This study was performed to test the viability of administering a single 40 mg dose of Folltropin-V® (FSH, Bioniche Animal Health) diluted in SRF (MAP-5 50, Sodium Hyaluronate, Bioniche Animal Health) on day 5 of a recipient synchronization protocol to beef cows to evaluate its effect on recipient fertility. All recipients were administered an estradiol 17beta (2.5 mg, IM) and progesterone (50 mg, IM) combination injection on day 0 and a CIDR® (progesterone 1.34 g, Pfizer Animal Health) was inserted. Lutalyse® (dinoprost tromethamine, Pfizer Animal Health, 25 mg, IM) was administered at the time of CIDR removal on day 7, and estradiol 17beta (1 mg, IM) was administered on day 8. On day 16, the presence of at least one corpus luteum (CL), detected via ultrasound, resulted in the recipient receiving an embryo (both fresh and frozen-thawed embryos were used). Embryos were not transferred into cows that did not show the presence of a CL. Dependent variables for which data were collected included circulating progesterone levels at the time of transfer, number of CLs and CL diameter, circumference, and area; measured in millimeters. The study (n=572) consisted of a treatment group (n=268) and a control group (n=304), and included both Bos indicus (Brahman influenced) crossbred (n=115) and Bos taurus (Angus based) cows (n=457). Pregnancy rates for Treated recipients (40.67%A) and Control recipients (52.96%B) differed (P<.05). There was no difference in the mean number of CLs per recipient for Treated (1.14 +/- .03) and Control (1.10 +/- .02) cows, nor was there a difference in progesterone (P4) at the time of transfer for Treated (3.14 +/- .40 ng/mL) and Control (3.23 +/- .18 ng/mL) recipients. Overall, the inclusion of Folltropin-V® diluted in hyaluronan in a FTET synchronization protocol did not improve the fertility of beef recipients.

No description available.

Master of Science / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / We hypothesized that administration of human chorionic gonadotropin (hCG) to recipients at embryo transfer (ET) would induce accessory corpora lutea (CL), increase circulating progesterone concentrations, and reduce early embryonic loss. At three locations, purebred and crossbred Angus, Simmental, and Hereford recipients (n = 719) were assigned alternately to receive i.m. 1,000 IU hCG or 1 ml saline (control) at ET. Fresh or frozen-thawed embryos were transferred on d 5.5 to 8.5 (median = d 7) of the estrous cycle to recipients having a palpable CL. Recipients received a body condition score (BCS) at ET. Pregnancy diagnoses occurred by transrectal ultrasonography 28 to 39 d (median = d 35) and reconfirmed 58 to 77 d (median = d 67) post-estrus. At one location (n = 108), ovaries were examined to count the number of CL at pregnancy diagnosis. More (P < 0.001) pregnant hCG-treated cows (69.0%) had multiple CL than pregnant controls (0%). Serum progesterone (ng/mL) determined at two locations (n=471) at both pregnancy diagnoses in pregnant cows was greater (P ≤ 0.05) after hCG treatment than in controls (first: 8.1 ± 0.9 vs. 6.1 ± 0.8; second: 8.8 ± 0.9 vs. 6.6 ± 0.7), respectively. Transfer pregnancy rates were analyzed using logistic regression. Unadjusted pregnancy rates at the first diagnosis was 61.8 vs. 53.9% for hCG vs. controls. At the second diagnosis, pregnancy rates were 59.0 vs. 51.4%, respectively. Factors affecting pregnancy rates were treatment (P = 0.03), embryo type (P = 0.02), and BCS (P = 0.08). Odds ratios indicated that greater pregnancy rates occurred in recipients receiving hCG treatment, receiving a fresh embryo (66.3 vs.55.5%), and when BCS >5 vs. ≤5 (62.3 vs. 55.3%). We concluded that hCG at ET increased incidence of accessory CL, increased progesterone in pregnant recipients, and increased transfer pregnancy rates.

As receptoras de embriões bovinos devem possuir um ambiente uterino adequado para o desenvolvimento do embrião, estabelecimento da gestação e nascimento de um bezerro saudável. Apesar dos avanços tecnológicos, a perda embrionária ainda permanece elevada, resultando em taxas de prenhez reduzidas após a transferência de embriões (TE). A progesterona (P4) apresenta papel importante nos processos de estabelecimento e manutenção de gestação em fêmeas bovinas. Diversos estudos evidenciaram que o tratamento com P4 injetável de longa ação (P4LA) após a inseminação artificial em tempo fixo (IATF) pode favorecer o estabelecimento da gestação e aumentar as taxas de concepção. Assim, o objetivo do presente estudo foi verificar se o tratamento com P4LA após a ovulação sincronizada em receptoras de embriões produzidos in vitro (PIV) resultará em aumento na concentração de P4 e na taxa de prenhez, sem comprometer o desenvolvimento do corpo lúteo (CL). Para isso, o presente estudo foi dividido em dois experimentos. O Experimento 1 foi realizado no Brasil com 41 vacas Bos indicus (Nelore) tratadas com um dispositivo intravaginal de P4 e 2 mg de benzoato de estradiol (BE) i.m. no D-10. No D-2, o dispositivo foi removido e as vacas receberam 300 UI de gonadotrofina coriônica equina (eCG) e 0,530 mg de cloprostenol sódico (PGF). No D-1, foi administrado 1 mg de BE para a indução da ovulação. Das 41 vacas, 36 (87,8%) ovularam e foram aleatoriamente distribuídas em três grupos experimentais: Controle (n = 13), sem tratamento adicional; P4D4 (n = 11), administração de 150 mg de P4LA i.m no D4; e P4D7 (n = 12), administração de 150 mg de P4LA i.m. no D7. Foram realizadas avaliações ultrassonográficas Color Doppler para verificar a perfusão do CL e colheitas de sangue para análise da concentração de P4 do D4 ao D7 a cada 24 h e do D7 ao D25 a cada 48 h. Verificou-se interação tratamento*tempo (P = 0,004) para concentração plasmática de P4, com aumento transitório na concentração de P4 24h após a administração de P4LA no D4 e D7. Entretanto, não ocorreu interação tratamento*tempo para o diâmetro do CL (P = 0,59) e tampouco efeito dos tratamentos no fluxo sanguíneo central (FSC; P = 0,97) e periférico (FSP) do CL (P = 0,97). No experimento 2, o mesmo protocolo de sincronização descrito no experimento 1 foi empregado em 787 receptoras mestiças sincronizadas para TE na República Dominicana. Destas, 69,9% apresentaram CL &gt; 14 mm no D4 e foram distribuídas aleatoriamente nos 3 grupos experimentais: Controle (n = 168), P4D4 (n = 201) e P4D7 (n = 181). A TE de embrião PIV foi realizada no D7 e o diagnóstico de gestação foi realizado por ultrassonografia no D30 e D60. A taxa de concepção aos 30 dias [Controle: 41,4% (70/168); P4D4: 42,3% (89/201); P4D7: 41,2% (80/181); P = 0,41], aos 60 dias [Controle: 36,3% (61/168); P4D4: 39,8% (80/201); P4D7: 38,1% (69/181); P = 0,49] e a perda gestacional [Controle: 5,4% (9/168); P4D4: 4,5% (9/201); P4D7: 6,1% (11/181); P = 0,73] não diferiram entre os tratamentos. Em conclusão, apesar do aumento da concentração plasmática de P4 após os tratamentos com P4LA no D4 ou D7, não foi observado efeito positivo desses tratamentos na taxa de prenhez e na perda gestacional de receptoras de embriões bovinos. Além disso, os tratamentos com P4LA não comprometeram o desenvolvimento do CL. / The recipients of bovine embryos must have a suitable uterine environment for embryo development, establishment of gestation and birth of a healthy calf. Despite technological advances, embryo loss remains high, resulting in reduced pregnancy rates after embryo transfer (ET). Progesterone (P4) plays an important role in the processes of establishing and maintaining gestation in bovine females. Several studies have shown that treatment with long-acting injectable progesterone (P4LA) after timed artificial insemination (TAI) can help the establishment of pregnancy and increase conception rates. Thus, the aim of the present study was to verify whether treatment with P4LA after synchronized ovulation in recipients of in vitro produced (IVP) embryos would increase P4 plasmatic concentration and pregnancy rate, without interfering with the development of corpus luteum (CL). The study was divided in two experiments. Experiment 1 was conduced in Brazil with 41 Bos indicus cows (Nelore) synchronized with a P4 intravaginal device and 2 mg of estradiol benzoate (EB) i.m. on D-10. On D-2, the device was removed and cows received 300 IU of equine chorionic gonadotropin (eCG) and 0.530 mg of sodium Cloprostenol (PGF) i.m. On D-1, 1 mg of EB was administered to induce ovulation. From the 41 cows, 36 (87.8%) ovulated and were randomly distributed in three experimental groups: Control (n = 13), without any additional treatment; P4D4 (n = 11) administration of 150 mg of P4LA i.m on D4; and P4D7 (n = 12), administration of 150 mg of P4LA i.m. on D7. The CL blood flow was evaluated using Color Doppler ultrasonography and blood sampling was collected for analysis of P4 concentration from D4 to D7 every 24 h and from D7 to D25 every 48 h. Interaction Treatment*Time (P = 0.004) was found for plasma P4 concentration, with a transient increase of P4 concentration 24h after treatments with P4LA on D4 and D7. However, there was neither an interaction Treatment*Time for CL diameter (P = 0.59), nor an effect of CL central (P = 0.97) and peripheral blood flow (P = 0.97). In experiment 2, the same synchronization protocol described in experiment 1 was used in 787 recipients synchronized for ET in Dominican Republic. From those, 69.9% had CL &gt; 14 mm on D4 and were randomly assigned to the following experimental groups: Control (n = 168), P4D4 (n = 201), and P4D7 (n = 181). The IVP embryos were transferred on D7 and the gestation diagnosis was performed on D30 and D60 by ultrasonography. The conception rate at 30 days [Control: 41.4% (70/168); P4D4: 42.3% (89/201); P4D7: 41.2% (80/181); P = 0.41] at 60 days [Control: 36.3% (61/168); P4D4: 39.8% (80/201); P4D7: 38.1% (69/181); P = 0.49] and gestational loss [Control: 5.4% (9/168); P4D4: 4.5% (9/201); P4D7: 6.1% (11/181); P = 0.73] did not differ between treatments. In conclusion, despite the increase in plasma P4 concentration after treatment with P4LA on D4 and D7, no positive effect of using P4LA was observed on conception rate and gestational loss of bovine embryo recipients. In addition, P4LA treatments did not compromise the development of CL.

A introdução de novas linhagens de camundongos em biotérios deve ser realizada com cautela devido aos riscos de introdução de doenças na colônia, motivo pelo qual alguns biotérios adotam como padrão introduzir novos animais somente após o processo de rederivação, que pode ser realizado por transferência embrionária, histerectomia ou cross-fostering. A rederivação é adotada para diversas espécies de animais de laboratório e constitui uma forma de reduzir o risco de surtos de doenças em um biotério com a consequente interferência na pesquisa e perdas, tanto do status sanitário como do bem-estar dos animais. Dentre esses métodos, a transferência de embriões nos estágios de pré-implantação é considerada mais segura que os outros métodos utilizados para se rederivar linhagens de camundongos por reduzir os riscos da transmissão vertical de patógenos. Essa técnica é adotada em diversos biotérios internacionais, porém não é rotina em biotérios do Brasil. O objetivo desse trabalho foi implantar a técnica de rederivação por transferência de embriões no estágio de duas células, no biotério de camundongos livres de patógenos específicos (SPF) do Departamento de Imunologia do Instituto de Ciências Biomédicas da Universidade de São Paulo. As transferências embrionárias foram realizadas para introdução de linhagens de camundongos procedentes de diferentes biotérios convencionais sem barreiras. Os embriões foram obtidos pelos métodos naturais, por meio do acasalamento das fêmeas doadoras superovuladas, com machos de genótipo ou fundo genético igual. Os embriões foram coletados por flushing e lavados em meio estéril. Realizou-se a transferência de embriões no estágio de duas células para fêmeas receptoras livres de patógenos específicos (SPF), pseudoprenhas no 0,5 dia pós coito (d.p.c.). Todos os procedimentos cirúrgicos foram realizados em condições assépticas. Total de 881 embriões, destes, 625 foram transferidos para rederivação de 18 linhagens de camundongos. Nasceram 148 filhotes vivos, dos quais 140 foram desmamados. Os seguintes patógenos foram eliminados pela técnica: vírus da hepatite murina, norovírus, Corynebacterium kutscheri, Staphylococcus aureus, Streptococcus beta hemolítico, Bordetella bronchiseptica e protozoários intestinais. Os dados apresentados foram obtidos da rotina do biotério num período de três anos. A implantação da técnica possibilitou a introdução de novas linhagens na criação, em condições sanitárias satisfatórias, com a consequente disponibilização de modelos animais com nível sanitário adequado à experimentação para a comunidade científica. / The introduction of new strains of mice should be performed carefully to avoid breaking sanitary barriers of specific pathogen free (SPF) animal facilities. In order to meet this need, animals should be rederived by embryo transfer, hysterectomy or cross-fostering and then maintained under strict biosecurity practices. Rederivation is a technique used in different species of laboratory animals to protect them from infectious agents, minimize risks of disease outbreaks and thus avoid research interference caused by loss of the health status and welfare of the animals. Among these techniques, embryo transfer at two cell stage should rather be implemented because it avoids post implantational vertical transmissions of infections. It is routine to introduce animals by embryo transfer in most animal facilities in the United States of America and Europe, however it is not seen in Brazilians rodents vivariums. The objective was to implement mice embryo transfer technique in the animal facility of the Department of Immunology of the Institute of Biomedical Science of the University of Sao Paulo, Brazil. Embryo transfers were performed to rederive strains of mice received from different sources. Fertilized eggs at two-cell stage were obtained by mating superovulated females with fertile males that had either the same genotype or the same genetic background. Embryos were collected by flushing of the oviducts and washed in sterile medium. Under general anesthesia, embryos were transferred into the oviducts of specific pathogen free (SPF) pseudo pregnant female mice at 0,5 - day post coitus (d.p.c.). All the surgical procedures were performed under asseptic conditions. Total of 881 embryos, out of these, 625 embryos at two-cell stage were transfered to rederive 18 mouse strains. It was born 148 pups, of which 140 were reared. The following mouse pathogens were eliminated by embryo transfer technique: Mouse hepatitis virus, Norovirus, Corynebacterium kutscheri, Staphylococcus aureus, Beta-Haemolytic Streptococci, Bordetella bronchiseptica and intestinal protozoa. Data were collected from routine laboratory in a period of two years. The improvement in the microbiological status of mice allowed their expansion in our SPF facility and the distribution of better models to the scientific community.

As receptoras de embriões bovinos devem possuir um ambiente uterino adequado para o desenvolvimento do embrião, estabelecimento da gestação e nascimento de um bezerro saudável. Apesar dos avanços tecnológicos, a perda embrionária ainda permanece elevada, resultando em taxas de prenhez reduzidas após a transferência de embriões (TE). A progesterona (P4) apresenta papel importante nos processos de estabelecimento e manutenção de gestação em fêmeas bovinas. Diversos estudos evidenciaram que o tratamento com P4 injetável de longa ação (P4LA) após a inseminação artificial em tempo fixo (IATF) pode favorecer o estabelecimento da gestação e aumentar as taxas de concepção. Assim, o objetivo do presente estudo foi verificar se o tratamento com P4LA após a ovulação sincronizada em receptoras de embriões produzidos in vitro (PIV) resultará em aumento na concentração de P4 e na taxa de prenhez, sem comprometer o desenvolvimento do corpo lúteo (CL). Para isso, o presente estudo foi dividido em dois experimentos. O Experimento 1 foi realizado no Brasil com 41 vacas Bos indicus (Nelore) tratadas com um dispositivo intravaginal de P4 e 2 mg de benzoato de estradiol (BE) i.m. no D-10. No D-2, o dispositivo foi removido e as vacas receberam 300 UI de gonadotrofina coriônica equina (eCG) e 0,530 mg de cloprostenol sódico (PGF). No D-1, foi administrado 1 mg de BE para a indução da ovulação. Das 41 vacas, 36 (87,8%) ovularam e foram aleatoriamente distribuídas em três grupos experimentais: Controle (n = 13), sem tratamento adicional; P4D4 (n = 11), administração de 150 mg de P4LA i.m no D4; e P4D7 (n = 12), administração de 150 mg de P4LA i.m. no D7. Foram realizadas avaliações ultrassonográficas Color Doppler para verificar a perfusão do CL e colheitas de sangue para análise da concentração de P4 do D4 ao D7 a cada 24 h e do D7 ao D25 a cada 48 h. Verificou-se interação tratamento*tempo (P = 0,004) para concentração plasmática de P4, com aumento transitório na concentração de P4 24h após a administração de P4LA no D4 e D7. Entretanto, não ocorreu interação tratamento*tempo para o diâmetro do CL (P = 0,59) e tampouco efeito dos tratamentos no fluxo sanguíneo central (FSC; P = 0,97) e periférico (FSP) do CL (P = 0,97). No experimento 2, o mesmo protocolo de sincronização descrito no experimento 1 foi empregado em 787 receptoras mestiças sincronizadas para TE na República Dominicana. Destas, 69,9% apresentaram CL &gt; 14 mm no D4 e foram distribuídas aleatoriamente nos 3 grupos experimentais: Controle (n = 168), P4D4 (n = 201) e P4D7 (n = 181). A TE de embrião PIV foi realizada no D7 e o diagnóstico de gestação foi realizado por ultrassonografia no D30 e D60. A taxa de concepção aos 30 dias [Controle: 41,4% (70/168); P4D4: 42,3% (89/201); P4D7: 41,2% (80/181); P = 0,41], aos 60 dias [Controle: 36,3% (61/168); P4D4: 39,8% (80/201); P4D7: 38,1% (69/181); P = 0,49] e a perda gestacional [Controle: 5,4% (9/168); P4D4: 4,5% (9/201); P4D7: 6,1% (11/181); P = 0,73] não diferiram entre os tratamentos. Em conclusão, apesar do aumento da concentração plasmática de P4 após os tratamentos com P4LA no D4 ou D7, não foi observado efeito positivo desses tratamentos na taxa de prenhez e na perda gestacional de receptoras de embriões bovinos. Além disso, os tratamentos com P4LA não comprometeram o desenvolvimento do CL. / The recipients of bovine embryos must have a suitable uterine environment for embryo development, establishment of gestation and birth of a healthy calf. Despite technological advances, embryo loss remains high, resulting in reduced pregnancy rates after embryo transfer (ET). Progesterone (P4) plays an important role in the processes of establishing and maintaining gestation in bovine females. Several studies have shown that treatment with long-acting injectable progesterone (P4LA) after timed artificial insemination (TAI) can help the establishment of pregnancy and increase conception rates. Thus, the aim of the present study was to verify whether treatment with P4LA after synchronized ovulation in recipients of in vitro produced (IVP) embryos would increase P4 plasmatic concentration and pregnancy rate, without interfering with the development of corpus luteum (CL). The study was divided in two experiments. Experiment 1 was conduced in Brazil with 41 Bos indicus cows (Nelore) synchronized with a P4 intravaginal device and 2 mg of estradiol benzoate (EB) i.m. on D-10. On D-2, the device was removed and cows received 300 IU of equine chorionic gonadotropin (eCG) and 0.530 mg of sodium Cloprostenol (PGF) i.m. On D-1, 1 mg of EB was administered to induce ovulation. From the 41 cows, 36 (87.8%) ovulated and were randomly distributed in three experimental groups: Control (n = 13), without any additional treatment; P4D4 (n = 11) administration of 150 mg of P4LA i.m on D4; and P4D7 (n = 12), administration of 150 mg of P4LA i.m. on D7. The CL blood flow was evaluated using Color Doppler ultrasonography and blood sampling was collected for analysis of P4 concentration from D4 to D7 every 24 h and from D7 to D25 every 48 h. Interaction Treatment*Time (P = 0.004) was found for plasma P4 concentration, with a transient increase of P4 concentration 24h after treatments with P4LA on D4 and D7. However, there was neither an interaction Treatment*Time for CL diameter (P = 0.59), nor an effect of CL central (P = 0.97) and peripheral blood flow (P = 0.97). In experiment 2, the same synchronization protocol described in experiment 1 was used in 787 recipients synchronized for ET in Dominican Republic. From those, 69.9% had CL &gt; 14 mm on D4 and were randomly assigned to the following experimental groups: Control (n = 168), P4D4 (n = 201), and P4D7 (n = 181). The IVP embryos were transferred on D7 and the gestation diagnosis was performed on D30 and D60 by ultrasonography. The conception rate at 30 days [Control: 41.4% (70/168); P4D4: 42.3% (89/201); P4D7: 41.2% (80/181); P = 0.41] at 60 days [Control: 36.3% (61/168); P4D4: 39.8% (80/201); P4D7: 38.1% (69/181); P = 0.49] and gestational loss [Control: 5.4% (9/168); P4D4: 4.5% (9/201); P4D7: 6.1% (11/181); P = 0.73] did not differ between treatments. In conclusion, despite the increase in plasma P4 concentration after treatment with P4LA on D4 and D7, no positive effect of using P4LA was observed on conception rate and gestational loss of bovine embryo recipients. In addition, P4LA treatments did not compromise the development of CL.

A introdução de novas linhagens de camundongos em biotérios deve ser realizada com cautela devido aos riscos de introdução de doenças na colônia, motivo pelo qual alguns biotérios adotam como padrão introduzir novos animais somente após o processo de rederivação, que pode ser realizado por transferência embrionária, histerectomia ou cross-fostering. A rederivação é adotada para diversas espécies de animais de laboratório e constitui uma forma de reduzir o risco de surtos de doenças em um biotério com a consequente interferência na pesquisa e perdas, tanto do status sanitário como do bem-estar dos animais. Dentre esses métodos, a transferência de embriões nos estágios de pré-implantação é considerada mais segura que os outros métodos utilizados para se rederivar linhagens de camundongos por reduzir os riscos da transmissão vertical de patógenos. Essa técnica é adotada em diversos biotérios internacionais, porém não é rotina em biotérios do Brasil. O objetivo desse trabalho foi implantar a técnica de rederivação por transferência de embriões no estágio de duas células, no biotério de camundongos livres de patógenos específicos (SPF) do Departamento de Imunologia do Instituto de Ciências Biomédicas da Universidade de São Paulo. As transferências embrionárias foram realizadas para introdução de linhagens de camundongos procedentes de diferentes biotérios convencionais sem barreiras. Os embriões foram obtidos pelos métodos naturais, por meio do acasalamento das fêmeas doadoras superovuladas, com machos de genótipo ou fundo genético igual. Os embriões foram coletados por flushing e lavados em meio estéril. Realizou-se a transferência de embriões no estágio de duas células para fêmeas receptoras livres de patógenos específicos (SPF), pseudoprenhas no 0,5 dia pós coito (d.p.c.). Todos os procedimentos cirúrgicos foram realizados em condições assépticas. Total de 881 embriões, destes, 625 foram transferidos para rederivação de 18 linhagens de camundongos. Nasceram 148 filhotes vivos, dos quais 140 foram desmamados. Os seguintes patógenos foram eliminados pela técnica: vírus da hepatite murina, norovírus, Corynebacterium kutscheri, Staphylococcus aureus, Streptococcus beta hemolítico, Bordetella bronchiseptica e protozoários intestinais. Os dados apresentados foram obtidos da rotina do biotério num período de três anos. A implantação da técnica possibilitou a introdução de novas linhagens na criação, em condições sanitárias satisfatórias, com a consequente disponibilização de modelos animais com nível sanitário adequado à experimentação para a comunidade científica. / The introduction of new strains of mice should be performed carefully to avoid breaking sanitary barriers of specific pathogen free (SPF) animal facilities. In order to meet this need, animals should be rederived by embryo transfer, hysterectomy or cross-fostering and then maintained under strict biosecurity practices. Rederivation is a technique used in different species of laboratory animals to protect them from infectious agents, minimize risks of disease outbreaks and thus avoid research interference caused by loss of the health status and welfare of the animals. Among these techniques, embryo transfer at two cell stage should rather be implemented because it avoids post implantational vertical transmissions of infections. It is routine to introduce animals by embryo transfer in most animal facilities in the United States of America and Europe, however it is not seen in Brazilians rodents vivariums. The objective was to implement mice embryo transfer technique in the animal facility of the Department of Immunology of the Institute of Biomedical Science of the University of Sao Paulo, Brazil. Embryo transfers were performed to rederive strains of mice received from different sources. Fertilized eggs at two-cell stage were obtained by mating superovulated females with fertile males that had either the same genotype or the same genetic background. Embryos were collected by flushing of the oviducts and washed in sterile medium. Under general anesthesia, embryos were transferred into the oviducts of specific pathogen free (SPF) pseudo pregnant female mice at 0,5 - day post coitus (d.p.c.). All the surgical procedures were performed under asseptic conditions. Total of 881 embryos, out of these, 625 embryos at two-cell stage were transfered to rederive 18 mouse strains. It was born 148 pups, of which 140 were reared. The following mouse pathogens were eliminated by embryo transfer technique: Mouse hepatitis virus, Norovirus, Corynebacterium kutscheri, Staphylococcus aureus, Beta-Haemolytic Streptococci, Bordetella bronchiseptica and intestinal protozoa. Data were collected from routine laboratory in a period of two years. The improvement in the microbiological status of mice allowed their expansion in our SPF facility and the distribution of better models to the scientific community.

Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-25Bitstream added on 2014-06-13T19:26:25Z : No. of bitstreams: 1 martins_amvc_dr_botfm_prot.pdf: 1428441 bytes, checksum: e687eeb3da2bd5d051917dea14402c3d (MD5) / Universidade Estadual Paulista (UNESP) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / O objetivo deste estudo foi determinar a importância do local de transferência embrionária (metade superior ou inferior da cavidade endometrial) nas taxas de implantação e gestação. Metodologia: Foram randomizadas 400 transferências embrionárias, guiadas por ultra-sonografia em dois grupos, de acordo com a distância entre a camada de basal de endométrio fúndico e a ponta do cateter de transferência, no ato do procedimento. O Grupo 1 (n200) correspondeu às transferências sendo realizadas a uma distância <50% do comprimento da cavidade endometrial, isto é, na metade superior da cavidade. O Grupo II (n=200) correspondeu às transferências realizadas a uma distância 50% do comprimento da cavidade endometrial, isto é, na metade inferior da cavidade. Os dados foram avaliados pelos testes t de Student, Mann-Whitney e exato de Fisher. Resultados: As características gerais da população estudada e das transferências tiveram distribuição igualitária (p>0,05) entre Grupos l e II. Não houve diferença estatística entre os grupos com relação às taxas de implantação embrionária (Grupo 1: 16,0%; Grupo II: 16,4% p=0,86) e gestação (Grupo 1: 35,0%; Grupo II: 29,5% p=0,28). Conclusão: As taxas de implantação e gestação são similares, quando os embriões são colocados na metade superior ou inferior da cavidade endometrial. / The objective of the present study was to determine the importance of the site of embryo transfer (upper or lower haif endometrial cavity) on implantation and clinical pregnancy rates. Methods: A total of 400 transfers guided by ultrasound were randomly assigned to two groups according to the distance between the basal layer of fundal endometrium and the catheter tip at the time of embryo placement. Group 1 (n=200) consísted of transfers corresponding to a distance of <50% of the endometrial cavity length, i.e. transfer in upper haif of the cavity; and group li (n=200) consisted of transfers corresponding to a distance of 50% of the endometrial cavity Iength, ie. transfer in lower haif of cavity. The Student's t-test, Mann-Whitney test and Fishers exact test were used where appropriate. Results: The general characteristics of the study population and the main transfer cycle characteristics had an equal distribution (p>0.05) between groups 1 and II. No significant difference in implantation (Group 1: 16.0%; Group II: 16.4% p=0.86) or pregnancy rates (Group 1: 35.0%; Group II: 29.5% p=0.28) was observed between groups 1 and II. Conclusion: The implantation or pregnancy rates were similar whether the embryos were deposited in the upper or lower half of the endometrial cavity.