Transferência Nuclear de Células Somáticas interespecífica (TNCSi) na conservação de cervídeos em risco de extinção / Interspecific Somatic Cell Nuclear Transfer (TNCSi) in the conservation of endangered species of deer

A Transferência Nuclear de Células Somáticas interespecífica (TNCSi) envolve a transferência de um núcleo ou célula de uma espécie para o citoplasma de um oócito enucleado de outra espécie. Após ativação, os complexos carioplasto-citoplasto reconstruídos podem ser cultivados in vitro até o blastocisto, o estádio final de desenvolvimento pré-implantação. Esta pode ser uma estratégia interessante na tentativa de conservação de espécies em risco de extinção. Esta revisão tem por objetivo apresentar alguns detalhes da técnica e relatar a experiência de nosso grupo usando como modelo o veado-catingueiro.(AU)

Interspecies Somatic Cell Nuclear Transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed caryoplast-cytoplast complexes can be cultured in vitro to blastocyst, the final stage of preimplantation development. This may be an interesting strategy in attempting to conserve endangered species. The objective of this review is to present some details of the technique and to report the experience of our group using as model the brown brocket deer.(AU)

Transferência Nuclear de Células Somáticas interespecífica (TNCSi) na conservação de cervídeos em risco de extinção / Interspecific Somatic Cell Nuclear Transfer (TNCSi) in the conservation of endangered species of deer

A Transferência Nuclear de Células Somáticas interespecífica (TNCSi) envolve a transferência de um núcleo ou célula de uma espécie para o citoplasma de um oócito enucleado de outra espécie. Após ativação, os complexos carioplasto-citoplasto reconstruídos podem ser cultivados in vitro até o blastocisto, o estádio final de desenvolvimento pré-implantação. Esta pode ser uma estratégia interessante na tentativa de conservação de espécies em risco de extinção. Esta revisão tem por objetivo apresentar alguns detalhes da técnica e relatar a experiência de nosso grupo usando como modelo o veado-catingueiro.

Interspecies Somatic Cell Nuclear Transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed caryoplast-cytoplast complexes can be cultured in vitro to blastocyst, the final stage of preimplantation development. This may be an interesting strategy in attempting to conserve endangered species. The objective of this review is to present some details of the technique and to report the experience of our group using as model the brown brocket deer.

Premissas e promessas da clonagem interespecífica de espécies em risco de extinção / Premises and promises of interspecific cloning in endangered species

Nos últimos anos, a transferência nuclear de células somáticas interespecífica (TNCSi) tem recebidodestaque devido à possibilidade de resgatar material genético de animais extintos e de auxiliar na multiplicaçãode espécies em vias de extinção. No entanto, apesar dos avanços obtidos até o momento, a TNCSi aindaapresenta baixa eficiência por possuir, por exemplo, altas taxas de perdas embrionárias e mortalidade após onascimento. Esses eventos estão relacionados com uma reprogramação anormal, que inclui a capacidade dooócito de modificar o estado de uma célula somática diferenciada para um estado de pluripotência. Assim, estarevisão tem por objetivo descrever o uso dessa biotécnica em diferentes espécies, relatar aspectos molecularesque comprometem o sucesso do desenvolvimento embrionário, bem como mostrar sua importância na medicina por meio da clonagem terapêutica.(AU)

In last years, the interspecific Somatic Cell Nuclear Transfer (iSCNT) has been highlighted due to thepossibility of recovering genetic material of extinct animals and aid to multiplied endangered species. However,despite the progress achieved to date, the iSCNT still has low efficiency by having, for example, high rates toboth embryonic loss and mortality after birth. These events are related to an abnormal reprogramming, whichincludes the oocyte's ability to modify the state of a differentiated somatic cell to a state of pluripotency. So thisreview is to describe the use of this biotechnical in different species, reporting molecular aspects thatcompromise the success of embryonic development as well as show their importance in medicine by therapeuticcloning(AU)

Premissas e promessas da clonagem interespecífica de espécies em risco de extinção / Premises and promises of interspecific cloning in endangered species

Nos últimos anos, a transferência nuclear de células somáticas interespecífica (TNCSi) tem recebidodestaque devido à possibilidade de resgatar material genético de animais extintos e de auxiliar na multiplicaçãode espécies em vias de extinção. No entanto, apesar dos avanços obtidos até o momento, a TNCSi aindaapresenta baixa eficiência por possuir, por exemplo, altas taxas de perdas embrionárias e mortalidade após onascimento. Esses eventos estão relacionados com uma reprogramação anormal, que inclui a capacidade dooócito de modificar o estado de uma célula somática diferenciada para um estado de pluripotência. Assim, estarevisão tem por objetivo descrever o uso dessa biotécnica em diferentes espécies, relatar aspectos molecularesque comprometem o sucesso do desenvolvimento embrionário, bem como mostrar sua importância na medicina por meio da clonagem terapêutica.

In last years, the interspecific Somatic Cell Nuclear Transfer (iSCNT) has been highlighted due to thepossibility of recovering genetic material of extinct animals and aid to multiplied endangered species. However,despite the progress achieved to date, the iSCNT still has low efficiency by having, for example, high rates toboth embryonic loss and mortality after birth. These events are related to an abnormal reprogramming, whichincludes the oocyte's ability to modify the state of a differentiated somatic cell to a state of pluripotency. So thisreview is to describe the use of this biotechnical in different species, reporting molecular aspects thatcompromise the success of embryonic development as well as show their importance in medicine by therapeuticcloning

Reproductive parameters and the use of MOET in transgenic founder goat carrying the human granulocyte colony-stimulating factor (hG-CSF) gene

This study aimed to monitor estrous cycle parameters of a human granulocyte colony-stimulating factor (hG-CSF)-transgenic founder female goat and to perform superovulation and embryo recovery (surgical or transcervical method) for further transfer to recipients to quickly obtain offspring. Two experiments were performed using a transgenic (TF) and a non-transgenic (NTF) female. In experiment 1, three estrous cycles were monitored for the following parameters: estrus behavior, progesterone concentration and ovarian activity. In experiment 2, two superovulation/embryo recovery sessions were performed and the recovered embryos were transferred to previously prepared recipients. Data were compared by either t test or Fisher's exact test. The mean interval between natural estrus was 20.7 ± 0.6 and 19.7 ± 0.6 (P > 0.05) days for the TF and NTF, respectively. Progesterone concentrations and ovarian activity were normal and similar between goats. The ovulation rate was similar between TF and NTF (12.0 ± 1.4 vs. 18.0 ± 4.2 CL; P > 0.05). No significant differences in embryo recovery rate (P > 0.05) were observed between the surgical and transcervical methods for TF (69.2 vs. 72.7%) or NTF (100.0 vs. 86.7%). Sixteen embryos from the TF were transferred to recipients, and eight kids were born. Among these kids, the transgene was identified in three (two males and one female), resulting in a transgenesis rate of 37.5%. In summary, the TF is a true founder, since she proved fertility and capacity of transmitting the hG-CSF transgene to progeny, suggesting that the analyzed reproductive traits were not compromised by the presence of the transgene.

Reproductive parameters and the use of MOET in transgenic founder goat carrying the human granulocyte colony-stimulating factor (hG-CSF) gene

This study aimed to monitor estrous cycle parameters of a human granulocyte colony-stimulating factor (hG-CSF)-transgenic founder female goat and to perform superovulation and embryo recovery (surgical or transcervical method) for further transfer to recipients to quickly obtain offspring. Two experiments were performed using a transgenic (TF) and a non-transgenic (NTF) female. In experiment 1, three estrous cycles were monitored for the following parameters: estrus behavior, progesterone concentration and ovarian activity. In experiment 2, two superovulation/embryo recovery sessions were performed and the recovered embryos were transferred to previously prepared recipients. Data were compared by either t test or Fisher's exact test. The mean interval between natural estrus was 20.7 ± 0.6 and 19.7 ± 0.6 (P > 0.05) days for the TF and NTF, respectively. Progesterone concentrations and ovarian activity were normal and similar between goats. The ovulation rate was similar between TF and NTF (12.0 ± 1.4 vs. 18.0 ± 4.2 CL; P > 0.05). No significant differences in embryo recovery rate (P > 0.05) were observed between the surgical and transcervical methods for TF (69.2 vs. 72.7%) or NTF (100.0 vs. 86.7%). Sixteen embryos from the TF were transferred to recipients, and eight kids were born. Among these kids, the transgene was identified in three (two males and one female), resulting in a transgenesis rate of 37.5%. In summary, the TF is a true founder, since she proved fertility and capacity of transmitting the hG-CSF transgene to progeny, suggesting that the analyzed reproductive traits were not compromised by the presence of the transgene.(AU)

Mensurações ultrassonográficas da cisterna da glândula mamária de caprino transgênico / Ultrasound measurements of the mammary gland of transgenic hormone-induced lactating goat

Milk production of transgenic does was evaluated by ultrasound measurements of the mammary gland. Two Canindé goats, which were nine months of age were used in the trial, one non-transgenic or other transgenic for hG-CSF. For hormone-induced lactation, animals were given estradiol (0.25mg/kg, IM), progesterone (0.75mg/kg, IM), and prednisolone (0.4mg/kg, IM). Ultrasonographic exams were carried out during milking, using a Falcon 100 ultrasound equipment with a 5MHz convex probe and were performed by the same operator. The results were expressed as mean±standard error. The maximum greater length and shorter length of the cistern were respectively 5.14cm and 1.36cm for the transgenic animal and 7.28cm and 2.25cm for non-transgenic, which is consistent with the maximum milk volume produced. The relationship between the average area of cisterns and milk yield was expressed as a linear correlation curve, with a correlation coefficient significantly positive for both transgenic (Y=-1.1314+10.8538*x; r=0.97) and non-transgenic (Y=-21.7551+18.3634*x; r=0.97) animals. In conclusion, the ultrasound is a practice and appropriate technique to evaluate the cisterns in ruminant udders in transgenic animal.(AU)

Oocytoe production and in vitro maturation in Canindé goats following hormonal ovarian stimulation

This study e valuated the effect of hormonal ovarian stimulation regimes on the quantity and quality of the cumulus - oocyte complexes (COCs) recovered by laparoscopy and their subsequent in vitro matur ation (IVM) . Eighteen cyclic Canindé goats received a vaginal sponge with 60 mg m edroxyprogesterone acetate for 11 days, together with a n injection of 50 μg d - cloprostenol on the 8 th day , along with additional treatment re g imens , as follows : i) five doses (5D), 120 mg of NIH - FSH - P1 in five injections at 12 h inter vals; ii) three doses (3D), 120 mg of NIH - FSH - P1 in three injections at 24 h i nterv als; iii) single dose (1D), 70 mg of NIH - FSH - P 1 and 200 IU of eCG at 36 h prior to sponge rem oval. Three sessions of hormonal treatment/oocyte recover y w ere performed and goats ( n = 6 / group) were allocated to different grou ps . The oocytes were collect ed by laparoscopy at the time of sponge removal and the IVM of the oocytes was monitored. A total of 14.8 ± 0.8 follicles were aspirated per animal with 11.1 ± 0.6 COCs being rec overed , resulting in a recovery rate of 74.7 % ( 577/772 ) . The mean number of CO Cs/goat did not differ (P > 0.05) between treatments (11.7 ± 1.1, 10.7 ± 0.9 and 10.8 ± 1.0 for the 5D, 3D and 1D groups, respectively). R ecovery rate was higher (P < 0.05) in the 5 D group ( 84.1 % ; 211/251 ) compared to the 3 D ( 68.2 % ; 182/267 ) and 1D group s (72.4% ; 184/254 ). T he lowest (P < 0.05) maturation rate was recorded in the 3D group (32.1% ; 27/84 ), while the rate for the 5D and 1D groups was 49.1% ( 53/108 ) and 46.2% ( 42/91 ) , respectively. Finally, taking into account the main performance results of th e three treatments, it i s advisable to use the 5D regime in future Canindé breed preservation programs based on laparoscopy technology as a means to recover oocytes .

Oocytoe production and in vitro maturation in Canindé goats following hormonal ovarian stimulation

This study e valuated the effect of hormonal ovarian stimulation regimes on the quantity and quality of the cumulus - oocyte complexes (COCs) recovered by laparoscopy and their subsequent in vitro matur ation (IVM) . Eighteen cyclic Canindé goats received a vaginal sponge with 60 mg m edroxyprogesterone acetate for 11 days, together with a n injection of 50 μg d - cloprostenol on the 8 th day , along with additional treatment re g imens , as follows : i) five doses (5D), 120 mg of NIH - FSH - P1 in five injections at 12 h inter vals; ii) three doses (3D), 120 mg of NIH - FSH - P1 in three injections at 24 h i nterv als; iii) single dose (1D), 70 mg of NIH - FSH - P 1 and 200 IU of eCG at 36 h prior to sponge rem oval. Three sessions of hormonal treatment/oocyte recover y w ere performed and goats ( n = 6 / group) were allocated to different grou ps . The oocytes were collect ed by laparoscopy at the time of sponge removal and the IVM of the oocytes was monitored. A total of 14.8 ± 0.8 follicles were aspirated per animal with 11.1 ± 0.6 COCs being rec overed , resulting in a recovery rate of 74.7 % ( 577/772 ) . The mean number of CO Cs/goat did not differ (P > 0.05) between treatments (11.7 ± 1.1, 10.7 ± 0.9 and 10.8 ± 1.0 for the 5D, 3D and 1D groups, respectively). R ecovery rate was higher (P < 0.05) in the 5 D group ( 84.1 % ; 211/251 ) compared to the 3 D ( 68.2 % ; 182/267 ) and 1D group s (72.4% ; 184/254 ). T he lowest (P < 0.05) maturation rate was recorded in the 3D group (32.1% ; 27/84 ), while the rate for the 5D and 1D groups was 49.1% ( 53/108 ) and 46.2% ( 42/91 ) , respectively. Finally, taking into account the main performance results of th e three treatments, it i s advisable to use the 5D regime in future Canindé breed preservation programs based on laparoscopy technology as a means to recover oocytes .(AU)

Clonagem em ruminantes: progressos e perspectivas atuais / Cloning in ruminants: progress and current perspectives

Desde o nascimento da ovelha Dolly, grupos de pesquisas têm empregado a técnica de transferência nuclear (TN) ou clonagem para a produção de embriões e crias em diferentes espécies de ruminantes. Em geral, a transferência nuclear consiste em transferir núcleos de células doadoras para o interior de oócitos enucleados, resultando na produção de indivíduos geneticamente idênticos ao animal doador de núcleo. Contudo, a eficiência da técnica ainda é baixa devido a inúmeros fatores. Assim, o objetivo deste trabalho é realizar uma revisão sobre a transferência nuclear em suas várias etapas, destacando os principais fatores que interferem no seu sucesso e discutir as perspectivas atuais provenientes do surgimento de metodologias e adaptações aos protocolos experimentais.(AU)

Since the birth of Dolly, research groups have employed the nuclear transfer (NT) or cloning to embryo and offspring production in many ruminant species. In general, this technique consists on nuclear transfer of donor cells into enucleated oocytes, resulting on the production of genetic identical individual to the nucleus donor animal. However, there are many factors that reduce the efficiency of this technique. Thus, the objective of this manuscript is to describe several steps of the technique with emphasis to the main factors that interfere on its success and to discuss current perspectives considering some new approaches in the methodology and experimental protocols.(AU)

Clonagem em ruminantes: progressos e perspectivas atuais / Cloning in ruminants: progress and current perspectives

Desde o nascimento da ovelha Dolly, grupos de pesquisas têm empregado a técnica de transferência nuclear (TN) ou clonagem para a produção de embriões e crias em diferentes espécies de ruminantes. Em geral, a transferência nuclear consiste em transferir núcleos de células doadoras para o interior de oócitos enucleados, resultando na produção de indivíduos geneticamente idênticos ao animal doador de núcleo. Contudo, a eficiência da técnica ainda é baixa devido a inúmeros fatores. Assim, o objetivo deste trabalho é realizar uma revisão sobre a transferência nuclear em suas várias etapas, destacando os principais fatores que interferem no seu sucesso e discutir as perspectivas atuais provenientes do surgimento de metodologias e adaptações aos protocolos experimentais.

Since the birth of Dolly, research groups have employed the nuclear transfer (NT) or cloning to embryo and offspring production in many ruminant species. In general, this technique consists on nuclear transfer of donor cells into enucleated oocytes, resulting on the production of genetic identical individual to the nucleus donor animal. However, there are many factors that reduce the efficiency of this technique. Thus, the objective of this manuscript is to describe several steps of the technique with emphasis to the main factors that interfere on its success and to discuss current perspectives considering some new approaches in the methodology and experimental protocols.

Sincronização do estro, indução da ovulação e fertilidade de ovelhas deslanadas após tratamento hormonal com gonadotrofina coriônica eqüina

The aim of this experiment was to evaluate the effect of different doses of eCG on reproductive performance of ewes. The estrus was synchronized with vaginal sponges with 30mg FGA during 12 days. At the time of sponge removal, ewes were distributed in three groups: 0 (n=26), 200IU eCG (n=30) and 400IU eCG (n=30). The estrus was detected by aid of a vasectomized ram. The ewes were inseminated by laparoscopy, 60h after sponge removal. Blood samples were collected at 5 and 18 days after insemination, in order to determine progesterone concentration. It was observed 70.9% of ewes in estrus after the end of treatment. The females that received eCG showed higher (P 0.05) percentage of estrus: 96.7% (400IU) and 76.7% (200IU) versus 34.6% (0 IU). The interval between the end of treatment and estrus onset was higher (P 0.05) in the group that did not receive eCG (54.7±6.3h) in comparison to the groups that received 200IU (45.9±7.8 h) and 400 IU eCG (40.4±10.3h). A lower (P 0.05) number of ovulating and pregnant ewes in the group that did not receive eCG was observed. The eCG withdraw resulted in negative effect on reproductive performance of ewes.

Utilizou-se a sincronização do estro com esponjas vaginais de 30mg de acetato de fluorogestona durante 12 dias para avaliar o efeito de diferentes doses de eCG sobre o desempenho reprodutivo de ovelhas. Na retirada das esponjas as ovelhas foram divididas em três grupos para receberem 0 (n=26), 200 (n=30) ou 400UI (n=30) de eCG. O estro foi detectado a cada 12h utilizando-se um rufião. As fêmeas foram inseminadas por laparoscopia 60h após a retirada das esponjas. Realizaram-se colheitas de sangue aos 5 e 18 dias pós-inseminação para dosagem de progesterona e determinação do número de ovulações e prenhezes, respectivamente. A fertilidade foi verificada por ecografia aos 60 dias e ao parto. Das 86 ovelhas 70,9% apresentaram estro. Essa porcentagem foi maior (P 0,05) nas fêmeas tratadas com eCG: 96,7% (400UI) e 76,7% (200UI) versus 34,6% (0 UI). O intervalo entre o final do tratamento e o início do estro foi maior (P 0,05) no grupo sem eCG, 54,7±6,3h versus 45,9±7,8h para 200UI e 40,4±10,3h para 400UI. Verificou-se menor (P 0,05) número de ovulações e prenhezes no grupo sem eCG. A não aplicação de eCG influiu negativamente no desempenho reprodutivo de ovelhas deslanadas.