ENUCLEAÇÃO DE OVÓCITOS MAMÍFEROS DESTINADOS À TRANSFERÊNCIA NUCLEAR

A clonagem por Transferência Nuclear (TN) permite reprogramar célulassomáticas de mamíferos. Essa técnica é realizada pela remoção do DNA doovócito e posterior introdução, neste mesmo ovócito, de um núcleo de umacélula doadora. Apesar das diversas aplicações em pesquisas científicas básicase na pecuária, a TN permanece laboriosa e pouco eficiente. No processo deTN, a enucleação é uma etapa crítica e falhas na sua realização tornam inviávelo posterior desenvolvimento embrionário. Em contrapartida, a remoção doscromossomos do gameta diminui o volume da célula e reduz sua capacidadede desenvolvimento. Avanços nesta metodologia podem simplificar o processode TN, aumentando a escala de produção de embriões. Recente pesquisamostrou evidências de que a actinomicina D, um antibiótico conhecido comoinibidor de transcrição e replicação de DNA, pode ser uma alternativa paraenucleação convencional. No entanto, novos experimentos são necessáriospara comprovação desta hipótese

PLURIPOTENCY AND CELLULAR REPROGRAMMING

O desenvolvimento em mamíferos começa após a fecundação e singamia dos genomas haplóides. O recém formado zigoto passa por múltiplas divisões celulares e torna–se um embrião composto de células pluripotentes, que darão origem a todos os tecidos encontrados nos animais adultos. As linhagens de células–tronco embrionárias (CTE) são cultivos ex vivo de células pluripotentes provenientes de embriões. As CTE oferecem novas oportunidades para investigar o desenvolvimento em mamíferos, para criar novos modelos de doenças humanas, e possivelmente para oferecer células para transplantes. O nascimento de animais clonados pela transferência nuclear de células somáticas para ovócitos demonstrou a reversibilidade da diferenciação celular, um processo denominado reprogramação celular. Outros métodos para obtenção de células reprogramadas indiferenciadas a partir de núcleos somáticos foram descritos, baseados na fusão celular ou na expressão exógena de determinados genes. A reprogramação celular permitiu o isolamento de células pluripotentes de pacientes para investigação da etiologia de doenças humanas e prospeção de novos medicamentos. Apesar do progresso monumental no entendimento do desenvolvimento inicial em mamíferos, a complexidade da pluripotência e reprogramação celular ainda são pouco entendidos. Esta revisão tem como objetivo descrever as principais descobertas nestes campos científicos, citar limitações técnicas e biológicas destas linhas de pesquisas, e prover possíveis soluções para contornar tais dificuldades

THE EVOLVING PICTURE IN OBTAINING GENETICALLY MODIFIED LIVESTOCK

Animais de produção geneticamente modificados são aqueles que tiveram uma sequência de DNA endógena modificada ou um DNA exógeno introduzido em seu genoma. Animais de produção geneticamente modificados apresentam grande potencial como modelo para estudo de doenças humanas, para produção mais eficiente de carne e derivados do leite, para xenotransplante e para produção de produtos sob grande demanda para saúde humana. Diferentes abordagens têm sido descritas para obtenção de animais de produção geneticamente modificados, as quais apresentam eficiências, vantagens e limitações variáveis. O objetivo da revisão é descrever o histórico da obtenção de animais de produção geneticamente modificados, os principais obstáculos, abordagens atuais e perspectiva futuras sobre a tecnologia

DESENVOLVIMENTO DA CRIOPRESERVAÇÃO DE EMBRIÕES EM CAPRINOS

A criopreservação de embriões permite a conservação do material genético de caprinos por períodos indefinidos de tempo. O método de congelação tradicional e a vitrificação são os principais métodos de criopreservação de embriões, diferindo pela velocidade de resfriamento e concentrações de crioprotetores. Além dos aspectos técnicos, o sistema de produção de embriões (in vitro, in vivo, micro-manipulação), a qualidade morfológica e o estádio de desenvolvimento são fatores determinantes para a eficiência da criopreservação. O objetivo desta revisão foi apresentar um breve histórico, aplicações, principais investigações e algumas perspectivas sobre a criopreservação de embriões em caprinos

Induction of Ovulation in Mangalarga Marchador Mares by hCG or GnRH

Background: Induction of ovulation is a key procedure for horse assisted reproduction technologies, such as for artificial insemination (AI) and embryo transfer. The application of hCG remains as the primary ovulation-inducing agent for horse assisted reproduction, but alternatives are in demand to avoid its adverse effects, such as loss of ovulation-inducing efficiency over multiple applications by hCC-antibody production. Despite reports on alternative ovulation-inducing agents, pair-wise comparisons of such agents under similar conditions have been limited. Under such scenario, the work was aimed to determine the efficiency of both hCG and Buserelin at inducing ovulation in Mangalarga Marchador mares raised in the Northeast of Brazil under an AI program.Materials, Methods & Results: Mares were initially selected based on their reproductive performance, the absence of clinical-reproductive alterations and adequate body condition score. Mares in diestrus were randomly distributed in three experimental conditions, received 5 mg of Dinoprost and were monitored daily for estrus detection. After estrus detection, ovaries were monitored by ultrasonography, in 12-h intervals, until the follicle reached 35 mm. At this time-point, ovulation was induced with 0.042 mg of Buserelin endovenously, with 3,000 IU hCG by an intramuscular shot, and control mares received 2 mL of saline solution, also by an intramuscular shot. Both hCG and Buserelin displayed similar efficiencies (P > 0.05) for induction of ovulation and that both agents were effective (P 0.05) from those found in the Control. All ovulations occurred within a 72-h period after treatment. It can be observed that in mares treated with hCG or Buserelin, ovulations occurred both in more mares (P 0.05) between hCG and Buserelin groups, and that pregnancy of mares treated with ovulation-inducing factors was similar to the control.Discussion: The majority of ovulations in mares occurred within initial 48-h after treatment for both hCG and GnRH, suggesting a similar potential for horse assisted reproduction. Both hCG and Buserelin are two commonly used agents for induction of ovulation in mares. As described here, the majority of ovulations occurred within initial 48-h after treatment, a fact which can be attributed to hCG and GnRH activity, since it can happen in intervals from 36 to 48-h after treatment. Pregnancy rates did not differ among groups. These results are under the working hypothesis that hCG and Buserelin would display similar efficiencies on pregnancy rates. Despite the understanding of hCG activity on induction of ovulation due to its high specificity toward LH receptors and results from a direct effect on diminishing estradiol concentration, increasing LH, and further inducing ovulation within 48-h after treatment. In contrast, Buserelin has a similar functional property but acts upon LH synthesis and its release. In conclusion, ovulation in mares can be induced with both hCG and Buserelin, and both ovulation-inducing agents do not affect pregnancy rates

Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

Background: fertility rates using horse frozen-thawed semen remain lower than in other livestock species. This fact further suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is a substantial influence of genetic factors and diluent choice upon horse cryopreservation outcome. Collectively, these genetic and technical properties of horse semen could be explored to identify factors or conditions that may increase semen viability after freeze-thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: frozen-thawed semen was evaluated for motility, membrane integrity, and sperm DNA fragmentation using the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h. Discussion: frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Sperm DNA fragmentation was quite low for both groups, as described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions, since it is caused by multiple origins such as genetic factors. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability

Influence of Male to Female Ratio on Hormone Profiles and Reproductive Performance of Anestrus Postpartum Ewes Subjected to the Male Effect

Background: Complete isolation of genders allows intense estrous induction and synchronization once rams are introduced in ewe flocks at the onset of the breeding season (BS). This management practice, defined as the male effect, results from a neuroendocrine process mediated by pheromones. The male effect is a straightforward procedure to induce estrous in noncycling ewes, but conditions for its use have not been fully explored. Thus, this study aimed to evaluate hormone levels and ovarian activity of postpartum ewes in anestrus which are subjected to the male effect under different male to female ratios.Material, Methods & Results: Pospartum females were selected according to body condition score and cyclicity status. Females were kept apart from males during 30 days at a distance of 10 m. Anestrus and ovulation were determined by P4 measures on days 10, 20 and 30 after isolation from males. After P4 concentration diagnosis, anestrus ewe (n = 99) were subjected to male to ewe ratios (MFR) of 1:20 (MFR20), 1:30 (MFR30) and 1:40 (MFR40). Santa Inês rams  (n = 3) of proven fertility were used. Three females of each group were randomly subjected to blood collection for LH concentration analysis. Ovarian activity was performed by ultrasonography after estrus manifestation in six ewe of each group. Estrus events were observed twice a day during the BS of 35 days, and estrus were considered synchronized when it occured within the initial five days of the BS. Pregnancy diagnosis was performed by ultrasonography on days 35 and 60 after the last mating. All ewe were in a non-cycling condition before BS onset, based upon P4 analysis. After initiation of the BS, P4 concentrations increased for all groups. Irrespectively of male to female ratio, male effect induced LH pre-ovulatory peaks within the initial 26 to 86 h of the BS. Synchronization of estrus reached 50% for MFR20, 40% for MFR30 and 20% for MFR40 for all ewe. Moreover, overall estrus incidence was 100% (MFR20), 90% (MFR30) and 65% (MFR40) within the initial 15 days of the BS. However, incidence of ewe that had repeated estrus events was lower for MFR20 than for MFR30. Follicular growth and number of ovulations was similar between groups. Conception rates on first service was higher than that of second service for MFR20 and MFR30, although there was no difference between services for MFR40. In contrast, overall conception rates, delivery type and prolificacy were similar between groups.Discussion: P4 increased to cyclicity levels after contact between genders, demonstrating the potential of the male effect to induce estrus in non-cycling ewes. Most ewe ovulated within three days after the male effect, possibly due to elevated basal LH levels. Moreover, the LH preovulatory peak varied within groups, possibly due to greater interactions between genders, which ultimately may have led to earlier ovulation anticipation under lower MFR. Estrus parameters were similar between groups, suggesting low or negligible effects of MFR. Ovulatory follicle size and growth and the number of ovulations were similar between all groups; previous reports have suggested that this may be due to a strong effect of their genetic background. Conception rates were higher at first than second services, demonstrating the potential of male effect. In conclusion, male to female ratio affects the efficiency of the male effect to induce and synchronize estrus in ewes under postpartum anestrus, but it does not affect conception rates and prolificacy

Evaluation of clinical and reproductive parameters in Mangalarga Marchador mares treated with different doses of Cloprostenol or Dinoprost

Avaliou-se a ação de doses reduzidas e convencionais de substâncias luteolíticas sobre parâmetros clínicos e reprodutivos de éguas. As femeas receberam intramuscularmente, 125 ?g (n = 20) e 250 ?g (n = 20) de Cloprostenol e 2.5 mg (n = 20) e 5.0 mg (n = 20) de Dinoprost. A temperatura retal e as frequências cardíaca e respiratória foram aferidas antes e após a administração desses luteolíticos, considerando-se ainda a ocorrência de sudorese, diarreia, cólica e prostração. Monitorou-se o estro e o desenvolvimento folicular até a ovulação, quando realizou-se a inseminção artificial. A gestação foi diagnosticada com 300 e confirmada no 60o dia. Apenas as éguas tratadas com 2,5 e 5,0 mg de Dinoprost apresentaram alteração (P < 0.05) da frequência respiratória e os demais parâmetros não foram alterados (P > 0.05). A sudorese ocorreu em 5% e 10% das éguas tratadas, respectivamente, com 2.5 mg e 5.0 mg de Dinoprost e a diarréia em apenas 5% daquelas que receberam 5.0 mg desse luteolítico. As porcentagens de estro e prenhez das éguas tratadas com 125 ?g de Cloprostenol (45%/35%) e 2.5 mg de Dinoprost (50%/30%) foram menores (P < 0.05) do que os daquelas que receberam 250 ?g de Cloprostenol (85%/70%) e 5 mg de Dinoprost (90%/75%). O estro e a prenhez das éguas Controle foram menores (P < 0.05) do que nas tratadas. Conclui-se que apesar de não promoverem alterações significativas dos parâmetros clínicos, as doses reduzidas não apresentam as mesmas eficiências dos tratamentos com doses convencionais para induzir o estro

Atividade dos genes relacionados à pluripotência em ovinos

Embriões antes da implantação possuem células pluripotentes, ou seja, células que apresentam a capacidade de se diferenciar em todos os tecidos que compõem o feto. O controle da pluripotência em nível molecular é estabelecido por diversos fatores, entre eles, os genes relacionados à pluripotência (GRPs). Estes genes contribuem para inibição do processo de diferenciação celular e manutenção da viabilidade de células pluripotentes. No entanto, apesar do crescente conhecimento sobre as funções dos GRPs em camundongos e humanos, pouco se conhece sobre a expressão espaço-temporal e funções dos GRPs em outras espécies, incluindo ovinos. Evidências em bovinos, humanos e camundongos demonstram que GRPs podem apresentar mecanismos de ação diferentes entre as espécies. O objetivo da revisão foi analisar a atividade dos GRPs em ovinos através do perfil de expressão espaço-temporal e funções, bem como apresentar alternativas para acelerar o entendimento da pluripotência na espécie

Reproductive efficiency of nellore (Bos indicus) cows subject to both ftai and homeopathic supplementation

The aim of this work was to determine the effect of homeopathic supplementation on both ovarian dynamics and conception rate in Nellore cows subjected to fixed-time artificial insemination (FTAI). Cows (n = 150) were randomly distributed to the control (CG) and the homeopathy group (HG). The HG cows were supplemented with Pró-cio in the mineral salt for 60 days and both experimental groups were further subjected to FTAI. Cows were evaluated for ovarian dynamics (n = 16), progesterone (P4) concentration (n = 16), and conception rates (n = 150). Ovarian dynamics determined by ultrasonography and showed similar findings for CG and HG, respectively. Thus follicular diameter (8.7 ± 1.0 mm vs. 10.0 ± 0.8 mm), mean pre-ovulatory follicle volume (0.46 ± 0.15 mL vs. 0.61 ± 0.12 mL), and mean follicular growth (3.65 ± 1.41 mm vs. 4.60 ± 1.21 mm) did not differ between groups. Moreover, corpus luteum diameter was similar between groups (CG: 16.28 ± 0.7 mm vs. HG: 15.6 ± 0.8 mm; P > 0.05), although P4 levels did differ (CG: 2.55 ± 0.85 ng mL-1 vs. HG: 6.52 ± 1.19 ng mL-1; P 0.05). In conclusion, the homeopathic supplementation Pró-cio increases P4 concentrations but does improve the reproductive efficiency of Nellore cows subject to FTAI