Ovum pick-up technique in recently weaned ewe lambs subjected to ovarian stimulation

Background: The purpose of this study was to evaluate the effectiveness and possible complications of the laparoscopic ovum pick-up (LOPU) technique and to optimize ovarian stimulation in recently weaned ewe lambs.Materials, Methods & Results: Thirty-six healthy lambs, aging 4-8 weeks, were used in this study and divided into six different groups of six animals. Intravaginal implants of medroxyprogesterone were used for follicular wave induction, and parenteral equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) were administered for ovarian stimulation. Ewes from group GCN did not receive any treatment. In group GCI, the animals received only intravaginal implants. For other groups, ovarian stimulation was carried out as follows: in Group G80U, ewes were treated with a single dose of 80 mg of FSHp; in Group G80M, ewes received 4 administrations of 20 mg FSH, in Group G160U, they were treated with a single dose of 160 mg FSH; and in Group G160M they received 4 administrations of 40 mg FSH). Quantitative and qualitative assessment of retrieved oocytes were carried out after LOPU. In vitro maturation (IVM) of oocytes was also quantitative and qualitatively analyzed. Surgical procedure was divided into 6 stages and overall and stage surgical times were assessed. Post-operative pain and plasma fibrinogen were also assessed. The mean overall surgical time...(AU)

Ovum pick-up technique in recently weaned ewe lambs subjected to ovarian stimulation

Background: The purpose of this study was to evaluate the effectiveness and possible complications of the laparoscopic ovum pick-up (LOPU) technique and to optimize ovarian stimulation in recently weaned ewe lambs.Materials, Methods & Results: Thirty-six healthy lambs, aging 4-8 weeks, were used in this study and divided into six different groups of six animals. Intravaginal implants of medroxyprogesterone were used for follicular wave induction, and parenteral equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) were administered for ovarian stimulation. Ewes from group GCN did not receive any treatment. In group GCI, the animals received only intravaginal implants. For other groups, ovarian stimulation was carried out as follows: in Group G80U, ewes were treated with a single dose of 80 mg of FSHp; in Group G80M, ewes received 4 administrations of 20 mg FSH, in Group G160U, they were treated with a single dose of 160 mg FSH; and in Group G160M they received 4 administrations of 40 mg FSH). Quantitative and qualitative assessment of retrieved oocytes were carried out after LOPU. In vitro maturation (IVM) of oocytes was also quantitative and qualitatively analyzed. Surgical procedure was divided into 6 stages and overall and stage surgical times were assessed. Post-operative pain and plasma fibrinogen were also assessed. The mean overall surgical time...

Reproductive efficiency of adult and prepubertal goats subjected to repeated follicular aspiration / Eficiência reprodutiva em cabras adultas e pré-púberes após repetidas sessões de aspiração folicular

The objective of this study was to assess the reproductive response of adult and prepubertal goats subjected to repeated laparoscopic ovum pick-up (LOPU). The study animals were divided into two groups, specifically, adult nanny goats (GA, n=10) and prepubertal nanny goats (GP, n=10), which were subjected to estrous synchronization and ovarian stimulation for LOPU. Both groups underwent six LOPU procedures at seven-day intervals and were subsequently subjected to controlled mating and pregnancy diagnosis to evaluate their future fertility. The study showed a reduction in the number of follicles visualized and in the amount and quality of the oocytes that were recovered and exposed to in vitro maturation. As indicated by the fertility test, however, no complications were found during the laparoscopic procedures that would impair the reproductive future of the animals. Therefore, a viable number of oocytes were obtained even with the decreased reproductive efficiency, proving that repeated LOPUs do not interfere with the reproductive of adult and prepubertal nanny goats. These results indicate a positive aspect of this procedure, allowing for increasing reproductive performance of this kind, when used for the production in vitro.(AU)

O objetivo deste estudo foi avaliar a resposta reprodutiva de cabras adultas e pré-púberes submetidas a repetidas aspirações foliculares por videolaparoscopia (LOPU). Os animais do estudo foram divididos em dois grupos, especificamente cabras adultas (GA, n = 10) e pré-púberes (GP, n = 10), que foram submetidas a sincronização de estro e estimulação ovariana para LOPU. Ambos os grupos foram submetidos a seis procedimentos LOPU em intervalos de sete dias e foram posteriormente submetidos à monta controlada e posterior diagnóstico de gestação para avaliar sua fertilidade. O estudo mostrou uma diminuição do número de folículos visualizados, na quantidade e na qualidade dos oócitos que foram recuperados e expostos à maturação in vitro. Tal como indicado pelo teste de fertilidade, no entanto, não foram observadas grandes complicações durante os procedimentos laparoscópicos que pudessem prejudicar o futuro reprodutivo dos animais. Portanto, foi obtido um número de oócitos viáveis, mesmo com a diminuição da eficiência reprodutiva, provando que repetidas LOPU não interferem na reprodutividade de animais adultos e pré-púberes. Estes resultados indicam um aspecto positivo desse procedimento, possibilitando um aumento da eficiência reprodutiva dessa espécie quando utilizado para a produção in vitro.(AU)

ENSAIO CLÍNICO COM CÉLULAS-TRONCO MESENQUIMAIS (CTM) NA RECUPERAÇÃO DE OVÁRIOS DE CABRAS SUBMETIDAS A MÚLTIPLAS ASPIRAÇÕES FOLICULARES POR LAPAROSCOPIA (LOPU)

Os problemas ligados à diminuição do potencial produtivo dos ovários estão entre os principais fatores limitantes à reprodução com isso, objetivou-se com este trabalho avaliar o efeito da infusão de células-tronco mesenquimais (CTM) em ovários de cabras submetidas a múltiplas aspirações foliculares por laparoscopia (LOPU). Foram utilizadas cabras sem padrão racial definido (SPRD), não lactantes, com idade média de três anos e escore da condição corporal (ECC) variando de 3,0 a 4,0 e distribuídas em dois experimentos, no primeiro com doze cabras (n=12) e no segundo com sete (n=7). No experimento um as cabras foram sincronizadas com o uso de esponjas intravaginais com acetato de medroxiprogesterona e aplicações de d-cloprostenol e a estimulação ovariana constituiu-se na administração de FSHp e eCG, em aplicações únicas, feita 36 horas anterior às LOPUs, e posteriormente as fêmeas foram submetidas a 5 sessões/animal de LOPU com intervalos de 12 dias. No experimento dois as cabras sincronizadas da mesma forma do experimento um e posteriormente divididas em dois tratamentos: T1-CTR (Controle 3 animais); T2 - CTM (ovários tratados com células tronco mesenquimais 4 animais), incluindo a terapia celular através de infusão ovariana com células tronco CTM, na concentração de 1 x 106 cel/mL, nanomarcadas com Qtracker® e diluídas em 0,2 mL de solução fisiológica (NacL a 0,9%) em duas aplicações: na última LOPU e 70 dias após. As avaliações quanto à funcionalidade ovariana/oocitária foi realizada 45 dias posterior à ultima infusão de CTM. Coletas de sangue para dosagens hormonais de estradiol e progesterona foram realizadas, antes da primeira, e após a quinta LOPU juntamente com biopsias do córtex ovariano. A avaliação histológica foi realizada após 115 dias da primeira infusão de CTM, a partir do abate dos animais e coleta dos ovários. Todas as análises foram executadas através do programa Statistical Analysis System (SAS Institute Inc., 2002). No experimento um, houve diferença significativa (P<0,05) na média de folículos aspirados com menor média na 3ª LOPU. Em relação à média de oócitos recuperados, não observou-se diferença significativa entre a 1ª e a 5ª LOPU (P>0,05), porem as médias dos folículos aspirados na 2ª, 3ª e 4ª LOPU reduziram significativamente em relação a 1ª LOPU (P<0,05). Quanto a taxa de recuperação oocitária, houve uma redução na 3ª e 4ª LOPU em relação à 1ª e 2ª LOPU (P<0,05) e na 5ª LOPU observou-se um aumento da recuperação oocitaria em relação à 3ª e 4ª LOPU (P<0,05). Quanto a qualidade, os CCOs de grau 2 reduziram entre a 1ª e a 4ª LOPU e os oócitos de grau 4 aumentaram na 2ª, 4ª e 5ª LOPU em relação a 1ª e 3ª LOPU(P<0,05). Houve uma redução (P<0,05) de estruturas viáveis na 2ª e 4ª LOPU (46,4% e 53,8% respectivamente), com retomada da viabilidade na 3ª e 5ª LOPU (80,76% e 61,2% respectivamente). As biópsias que antecederam as coletas e pós sucessivas aspirações evidenciaram tecidos ovarianos morfologicamente normais, com alterações discretas no parênquima ovariano. Não houve diferença (P>0,05) na dosagem de progesterona entre as sucessivas LOPUs porem houve um aumento (P<0,05) na dosagem de estradiol da 2º LOPU em relação às demais. No experimento 2, não houve diferença significativa (P>0,05) entre os folículos aspirados, oócitos recuperados e taxa de recuperação em relação aos grupos controle e grupos tratados com CTMs. Em relação a qualidade dos CCOs não ocorreu diferença significativa (P<0,05) entre as classificações das estruturas oocitárias. Nas avaliações histológicas foram observadas discretas alterações no parênquima ovariano, sem comprometimento do mesmo. Conclui-se que, cinco sessões de aspirações foliculares por laparoscopia com intervalos de doses dias não interfere morfofuncionalmente nos ovários de cabras e que a utilização de CTM não influenciou nos parâmetros qualitativos e quantitativos de oócitos recuperados por LOPU de cabras SPRD.

The problems related to the reduction of ovaries productive potential are among the main limiting factors for reproduction, therefore, the objective of this work was to evaluate the effect of the infusion of mesenchymal stem cells (CTM) on ovary of goats submitted to multiple follicular aspirations by laparoscopy (LOPU). Goats with undefined racial pattern (SPRD), non-lactating, with mean age of three years and body condition scoring (ECC) ranging from 3.0 to 4.0 were used and distributed in two experiments, in the first with twelve goats (n= 12) and in the second with seven (n = 7). In the first experiment, the goats were synchronized using intravaginal sponges with medroxyprogesterone acetate and d-cloprostenol applications, ovarian stimulation consisted of administration of FSHp and eCG in single applications 36 hours prior to LOPUs, and later females were submitted to 5 sessions/animal of LOPU at 12-day intervals. In the second experiment, the goats were synchronized in the same way of the first experiment and later divided into two treatments: T1-CTR (Control - 3 animals); T2-CTM (ovaries treated with mesenchymal stem cells - 4 animals), including cell therapy through ovarian infusion in two applications with 1 x 106 cel/mL of stem cells - CTM, nanoparticulated with Qtracker ® and diluted in 0.2 mL of physiological solution (0.9% NaCl), these two applications were performed at the last LOPU and 70 days after. Evaluations for ovarian / oocyte functionality were performed 45 days after the last CTM infusion. Blood collections for hormonal dosages of estradiol and progesterone were performed, before the first LOPU, and after the fifth LOPU simultaneously to biopsies of the ovarian cortex. The histological evaluation was performed after the animals slaughter and collection of the ovaries, 115 days after the first CTM infusion. All analyzes were performed through the Statistical Analysis System (SAS Institute Inc., 2002). In the first experiment, there was a significant difference (P <0.05) in the mean of the aspirated follicles with lower mean in the 3rd LOPU. Regarding the mean of oocytes recovered, no significant difference was observed between the 1st and 5th LOPU (P> 0.05), however the mean of follicles aspirated in 2nd, 3rd and 4th LOPU significantly reduced compared to 1st LOPU (P <0.05). Regarding the oocyte recovery rate, there was a reduction in the 3rd and 4th LOPU in relation to the 1st and 2nd LOPU (P <0.05) and in the 5th LOPU there was an increase in oocyte recovery in relation to the 3rd and 4th LOPU (P <0.05). As regards the quality, the grade 2 levels of the CCO system increased between 1st and 4th LOPU and grade 4 levels increased in 2nd, 4th and 5th LOPU in relation to 1st and 3rd LOPU (P <0.05). There was a reduction (P <0.05) of viable structures in the 2nd and 4th LOPU (46.4% and 53.8%, respectively), with recovery of viability in the 3rd and 5th LOPU (80.76% and 61.2% respectively). The biopsies that preceded the collections and subsequent successive aspirations revealed morphologically normal ovarian tissues, with discrete alterations in the ovarian parenchyma. There was no difference (P> 0.05) in the progesterone dosage between successive LOPU's, but there was an increase (P <0.05) in the estradiol dosage of the 2nd LOPU in relation to the others. In the second experiment, there was no significant difference (P> 0.05) between aspirated follicles, recovered oocytes and recovery rate in relation to the control groups and groups treated with CTM's. Regarding the quality of CCOs, there was no significant difference (P <0.05) between the classifications of oocyte structures. In the histological evaluations, discrete alterations in the ovarian parenchyma were observed, without any impairment. It was concluded that five sessions of follicular aspiration by laparoscopy with dose and day intervals did not interfere morphofunctionally in the ovaries of goats and that the use of CTM did not influence the qualitative and quantitative parameters of oocytes recovered by LOPU from SPRD goats.

Nascimento de bezerra gerada com auxílio das técnicas de punção folicular e fertilização in vitro no Estado de Minas Gerais

Ultrasound-assisted follicular puncture is a technique of oocyte recovery for in vitro fertilization and production of embryos. In order to adjust the methodology, a field trial was performed using lactating Holstein cows (n=8) as oocyte donors. Punctures were done with an ultrasound device equipped with a 7.5 MHz intravaginal sectorial probe. Oocytes recovered were matured, fertilized and cultured in vitro, and embryos produced transferred to recipients heifers. On July, 2000, the first calf produced by these techniques in Minas Gerais State, Brazil, was born.