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

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

Efeito de diferentes concentrações de progesterona em dispositivos vaginais sobre a dinâmica folicular de novilhas Holandesas (Bos taurus) submetidas à IATF

Avaliou-se o efeito de diferentes concentrações de P4 [0,75g (T1) e 1g (T2)] sobre a dinâmica folicular de novilhas púberes submetidas à IATF. No D0 do protocolo, as fêmeas do T1 (n=10) e do T2 (n=9) receberam P4 em dispositivos intravaginais e, intramuscularmente, 2mg de BE. No D7 aplicou-se 530?g de cloprostenol e 300 UI de eCG, no D8 removeu-se os dispositivos, no D9 administrou-se 1 mg de BE e no D10 realizou-se a inseminação. A sincronização da onda folicular ocorreu em 100% das fêmeas. O diâmetro (mm) do folículo dominante (FD), avaliado no D9, o diâmetro máximo do FD o diâmetro máximo do folículo ovulatório (FO) não havendo diferença (P>0,05) entre os tratamentos. A média de crescimento diário dos folículos foi maior (P<0,05) no T1 (1,6±0,12) do que no T2 (1,0±0,18). O diâmetro do FD das novilhas do T1 e do T2 não diferiu (P<0,05), tanto das que ovularam quanto das que não ovularam. O diâmetro do FD entre as novilhas que ovularam com o daquelas que não ovularam foi maior (P<0,05) tanto no T1 (14,8±0,6 vs 9,6±0,7) quanto no T2 (11,2±1,6 vs 7,7±0,03). A porcentagem de prenhez não diferiu entre T1 e T2.  Os resultados indicam que ambas as dosagens de P4 são eficientes para induzir a emergência da nova onda folicular, controlar o crescimento folicular e sincronizar a ovulação de novilhas púberes

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

Lessons learned from functional assessment of pluripotency-associated transcription factors during early embryogenesis and embryonic stem cells

Pluripotency-associated transcription factors (PATF) play significant roles during early embryogenesis and in embryonic stem (ES) cells, such as control of cell-cycle progression, modulation of cellular metabolism, and transcriptional control of differentiation-inducing factors. The review aims to describe the current understanding of how these PATFs contribute to the early embryo and the ES-cell phenotypes. By a selection of representative examples of such PATFs, their roles are described, and some interesting questions are presented concerning their activity in pluripotent cells which have yet to be addressed

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

Expression of RONIN and NANOG-associated proteins in goat parthenogenetic embryos

The expression of a subset of transcription factors is enriched in early preimplantation embryos, which contributes to their cellular plasticity. RONIN, NANOG and its associated proteins are PluripotencyAssociated Transcription Factors (PATF) that control relevant downstream pathways in pluripotent stem cells, but their activity in early embryos remained less understood. The work was aimed to determine the expression of RONIN and four NANOG-associated PATFs in goat preimplantation embryos. Goat embryos were produced in vitro by parthenogenetic activation. Gene transcripts of cleavage-stage embryos were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), while blastocysts were analyzed by both RTPCR and quantitative RT-PCR (RT-qPCR) assays. Gene transcripts of ZFP281, NAC1, and NR0B1 were detected in cleavage-stage embryos, while RONIN and OCT4 were not found expressed. Detection in blastocysts by RT-PCR confirmed the activity of NR0B1, RONIN, and OCT4. Moreover, all five PATF were detected in blastocysts by RT-qPCR (ZFP281, NAC1, RONIN, OCT4, and NR0B1). In conclusion, RONIN and NANOG-associated proteins are active during goat parthenogenetic preimplantation development and hold stage-specific expression patterns

Fertilização in vitro com sêmen sexado de bovinos da raça 5/8 girolando

O uso de sêmen sexado em conjunto com a produção in vitro (PIV) de embriões é um meio potencialmente eficiente na obtenção da descendência com sexo predeterminado. Há anos, os proprietários dos animais desejaram uma metodologia que pré determinasse o sexo da prole para seus rebanhos. As taxas de clivagem, mórula e blastocisto parecem ser afetadas não só pela debilitação provocada na sexagem, mas acredita-se que pelo reprodutor em questão, e por outros fatores que não foram ainda totalmente elucidados, influenciando diretamente na sua utilização com sucesso. O objetivo desse trabalho foi avaliar a influencia do tipo de sêmen (sexado/convencional) e do touro, nas taxas de blastocisto na PIV de oócitos bovinos obtidos em matadouro, e ainda comparar esses resultados com as análises da cinética espermática. Oócitos (N= 959) foram maturados, fertilizados com sêmen sexado e convencional de três touros da raça 5/8 Girolando. Uma palheta de cada tipo de sêmen foi avaliada com uso da “computer-assisted sêmen analysis” (CASA) e da microscopia de fluorescência. Foram realizadas três repetições durante o experimento. Os dados foram analisados pelo programa estatístico SPSS 16.0 empregando-se a análise de variância (ANOVA). O teste t-Student foi usado para detectar diferenças entre os grupos. O Qui-quadrado foi utilizado para análise dos resultados da produção de embriões. Para todas as análises, os valores foram considerados significativos (P< 0,05). Os resultados diferiram entre o sêmen convencional (31,06) e sexado (21,10%) para produção de blastocisto. Quando comparamos a produção de blastocisto individualmente nas amostras de sêmen sexado (27,69%; 17,93% e 25,56%, touros 1, 2 e 3 respectivamente) percebemos que T2 < T1 e T1=T3 e T2=T3. Concluimos que no presente trabalho o sêmen sexado foi menos eficiente na produção de blastocisto quando comparado ao sêmen convencional de forma geral e do mesmo touro. As análises da cinética espermática bem como as sondas fluorescentes foram compatíveis com o potencial fertilizante das amostras de sêmen sexado e convencional em touros da raça 5/8 Girolando.The use of sexed semen for in vitro embryo production (IVP) is a potentially effective means for obtaining the progeny with predetermined sex. For years, animal owners wanted a methodology that pre determine the sex of offspring for their herds. Rates of cleavage, morula and blastocyst seem to be affected not only by the damages caused by sexing, but it is believed that the bull factor, and other aspects that have not been fully elucidated, directly influencing its successful use. The aim of this study was to evaluate the influence of type of semen (sexed /conventional) and a bull factor over blastocyst rates when submitting bovine oocytes obtained from slaughterhouse to IVP, and also compare these results with analyzes of sperm kinetics. Oocytes (n = 959) were matured, fertilized with sexed and non-sexed semen from three 5/8 Girolando bulls. A straw of each type of semen was assessed with use of "computer-assisted semen analysis" (CASA) and fluorescence microscopy. Three replicates were performed during the experiment. Data were analyzed by SPSS 16.0 statistical program employing analysis of variance (ANOVA). The Student t test was used to detect differences between groups. The Chi-square test was used to analyze the results of embryo production. For all analyzes, values were considered significant (P <0.05). The results differed between sexed (21.10%) and non-sexed semen (31.06%) to blastocysts production. We conclude that in the present work sexed semen was less efficient in producing blastocyst when comparing non-sexed semen of the same bulls and when comparing semen types (sexed and non-sexed) from the same bull. Analyses of sperm kinetics and fluorescent probes were compatible with the fertilizing potential of samples of sexed and non-sexed semen of 5/8 Girolando bulls

PRODUCTION OF IN VITRO EMBRYO USING SEXED SPERM OF 5/8 GIROLANDO BULLS

We evaluated the "in vitro" blastocyst rate production using bovine sexed semen. Semen from three bulls was used to verify the individual's semen variation, cleavage rates and embryo production. In this study, we employed reproductive biotechnologies, computer analysis of post-thawed semen and fluorescent probes for sperm cells integrity analysis (plasma membrane, acrosome membrane and mitochondrial potential). A total of 959 oocysts went through in vitro maturation steps for in vitro fertilization and cultivation, being 473 with sexed semen and 486 with conventional semen. The cleavage rate was observed in blastocysts on D2 and D7. Data were analyzed by SPSS 16.0 software using analysis of variance (ANOVA), Student's t-test was used to detect differences between groups, and chi-square analysis for in vitro production results (P <0.05 ). The results differed between conventional (31.06%) and sexed semen (21.10%) in the obtainment of blastocyst. When the blastocyst production was individually compared in sexed semen samples (27.69%, 17.93% and 25.56%, bulls 1, 2 and 3, respectively) we verified T2 <T1 and T1 = T3 and T2 = T3. As for sperm kinetic analyzes, the sexed semen samples showed differences among bulls in curvilinear speed, linear speed and path velocity variables where T1 (117.7 ± 1.6 µm/s; 60.0 ± 0.3 µm/s, 73.6 ± 0.4 µm/s, respectively) showed the highest values when compared to T2 bulls (80.2 ± 2.3 µm/s, 47.0 ± 2.0 µm/s, 57.7 ± 0, 9 µm/s, respectively) and T3 (86.4 ± 5.7 µm/s, 46.2 ± 2.7 µm/s, 53.8 ± 2.8 µm/s, respectively). Analyses of sperm cell integrity did not differ among conventional semen samples, but in the sexed semen, membrane integrity was the variable that differ statistically among bulls once T1 (38 ± 2.7) differed from T3 (53 8 ± 1.8) (P = 0.009) but did not differ from T2 (44.1 ± 4.4). It is possible to conclude that sexed semen was less efficient in blastocyst production when compared with conventional semen. Analyses of kinetic and integrity were consistent with the fertilization potential of semen samples from 5/8 Girolando bulls

Role of Male effect on Reproductive Efficiency of Nulliparous Santa Inês and Morada Nova ewes raised in Different Regions

Background: The male effect is an attractive strategy to increase herd production by concentrating mating events and deliveries and further allowing the adoption of genetic improvement programs. It holds similar efficiency to those chemically based estrous synchronization methods, but has the advantage of being a natural method. The work was aimed to evaluate the influence of male effect on estrous induction and synchronization, pregnancy and prolificacy of nulliparous Santa Inês and Morada Nova ewes raised in Semiarid and Zona da Mata regions of Pernambuco state.Materials, Methods & Results: Santa Inês (n = 80) and Morada Nova (n = 80) females, with age from 11 to 12 months, after being evaluated and selected, were identified with plastic ear tags, weighted and maintained isolated from males, during 30 days before experiment onset, without any physical, visual, olfactive and auditive contact. Estrous events were observed twice a day (6:00 and 16:00 h) by trained personnel, during a breeding season of 60 days, and estrous were considered synchronized when detected, within first five days of breeding season. Rams of Santa Inês (n = 2) and Morada Nova (n = 2) breeds were selected based upon reproductive capacity by an andrology exam, and were marked on the externum bone region with a wax and ink (4:1) mixture, and were marked in female lots in order to identify females in estrous. After ten days of breeding season onset, rams were again marked with the same wax and ink mixture, but with a different ink color. Pregnancy diagnosis was performed on day 30 by ultrasonography and confirmed on day 60 after the last mating. The statistical analysis was performed using SAS, version 8. Differences of 5% (P 0.05). The total pregnancy was 42.50% on first, 64.70% on second service and total delivery was 86.20% singletons, 12.06% twins and 3.33% triples with prolificacy of 1.15 ± 0.31. On both Semiarid and Zona da Mata regions, the majority of estrous events occurred between the11th and 15th day of the breeding season for Santa Inês ewes and between 6th and 10th day for Morada Nova ewes.Discussion: The occurrence of estrous, for both breeds, in both regions, were detected throughout the breeding seasons, despite most estrous detections were within the initial fifteen days, which normally happens with cycling pluriparous females, in disagreement with findings in the literature that young females display lower reproductive performance on the first breeding season. However, the sexual inexperience of young females is not equivalent to lack of male receptivity, since then, could not be responsible for late estrous onset in a breeding season of young females. In agreement with this statement, and based on the data described here, it has been described that young ewes display estrous within the initial 18 days of breeding season onset. The estrous dispersion in biostimulation programs is normally due to female cyclicity, a physiological condition that lowers the sensibility to estradiol negative feedback response, but are still responsible to the presence of males