and evaluated about the above mentioned parameters. It was found that vitrification resulted in a lower percentage of viable sperms, normal morphology, total and progressive motilities (p0.05) compared to fresh semen samples. In conclusion, our results demonstrate that vitrification with coconut water extender with addition of 1% soy lecithin and 0.25M sucrose as cryoprotectants, has an excellent potential for routine canine sperm cryopreservation.(AU)

Indigenous ecological calendars and seasonal vector-borne diseases in the Colombian Amazon: an intercultural and interdisciplinary approach / Calendarios ecológicos indígenas y enfermedades estacionales transmitidas por vectores en la Amazonia colombiana: un enfoque intercultural e interdisciplinario

Traditional ecological knowledge of indigenous groups in the southeastern Colombian Amazon coincides in identifying the two main hydrological transition periods (wet-dry: August-November; dry-wet: March-April) as those with greater susceptibility to disease in humans. Here we analyze the association between indigenous knowledge about these two periods and the incidence of two vector-borne diseases: malaria and dengue. We researched seven "ecological calendars" from three regions in the Colombian Amazon, malaria and dengue cases reported from 2007 to 2019 by the Colombian National Institute of Health, and daily temperature and precipitation data from eight meteorological stations in the region from 1990-2019 (a climatological normal). Malaria and dengue follow a seasonal pattern: malaria has a peak from August to November, corresponding with the wet-dry transition (the "season of the worms" in the indigenous calendars), and dengue has a peak in March and April, coinciding with the dry-wet transition. Previous studies have shown a positive correlation between rainfall and dengue and a negative correlation between rainfall and malaria. However, as the indigenous ecological knowledge codified in the calendars suggests, disease prediction cannot be reduced to a linear correlation with a single environmental variable. Our data show that two major aspects of the indigenous calendars (the time of friaje as a critical marker of the year and the hydrological transition periods as periods of greater susceptibility to diseases) are supported by meteorological data and by the available information about the incidence of malaria and dengue.(AU)

Los conocimientos ecológicos tradicionales de grupos indígenas del sureste de la Amazonia colombiana coinciden en identificar dos principales periodos de transición hidrológica (seco-húmedo: agosto-noviembre; húmedo-seco: marzo-abril) como los de mayor susceptibilidad a enfermedades en humanos. Aquí analizamos la asociación entre el conocimiento indígena sobre estos dos periodos y la incidencia de dos enfermedades transmitidas por vectores: malaria y dengue. Investigamos siete calendarios ecológicos de tres regiones en la Amazonia colombiana, casos de dengue y malaria reportados de 2007 hasta 2019 por el Instituto Nacional de Salud de Colombia y datos diarios de temperatura y precipitación de ocho estaciones meteorológicas en la región, de 1990 a 2019 (una normal climatológica). Malaria y dengue siguen un patrón estacional, la malaria tiene un pico de agosto a noviembre, correspondiendo con la transición húmedo-seco (el "tiempo de gusano" según los calendarios indígenas), mientras que dengue tiene un pico de marzo a abril, coincidiendo con la transición seco-húmedo. Estudios previos mostraron una correlación positiva entre precipitación y dengue, y una correlación negativa entre precipitación y malaria. Sin embargo, como lo sugiere el conocimiento ecológico codificado en los calendarios indígenas, la predicción de enfermedades no puede reducirse a una correlación lineal con una sola variable medioambiental. Nuestros datos muestran que dos aspectos principales de los calendarios indígenas (el tiempo de friaje como un marcador crítico anual y los periodos de transición hidrológica como épocas de mayor susceptibilidad a enfermedades) están soportados por datos meteorológicos e información disponible acerca de la incidencia de malaria y dengue.(AU)

Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.(AU)

In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.(AU)

Diagnóstico precoce de acetonemia subclínica em vacas holandesas por mensuração de corpos cetônicos / Early diagnosis of subclinical acetonemia in holstein cows by measuring ketone bodies / Diagnóstico precoz de acetonemia subclínica en vacas holstein mediante la medición de cuerpos cetónicos

O trabalho foi realizado em uma fazenda de exploração leiteira em Castrolanda, no município de Castro ­ PR. O sistema de manejo é free-stall, com 220 vacas da raça Holandesa com RHA305 de 6.740 litros. Foram coletadas amostras de sangue de 18 vacas de pré-parto, 4 dias antes da data prevista para o parto; e nos dias 4, 7 e 12 pós-parto, mediante punção venosa coccígea, utilizando-se SnapTest digital Ketovet®, constituindo 72 amostras, no período de fevereiro a maio de 2020. Em 4 vacas o BHB do sangue total apresentou-se acima do limite para cetose subclínica no quarto dia após o parto e 17 apresentaram-se acima do limite no sétimo dia, declinando em seguida, principalmente devido às intervenções clínicas. As médias e desvios-padrão foram: D - 4: 0,89 ± 0,257061; D 4: 1,05 ± 0,283279; D 7: 1,81 ± 0,456131; e D 12: 1,19 ± 0,437762. O benefício do monitoramento de BHB foi a imediata intervenção clínica, evitando-se a severidade por instalação de quadro clínico e as enfermidades concomitantes.(AU)

The study was carried out on a dairy farm in Castrolanda, in the county of Castro - PR. The management system is free stall, with 220 Holstein cows with RHA305 of 6,740 liters. Blood samples were collected from 18 pre-calving cows, 4 days before the expected date of calving; and on days 4, 7 and 12 postpartum, by means of coccygeal venipuncture, using SnapTest digital Ketovet®, constituting 72 samples, from February to May 2020. In 4 cows the BHB of whole blood was shown above the limit for subclinical ketosis on the fourth day after delivery and 17 presented above the limit on the seventh day, then declining mainly due to clinical interventions. The means and standard deviations were D -4: 0.89 ± 0.257061; D 4: 1.05 ± 0.283279; D 7: 1.81 ± 0.456131; and D 12: 1.19 ± 0.437762. The benefit of monitoring BHB was immediate clinical intervention, avoiding clinical ketosis and concomitant illnesses.(AU)

El estudio se realizó en una explotación lechera de Castrolanda, en la comarca de Castro - PR. El sistema de manejo es estabulación libre, con 220 vacas Holstein con RHA305 de 6.740 litros. Se recogieron muestras de sangre de 18 vacas pre- parto, 4 días antes de la fecha prevista de parto; y en los días 4, 7 y 12 postparto, mediante venopunción coccígea, utilizando SnapTest digital Ketovet®, constituyendo 72 muestras, desde febrero a mayo de 2020. En 4 vacas la BHB de sangre total se mostró por encima del límite para cetosis subclínica en el cuarto día después del parto y 17 presentaron por encima del límite en el séptimo día, disminuyendo después debido principalmente a intervenciones clínicas. Las medias y desviaciones estándar fueron D -4: 0,89 ± 0,257061; D 4: 1,05 ± 0,283279; D 7: 1,81 ± 0,456131; y D 12: 1,19 ± 0,437762. El beneficio de monitorizar la BHB fue la intervención clínica inmediata, evitando la cetosis clínica y las enfermedades concomitantes.(AU)

Efeito residual de estratégias de adubação sobre características produtivas do capim mombaça no período seco / Residual effect of fertilization strategies on productive characteristics ofmombaça grass in the dry season / Efecto residual de las estrategias de fertilización sobre las características productivas de la hierba mombaça en el período seco

Objetivou-se avaliar o efeito residual de diferentes recomendações de adubação e momentos deaplicação de adubação nitrogenada após a desfolhação sobre as características morfogênicas, estruturais e produtivas do Megathyrsus maximuscv. Mombaça no período seco do ano. Utilizou-se o delineamento em blocos casualizados em esquema fatorial 4 x 3, com 4 repetições. Os tratamentos foram constituídos pelas recomendações de adubação: Manual 5ª aproximação (5AP), Manual do Pará (MP), Reposição de nutrientes (RP) e modelo adaptado de Michaelis e Menten (MM) em três momentos de aplicação da fonte de nitrogênio (N) após desfolhação (dia 0, 3 e 6). As adubações com N foram realizadas no período das águas e no período de transição. A estratégia 5AP com N no dia 0 apresentou os maiores valores para massa seca de lâmina foliar (MSLF) e número de folhas vivas e osmenores para taxa de senescência foliar e massa seca de material morto. Já a recomendação MP com N no dia 6 apresentou os maiores valores para MSLF. A adubação nitrogenada no final do período das águas, promove aumento do comprimento médio de lâmina foliar e do número de folhas vivas além de promover redução de material morto da forragem no período seco.(AU)

The objective was to evaluate the residual effect of different fertilization recommendations and nitrogen application times after defoliation on the morphogenic, structural and productive characteristics of Megathyrsus maximuscv. Mombaça in the dry period of the year. A randomized block design was used in a 4 x 3 factorial scheme, with 4 replications. The treatments were constituted by the fertilization recommendations: 5thApproach Guide(5AP), Pará Guide(PG), Nutrient Replacement(RP) and theMichaelis and Menten adapted model(MM) applied for three days after application of the nitrogen (N) source following defoliation (days 0, 3, and 6). Fertilizations with N were carried out in the rainy season and in the transition period. The 5AP strategy with N on day 0 showed the highest values for leaf blade dry mass (LBDM) and number of live leaves and the lowest values for leaf senescence rate and dead material dry mass. The PG recommendation with N on day 6 presented the higher values for LBDM. Nitrogen fertilization at the end of the wet season promotes an increase in the average length of leaf blade and in the number of live leaves, in addition to promoting a reduction in forage dead material in the dry season.(AU)

El objetivo fue evaluar el efecto residual de diferentes recomendaciones de fertilización y tiempos de aplicación de la fertilización nitrogenada después de la defoliación sobre las características morfogénicas, estructurales y productivas de Megathyrsus maximus cv. Mombasa en el período seco del año. Se utilizó un diseño de bloques al azar en un esquema factorial 4 x 3, con 4 repeticiones. Los tratamientos estuvieron constituidos por las recomendaciones de fertilización: Manual 5º aproximación (5AP), Manual do Pará (MP), Reposición de nutrientes (RP) y modelo adaptado de Michaelis y Menten (MM) en tres momentos de aplicación de la fuente de nitrógeno (N) después de la defoliación (día 0, 3 y 6). Las fertilizaciones con N se realizaron en la época de lluvias y en el período de transición. Laestrategia 5AP con N en el día 0 mostró los valores más altos de masa seca del limbo foliar (MSLF) y número de hojas vivas y los valores más bajos de tasa de senescencia foliar y masa seca de material muerto. La recomendación MP con N en el día 6 presentóvalores superiores para MSLF. La fertilización nitrogenada al final de la estación lluviosa promueve un aumento en la longitud promedio de la lámina foliar y el número de hojas vivas, además de promover una reducción en la materia muerta del forraje en laestación seca.(AU)

Rural depopulation on agricultural technological choice in China / Efeito do despovoamento rural na escolha tecnológica agrícola na China

Rural depopulation is an inevitable trend due to urbanization and industrial development, and it is a common phenomenon in industrialized countries. The classic theory of induced technological innovation proposed by HAYAMI & RUTTAN (1971) explains the change in agricultural technology during the process of rural depopulation. This paper tests the Hayami-Ruttan induced technological innovation theory from the perspective of agricultural technology choice in the process of rural depopulation. We sort out the influence mechanism of rural depopulation on the choice of agricultural technology from four aspects, namely, factor substitution effect, demand scale effect, conditional bottleneck effect, and agricultural comparative benefit. Using the difference between the rural registered population and the permanent population, we comprehensively compiled the data on the net outflow rate of the rural population from 2005 to 2018 in 313 prefecture-level cities, and reach conclusions through panel regression. The results showed that rural depopulation is not necessarily conducive to labor-saving technological choices, but is conducive to the land-saving technological choice. However, rural depopulation promotes labor-saving technological choices when the population reaches a certain level. The conclusion is not completely consistent with the theory of Hayami-Ruttan, it reflects an idiographic feature of the Chinese environment.

O despovoamento rural é uma tendência inevitável devido à urbanização e ao desenvolvimento industrial, e é um fenômeno comum em países industrializados. A teoria clássica da inovação tecnológica induzida proposta por Hayami & Ruttan (1971) explica a mudança na tecnologia agrícola durante o processo de despovoamento rural. Este artigo testa a teoria da inovação tecnológica induzida por Hayami-Ruttan sob a perspectiva da escolha da tecnologia agrícola no processo de despovoamento rural. Classificamos o mecanismo de influência do despovoamento rural na escolha da tecnologia agrícola a partir de quatro aspectos, a saber, efeito de substituição de fatores, efeito de escala de demanda, efeito de gargalo condicional e benefício comparativo agrícola. Usando a diferença entre a população rural registrada e a população permanente, compilamos de forma abrangente os dados sobre a taxa líquida de saída da população rural de 2005 a 2018 em 313 cidades em nível de prefeitura e chegamos a conclusões por meio de regressão em painel. Os resultados mostram que o despovoamento rural não é necessariamente propício para a escolha tecnológica de economia de mão de obra, mas é propício para a escolha tecnológica de economia de terra. No entanto, o despovoamento rural promove escolhas tecnológicas que economizam mão-de-obra quando a população atinge um certo nível. A conclusão não é totalmente consistente com a teoria de Hayami-Ruttan, ela reflete uma característica idiográfica do ambiente chinês.

Manual collapse of blastocoels in not effective in increasing the viability of vitrified equine embryos / Colabamento manual da blastocele não foi efetivo para aumentar a viabilidade de embriões equinos vitrificados

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 µm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 µm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.(AU)

Métodos de criopreservação de embriões têm sido utilizados com diversos objetivos. Maiores taxas de coleta embrio-nária em éguas com lavagem uterina realizada 7 a 8 dias pós ovulação. A criopreservação de embriões equinos com diâmetro <300 µm (6-6,5 dias após a ovulação) permite a obtenção de taxas de prenhez satisfatórias. Embriões com diâmetro >300 µm (7º dia pós-ovulação) somente são adequadamente criopreservados quando submetidos a colabamento da blastocele. Objetivando avaliar a viabilidade da punção da blastocele com equipamento de baixa sofisticação e custo, 22 embriões coletados no 8º. dia pós-ovulação (D8) foram alocados aos seguintes grupos: (1) micropunção com uma agulha 30 G assistida por micromanipula-dor antes da vitrificação (n=4); (2) microssecção manual por lâmina antes da vitrificação (n=6); (3) sem manipulação anterior à vitrificação (n=8); e (4) transferidos a fresco (n=4). Apesar de altas taxas de reexpansão após a criopreservação, os embriões manipulados previamente a vitrificação não resultaram em prenhez aos 25 dias. Tanto os embriões não micromanipulados, quanto os transferidos a fresco resultaram em prenhezes aos 25 dias. A microssecção manual não se mostrou eficiente como método para aumento da criotolerância de embriões grandes, necessitando um aprimoramento visando a obtenção de prenhezes.(AU)

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Relative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes / Relative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes

Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and nonvitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicl e stage prior and after vitrification by exposure to 10 µg/ml of bisbenzimide. A difference was observed in the COCs' mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.(AU)

Pesquisas sobre a vitrificação de gametas femininos estão sendo realizadas para o desenvolvimento de um método confiável de criopreservação dos complexos cumulus-oócitos (CCOs) na espécie equina. Apesar da implementação intensiva de biotecnologias reprodutivas em equinos, a vitrificação dos CCOs imaturos permanece em estágio experimental em relação à competência celular. O objetivo do estudo foi determinar o nível de transcrição relativo dos genes Proteína morfogenética óssea 15 (BMP15); Proteína X associada a Bcl-2 (BAX); e Caspase 3 (CASP3) em CCOs equinos antes e após a vitrificação. Folículos ovarianos foram aspirados de ovários coletados em matadouro. O total de 240 CCOs foi coletado e distribuído em grupos vitrificados (VIT, n=120) e não vitrificados (N-VIT, n=120). Os CCOs foram preservados e as expressões transcritas relativas de BMP15, BAX, CASP3 foram determinadas pela técnica de qRT-PCR sendo normalizadas em relação ao GAPDH. Além disso, 38 CCOs foram avaliados para determinar a configuração da cromatina no estágio de vesícula germinativa antes e após a vitrificação pela exposição a 10 µg/ml de bisbenzimida. Os resultados mostraram uma diferença no nível de abundância de mRNA dos CCOs para o gene BAX entre os grupos VIT (2,05 ± 0,47) e N-VIT (0,85 ± 0,08). Não houve diferença no nível de transcrição relativa do mRNA de CASP3 e BMP15 nos CCOs do grupo N-VIT (0,63 ± 0,20 e 1,55 ± 0,73, respectivamente) em comparação com VIT (0,64 ± 0,01 e 2,84 ± 2,20, respectivamente). Todos os CCOs foram considerados em estágio imaturo de desenvolvimento, embora os CCOs no grupo N-VIT apresentaram a configuração de cromatina condensada em maior número de células avaliadas em comparação com VIT (100% vs 60,7%, respectivamente). Demonstramos que BMP15 e CASP3 são detectados em CCOs imaturos em VIT e N-VIT. Conclui-se que o BAX é altamente expresso em CCOs equinos imaturos vitrificados sendo relacionado à sinalização de apoptose em células expostas ao processo de vitrificação.(AU)

First record of Ereymatermes rotundiceps (Termitidae: Nasutitermitinae) as inquiline of Embiratermes neotenicus (Termitidae: Syntermitinae) in the Colombian Amazon / Primer registro de Ereymatermes rotundiceps (Termitidae: Nasutitermitinae) como inquilino de Embiratermes neotenicus (Termitidae: Syntermitinae) en la Amazonia Colombiana

We report, for the first time, the presence of the termite Ereymatermes rotundiceps Constantino, 1991 as an inquiline of the termite Embiratermes neotenicus (Holmgren, 1906) in the Colombian Amazon, and provide new morphometric information on the species. Soldiers of E. rotundiceps were collected in epigeal nests built by E. neotenicus in pastures during the transition from the dry to the rainy season. The association between host and inquiline termites does not seem to be antagonistic, but the relationship between these two species still needs to be clarified.(AU)

Reportamos por primera vez la presencia de la termita Ereymatermes rotundiceps Constantino, 1991 como inquilino de Embiratermes neotenicus (Holmgren, 1906) en la Amazonia Colombiana y aportamos nueva información morfométrica para las especies. Soldados de E. rotundiceps fueron recolectados en nidos epigeos construidos por E. neotenicus en pasturas durante la transición de la estación seca a la lluviosa. La asociación entre las termitas hospederas e inquilinas parece no ser antagónica, sin embargo, las relaciones entre estas dos especies están aún por esclarecer.(AU)

Avanços na andrologia de animais selvagens / Advances in wild animal andrology

As tecnologias de reprodução assistida (TRA) são de fundamental importância para a conexão de indivíduos em diferentes localidades, facilitando assim o intercâmbio genético e favorecendo a variabilidade genética de uma espécie. Por esta razão, as TRAS podem ser ferramentas importantes para a conservação de espécies ameaçadas de extinção. Apesar dos esforços nas últimas décadas, o avanço no desenvolvimento de tais tecnologias está aquém à urgência de reverter processos de baixa variabilidade genética em algumas espécies. A necessidade de refinamento das técnicas para as particularidades fisiológicas e comportamentais de cada espécie, somada à raridade de acesso aos animais são os principais fatores relacionados as dificuldades em se avançar com as TRAS. As técnicas mais recentes desenvolvidas para a recuperação de espermatozoides em animais selvagens são a colheita farmacológica, com uso de alfa-2-agonistas e a criopreservação / vitrificação testicular com posterior cultivo. Pouco de avançou, no entanto, em relação aos métodos de criopreservação, prevalecendo associação clássica de TRIS-gema-glicerol. Discutimos, então os métodos usados para acesso ao gameta masculino em espécies selvagens e suas aplicações na conservação animal.(AU)

Assisted reproduction technologies (ART) are of fundamental importance for connecting individuals in different locations, thus facilitating genetic exchange and favoring the genetic variability of a species. For this reason, TRAS can be important tools for the conservation of endangered species. Despite efforts in recent decades, the advance in the development of such technologies is short of the urgency of reversing processes of low genetic variability in some species. The need to refine the techniques for the physiological and behavioral particularities of each species, added to the rarity of access to animals, are the main factors related to the difficulties in advancing with TRAS. The most recent techniques developed for sperm collection in wild animals are pharmacological collection, with the use of alpha-2-agonists and testicular cryopreservation / vitrification with subsequent cultivation. Little progress has been made, however, in relation to cryopreservation methods, prevailing the classic association of TRIS-yolk-glycerol. We therefore discuss the methods used to access the male gamete in wild species and their applications in animal conservation.(AU)

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.(AU)

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.(AU)

Life history trait variations and population dynamics of Calophysus macropterus (Siluriformes: Pimelodidae) in two river systems of the Colombian and Peruvian Amazon

The catfish Calophysus macropterus is heavily exploited in the Amazon basin, yet its life history characteristics are poorly documented, hampering proper fisheries management. In order to fill this gap, monthly sampling in the upper Putumayo River and in the Amazonas, lower Marañón and Ucayali (AMU) rivers were carried out over several years (2013-2015 in the AMU, 2013-2017 in the Putumayo) to provide detailed information about its reproduction, growth and mortality patterns (using length frequency data). Reproduction, which occurs during the dry and early flooding season, was only observed in the upper Putumayo River and not in the sampled portion of the AMU system, suggesting that the species reproduces closer to the Andes than other pimelodid catfishes. Size at first sexual maturity did not differ significantly among sex or among river systems, ranging from 25-28 cm SL. In both river systems, females had a faster growth than males and both genders also tended to have a better growth in the AMU than in the upper Putumayo. Mortality and exploitation estimate all indicated overexploitation of the species in both river systems. The implications of these results for fisheries management and conservation are discussed.(AU)

El bagre Calophysus macropterus está fuertemente explotado en la cuenca Amazónica. Sin embargo, su ciclo de vida está poco documentado, lo que dificulta su gestión pesquera. Para llenar este vacío, se realizaron muestreos mensuales en el alto río Putumayo alrededor de Puerto Leguízamo y en el Amazonas y los ríos bajo Marañón y Ucayali (AMU) cerca de Iquitos durante varios años (2013-2015 en AMU, 2013-2017 en Putumayo) para generar información sobre su reproducción, crecimiento y mortalidad. La reproducción, que se produce durante la estación seca y el inicio de la inundación, sólo se observó en el curso superior del río Putumayo y no en la parte muestreada del sistema de la AMU, lo que sugiere que la especie se reproduce más cerca de los Andes que otros pimelodidos. El tamaño de primera madurez sexual no difirió entre sexos o entre sistemas fluviales, oscilando entre 25-28 cm. En ambos sistemas fluviales, las hembras tuvieron un crecimiento más rápido que los machos y ambos géneros también tendieron a tener un mejor crecimiento en el AMU que en el alto Putumayo. Estimaciones de mortalidad y explotación indicaron una sobreexplotación en ambos sistemas. Se discuten las implicaciones de estos resultados para la gestión y conservación de las pesquerías.(AU)

GPS, LiDAR and VNIR data to monitor the spatial behavior of grazing sheep

Traditional knowledge about the behavior of grazing livestock is about to disappear. Shepherds well know that sheep behavior follows non-random patterns. As a novel alternative to seeking behavioral patterns, this study quantified the grazing activities of two sheep flocks of Churra breed (both in the same area but separated by 10 years) based on Global Position System (GPS) monitoring and remote monitoring sensing techniques. In the first monitoring period (2009-10), geolocations were recorded every 5 min (4,240 records), while in the second one (2018-20), records were taken every 30 min (7,636 records). The data were clustered based on the day/night and the activity (resting, moving, or grazing). An airborne LiDAR dataset was used to study the slope, aspect, and vegetation height. Four visible-infrared orthophotographs were mosaicked and classified to obtain the land use/land cover (LU/LC) map. Then, GPS locations were overlain on the terrain features, and a Chi-square test evaluated the relationships between locations and terrain features. Three spatial statistics (directional distribution, Kernel density, and Hot Spot analysis) were also calculated. Results in both monitoring periods suggested that the spatial distribution of free-grazing ewes was non-random. The flocks showed strong preferences for grazing areas with gentle north-facing slopes, where the herbaceous layer formed by pasture predominates. The geostatistical analyses of the sheep locations corroborated those preferences. Geotechnologies have emerged as a potent tool to demonstrate the influence of environmental and terrain attributes on the non-random spatial behavior of grazing sheep.

Aspects of the population dynamics of Anomalocardia brasiliana: subsidies for sustainable fishing in the west coast of Ceará, Brazil / Aspectos da dinâmica populacional de Anomalocardia brasiliana: subsídios para a pesca sustentável no Litoral Oeste do Ceará, Brasil

Shellfish gathering is an artisanal activity and a means of family subsistence for several coastal communities along the Brazilian coast and Anomalocardia brasiliana is one of the main exploited mollusk species. This paper aimed to obtain information about the temporal distribution, growth, yield, and gonadosomatic index of the bivalve mollusk A. brasiliana in a natural stock to provide subsidies for the sustainable extraction of this resource. The study was carried out in an area of the Acaraú River estuary, on Volta do Rio Beach, municipality of Acaraú, Ceará, Brazil. Samples were collected monthly for one year at spring low tide, using the random squares method. Water and sediment temperature and salinity were measured in situ. Sediment samples were collected for granulometric analysis. The specimens were individually quantified, measured, and weighed. A total of 8,507 A. brasiliana specimens were sampled, and the highest density recorded was 2,534 individuals/m2, whose longest shell length reached between 21 and 25 mm, while the mean meat yield was 12.42%. The means of biometric variables of individuals showed significant differences during the sample period. Frequency histograms evidenced the presence of adult and juvenile individuals, showing a continuous reproductive cycle in the studied natural stock. These results can assist the sustainable shellfish gathering activity on Volta do Rio Beach. We recommend that A. brasiliana is above 20 mm in length to be captured and the reduction in the capture effort from April to September, when the highest density of juveniles was observed, to guarantee the sustainability of this stock.

A mariscagem é uma atividade artesanal e meio de subsistência familiar de diversas comunidades litorâneas ao longo da costa brasileira, sendo Anomalocardia brasiliana uma das principais espécies de molusco exploradas. Esse trabalho objetivou obter informações sobre a distribuição temporal, crescimento, rendimento e o índice gonadossomático do molusco bivalve A. brasiliana em um estoque natural a fim de fornecer subsídios para a extração sustentável deste recurso. O estudo foi realizado em uma área do estuário do rio Acaraú, na Praia Volta do Rio, município de Acaraú, Ceará, Brasil. As coletas foram realizadas mensalmente durante um ano na maré baixa de sizígia, utilizando o método dos quadrados aleatórios. A temperatura da água, do sedimento e a salinidade foram aferidas in situ. Foram coletadas amostras de sedimento para análise granulométrica. Os espécimes foram quantificados, mensurados e pesados individualmente. Foram amostrados um total de 8.507 exemplares de A. brasiliana, a maior densidade registrada teve 2.534 indivíduos/m2, aqueles com maior comprimento de concha tinham entre 21 e 25 mm, o rendimento da carne apresentou um valor médio de 12,42%. As médias das variáveis biométricas dos indivíduos apresentaram diferenças significativas durante o período amostral. Os histogramas de frequência evidenciaram a presença de indivíduos adultos e juvenis, indicando um ciclo reprodutivo contínuo no estoque natural estudado. Estes resultados auxiliarão no exercício da mariscagem sustentável na praia da Volta do Rio. Recomenda-se a captura de A. brasiliana com comprimento acima de 20 mm e a redução do esforço de captura nos meses de abril a setembro, período em que foi observada a maior densidade de juvenis, a fim de garantir a sustentabilidade desse estoque.

Population dynamics and seasonality of Euspilotus (Hesperosaprinus) azureus Sahlberg (Coleoptera: Histeridae: Saprininae) / Dinâmica populacional e sazonalidade de Euspilotus (Hesperosaprinus) azureus Sahlberg (Coleoptera: Histeridae: Saprininae)

The effect of climatic conditions on the structure of populations has been perceived for different orders of insects and families of beetles. Here we described the population dynamics of Euspilotus azureus (Sahlberg, 1823) and determined its relationship with climatic variables. The specimens were collected monthly for one year in an area of Seasonal Semideciduous Forest, in Viçosa, Brazil. The seasonality of the population and of sexes was estimated through the absolute frequency of occurrence (AF) and the percentage frequency of occurrence (FO%) of the population and each sex, which were determined monthly. To test whether the changes in monthly AF of the population and of each sex are correlated with climatic variables, Spearman correlation tests were performed. To identify whether there are temporal fluctuations in the sex ratio, considering months and seasons, and within each season, we performed Chi-square tests. A total of 3,126 individuals (1,009 females and 2,117 males) were collected. The population had the highest abundance in spring and summer, and the lowest in autumn and winter. The highest AF and FO were found in December and January, and the lowest in June and July. The sex ratio of the population did not differ in the months of the year, but in the spring there was a marked predominance of males. For E. azureus, the temporal fluctuation in abundance of the population and in the sex ratio is related to the climatic variables of maximum temperature, humidity, and precipitation. Climatic variables, especially the temperature, provided a greater abundance of the population in the hottest and rainiest periods of the year for inducing reproductive activity and altering the population's sex ratio.

O efeito das condições climáticas na estrutura das populações tem sido percebido para diferentes ordens de insetos e famílias de besouros. Nós descrevemos a dinâmica populacional temporal de Euspilotus azureus (Sahlberg, 1823) e determinamos sua relação com as variáveis climáticas. Os espécimes foram coletados durante um ano em uma área de Floresta Semidecidual Sazonal, em Viçosa, Brasil. A sazonalidade da população e dos sexos foi estimada através da frequência de ocorrência absoluta (FA) e da frequência de ocorrência percentual (FO%) da população, as quais foram determinadas mensalmente. Para testar se as mudanças mensais na FA da população e de cada sexo estão correlacionadas com as variáveis climáticas, testes de correlação de Spearman foram realizados. Para identificar se há oscilações temporais na razão sexual considerando meses e estações, e dentro de cada estação, foram realizados testes de Qui-quadrado. Um total de 3.126 indivíduos (1.009 fêmeas e 2.117 machos) foram coletados. A população teve maior abundância na primavera e verão, e menor no outono e inverno. As maiores FA e FO foram em dezembro e janeiro, e a menores em junho e julho. A razão sexual da população não diferiu nos meses do ano, mas na primavera houve uma predominância marcante de machos. Para E. azureus, a flutuação temporal na abundância da população e na razão sexual é explicada pelas variáveis climáticas de temperatura máxima, umidade e precipitação. As variáveis climáticas, especialmente a temperatura, propiciaram uma maior abundância da população nos períodos os mais quentes e chuvosos do ano por induzirem a atividade reprodutiva e a alterar da razão sexual da população.

Effect of cowpea (Vigna unguiculata L. Walp) sowing season on population dynamics of pest insects

Several factors limit cowpea quality and yield, such as the choice of a cultivar better adapted to the region, the ideal sowing season, as well as diseases and pests. Aimed to evaluate the effect of different sowing seasons on the population dynamics of pest insects of cowpea crops. The study was carried out during the dry season, the rainy season, and the season recommended for sowing by the Experimental Farm of the Federal University of Amazonas (FAEXP­UFAM), Brazil. A total of 33 weekly collections were carried out during the three sowing seasons, distributed in two ways according to the phenological stage of the crop: manual collection per linear meter of plantation and entomological scanning net. The faunistic analysis detected the pest insects Acromyrmex laticeps nigrosetosus, Cerotoma arcuata, Crinocerus sanctus, Euschistus heros, Horciasinus signoreti, Liriomyza sativae, and Nezara viridula. Of these species, H. signoreti stands out for being recorded for the first time as predominant in cowpea crops in the Brazilian state of Amazonas. Infections by hemipterans such as C. sanctus, E. heros, H. signoreti, and N. viridula were detected throughout the study, regardless of the sowing season, except for H. signoreti, which was dominant in all three seasons. The highest infestation of C. arcuata occurred during the recommended sowing season for cowpeas (June to August). According to the Jaccard index, plantation I e II share 86% of the species. This study provides important information to programs in insect control and management and to the agronomic decision-making process.

Uso da galactose na vitrificação de sêmen ovino em palhetas / Use of galactose to vitrify ram semen in straws

A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.

Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.

Efeito do resveratrol associado a sacarose em protocolo de vitrificação de tecido ovariano de vacas / Effect of resveratrol associated with sacarosis on ovarian tissue vitrification protocol in cows

The objective of this work was to evaluate the effect of the association of the antioxidant resveratrol with sucrose in a vitrification protocol of ovarian tissue in cows, on the morphology of preantral follicles. Ten ovaries of cows were used, collected in local slaughterhouses, fragmented and distributed to the following treatments: fresh control (Co); toxicity (T); (T0) zero toxicity/ only with base vitrification solution (SBV), (TS) toxicity with SBV plus sucrose, (TR) toxicity with SBV plus resveratrol, (TS+R) toxicity with SBV and sucrose plus resveratrol; and for glazing (V); (VS) vitrification with SBV and sucrose, (VR) vitrification with SBV and resveratrol, (VS+R) vitrification with SBV and sucrose plus resveratrol. Preantral follicles were quantified and classified according to morphology into normal and degenerated. The mean percentages between normal and degenerated follicles did not differ (p>0.05) in the following percentages, normal 51.4% and degenerated 48.60%. In the toxicity test there was a difference (p0.05), demonstrating that the vitrification technique is efficient, but the concentration of cryoprotectants used needs to be re-evaluated. Concluding that the natural antioxidant association resveratrol to sucrose in vitrification and rewarming protocols contributes with reservations for the morphological preservation of preantral follicles in cows.