Fixed-time artificial insemination protocols on brazilian locally adapted breed gilts on ovulatory response and embryo production

Abstract The aim of this study was to use estrus synchronization protocols to favor fixed-time artificial insemination and consequently fixed-time embryo collection, and increase embryo production using eCG, in gits. In a cross over design, nine Piau breed gilts were subjected to 18 days of oral progesterone; P4 group did not receive any further; GnRH group received 25µg of GnRH 104 hours after the final application of P4; and eCG+GnRH group received 1000IU of eCG 24 hours after the final P4 in addition to GnRH for subsequent embryo collection, that was performed six days after first AI, by laparotomy. Artificial insemination was performed after 12 and 24 hours of estrus in P4 group, and 128 and 144 hours in GnRH and eCG+GnRH groups. The number of CL (8.6±3.9; 8.3±2.1; 26.7±15.0) and anovulatory follicles (4.3±3.7; 3.9±3.9; 17.2±9.5) was higher in the eCG+GnRH gilts (P<0.05). However, the use of 1000 IU of eCG reduced (P<0.05) the number of total structures (5.2±3.6; 5.1±3.1; 1.7±2.7), viable embryos (5.0±3.5; 4.8±3.3; 0.4±0.7), freezable embryos (3.6±3.4; 3.3±3.8; 0.1±0.3) and recovery rate (63.7±38.9; 58.6±24.7; 5.38±9.5). P4 and GnRH protocols were effective in the production and recovery of embryos. However, the use of 1000 IU of eCG, 24 hours after P4, was not effective in promoting the production of embryos, although the animals had superovulated.

Comparison of cattle embryo collection between Ringer's lactate solution and Dulbecco's phosphatebuffered saline

The objective of this study was to determine the efficiency of Ringer's lactate solution (RL) and RL + 1% fetal bovine serum (FBS) and compare them with the efficiency of Dulbecco's phosphate-buffered saline (D-PBS). Twenty-two Wagyu female cattle were subjected to superovulation and were randomly distributed to form three groups: group 1 ­ uterine flushing with RL (n = 8), group 2 ­ uterine flushing with RL + 1% FBS (n = 7), and group 3 ­ uterine flushing with D-PBS (n = 7, control group). Cows received a CIDR® device containing 1.9 g of progesterone at random stages of the estrous cycle (day 0). Progesterone withdrawal occurred on day 8 in the morning. For heifers, 160 mg of porcine follicle-stimulating hormone (FSH-P) was used and for cows, 200 mg. Prostaglandin F2α was also injected on the eighth day of FSH-P administration. On day 9, in the morning, hCG was administered. Females were superovulated and inseminated twice in a fixed time for embryo transfer. On the 16th day, females were subjected to uterine flushing for embryo collection. We collected 76 embryos from 22 females subjected to superovulation, of which 52 were transferable and 24 had degenerated. The total of embryos collected was 23, 16, and 23 for groups 1, 2, and 3, respectively. The embryo recovery rates per group were 13.86±4.23, 15.39±4.61 and 27.16±13.33%, in groups 1, 2, and 3, respectively. The means for the total structures collected per female were 2.88±0.85, 3.00±1.23, and 4.57±1.72 in groups flushed with RL, RL + 1% FBS, and D-PBS, respectively. We conclude that Ringer's lactate solution and Ringer's lactate solution + 1% of FBS and Dulbecco's phosphate-buffered saline showed no significant differences in terms of embryo quality or quantity, suggesting that Ringer's lactate solution is an alternative for collecting embryos in cattle.

Fixed-time artificial insemination protocols on brazilian locally adapted breed gilts on ovulatory response and embryo production

The aim of this study was to use estrus synchronization protocols to favor fixed-time artificial insemination and consequently fixed-time embryo collection, and increase embryo production using eCG, in gits. In a cross over design, nine Piau breed gilts were subjected to 18 days of oral progesterone; P4 group did not receive any further; GnRH group received 25µg of GnRH 104 hours after the final application of P4; and eCG+GnRH group received 1000IU of eCG 24 hours after the final P4 in addition to GnRH for subsequent embryo collection, that was performed six days after first AI, by laparotomy. Artificial insemination was performed after 12 and 24 hours of estrus in P4 group, and 128 and 144 hours in GnRH and eCG+GnRH groups. The number of CL (8.6±3.9; 8.3±2.1; 26.7±15.0) and anovulatory follicles (4.3±3.7; 3.9±3.9; 17.2±9.5) was higher in the eCG+GnRH gilts (P<0.05). However, the use of 1000 IU of eCG reduced (P<0.05) the number of total structures (5.2±3.6; 5.1±3.1; 1.7±2.7), viable embryos (5.0±3.5; 4.8±3.3; 0.4±0.7), freezable embryos (3.6±3.4; 3.3±3.8; 0.1±0.3) and recovery rate (63.7±38.9; 58.6±24.7; 5.38±9.5). P4 and GnRH protocols were effective in the production and recovery of embryos. However, the use of 1000 IU of eCG, 24 hours after P4, was not effective in promoting the production of embryos, although the animals had superovulated.(AU)

Recent advances in superovulation in sheep / Recentes avanços na superovulação de ovelhas

Nearly three years ago our research team published a review of intrinsic determinants and predictors of superovulatory responses in ewes (18th International Congress on Animal Reproduction, Tours, France; 26-30 June 2016; Theriogenology 2016;86:130-143). Heres a summary of major advances in superovulatory treatments of ewes reported over the last three years and of some most anticipated future directions. The rate of genetic improvement within a synthetic population of animals is significantly enhanced by an application of superovulation and embryo transfer. However, the sheep industry has progressed rather slowly in terms of implementing these technologies in comparison to other species although sheep have been used in many studies that paved the way to the development of modern assisted reproductive technologies (ARTs). Recently, several improvements in determining ovarian responses and non-invasively collecting/transferring embryos have been reported but some most frustrating problems associated with hormonal ovarian superstimulation (e.g., unpredictable and highly variable individual responses) remain unresolved. This warrants continued studies of both the physiological basis of and novel approaches to superovulatory treatments in sheep.(AU)

Recent advances in superovulation in sheep / Recentes avanços na superovulação de ovelhas

Nearly three years ago our research team published a review of intrinsic determinants and predictors of superovulatory responses in ewes (18th International Congress on Animal Reproduction, Tours, France; 26-30 June 2016; Theriogenology 2016;86:130-143). Here’s a summary of major advances in superovulatory treatments of ewes reported over the last three years and of some most anticipated future directions. The rate of genetic improvement within a synthetic population of animals is significantly enhanced by an application of superovulation and embryo transfer. However, the sheep industry has progressed rather slowly in terms of implementing these technologies in comparison to other species although sheep have been used in many studies that paved the way to the development of modern assisted reproductive technologies (ARTs). Recently, several improvements in determining ovarian responses and non-invasively collecting/transferring embryos have been reported but some most frustrating problems associated with hormonal ovarian superstimulation (e.g., unpredictable and highly variable individual responses) remain unresolved. This warrants continued studies of both the physiological basis of and novel approaches to superovulatory treatments in sheep.

PROTOCOLO DE RELAXAMENTO CERVICAL PARA COLETA DE EMBRIÕES EM OVELHAS SANTA INÊS: EFICIÊNCIA, BEM ESTAR ANIMAL, QUALIDADE EMBRIONÁRIA E PROTEÔMICA DO LAVADO UTERINO

Este estudo teve como objetivos avaliar: a) um teste de triagem para seleção de doadoras de embrião aptas para coleta transcervical (TC); b) a eficiência das técnicas de coleta de embrião TC ou cirúrgica por laparotomia (LP) e seus efeitos sobre o bem estar animal; c) o efeito do protocolo hormonal de dilatação da cérvix sobre o ambiente uterino e embrião. No primeiro experimento, o resultado do teste de transposição da cérvix no estro foi correlacionado com o resultado do teste imediatamente antes da coleta de embriões, sendo este último, precedido pelo protocolo de dilatação cervical que consistiu em: benzoato de estradiol (100 µg, i.v.) e cloprostenol (0,12 mg, i.m.) 12 h antes da coleta de embriões, acrescido de ocitocina (100 UI, i.v.) 15 min antes da coleta. No segundo experimento, a frequência cardíaca, temperatura retal, concentrações séricas de proteínas totais, albumina, globulina, cortisol e glicose foram mensuradas em animais submetidos à TC ou LP. As taxas de recuperação de fluido e estruturas também foram mensuradas. No terceiro e quarto experimentos, os grupos foram formados por ovelhas tratadas com o protocolo de dilatação da cérvix (descrito acima) ou ovelhas controles (solução salina). No terceiro experimento, a função luteal foi analisada através de ultrassonografia Doppler e mensuração das concentrações de progesterona. Os embriões foram avaliados quanto à morfologia e expressão gênica. Uma análise proteômica do lavado uterino recuperado também foi realizada. No quarto experimento, os embriões recuperados foram fixados para o teste de TUNEL e outra parte foi cultivada in vitro e submetida à qRT-PCR. No primeiro experimento, o teste de transposição apresentou acurácia de 80,0% e índice kappa de 0,52. No segundo experimento, a coleta TC foi mais eficiente que LP (taxas de recuperação de fluido, P = 0.0002; e de estruturas, P = 0.0180). Durante e imediatamente após procedimentos, a temperatura retal foi maior na coleta TC, enquanto que a frequência cardíaca foi maior no pós-operatório tardio da coleta LP (P < 0.05). Houve um pico no decorrer e logo após os procedimentos nas mensurações de glicose (maior na coleta TC, P < 0.05) e cortisol (maior na coleta LP, P < 0.05). A técnica de coleta não afetou as concentrações de proteínas totais, albumina e globulina. Nos dois últimos experimentos, a qualidade morfológica, e índice apoptótico não foram afetados pelo protocolo, mas os corpos lúteos de animais controle tiveram maior perfusão sanguínea em relação a animais tratados (P = 0.002). As concentrações de progesterona foram reduzidas no grupo tratado às 6, 9 e 12 h após a administração do tratamento (P < 0.0001). A expressão dos genes BAX, BCL2, PRDX1, e HSP90 não foi afetada pelo protocolo. Contudo, os embriões do grupo tratado tiveram menos transcritos de NANOG e OCT4 que embriões do grupo controle (P = 0.008; P = 0.006, respectivamente). Após o cultivo, não houve diferenças entre os grupos em nenhum gene. No lavado uterino, o grupo tratado obteve 436 proteínas exclusivas, destas 16 foram significativamente mais expressas que no grupo controle (P < 0,05). No grupo controle, 126 proteínas exclusivas e cinco proteínas diferenciais foram encontradas (P < 0.05). Em conclusão, o teste de transposição cervical é uma opção de triagem para doadoras de embrião pela técnica TC, que é mais eficiente e, possivelmente, menos estressante que a técnica LP. Contudo, o protocolo hormonal para dilatação cervical promove luteólise e uma alteração temporária da expressão gênica dos embriões, além de um perfil proteico do fluido uterino modificado.

This study aimed to evaluate: a) a screening test to select suitable embryo donors for transcervical collection (TC); b) the efficiency of embryo collection techniques TC or surgical by laparotomy (LP), and their effects on animal welfare; c) the effect of the hormonal protocol for dilation of the cervix on the uterine environment and embryo. In the first experiment, the result of the cervix transposition test at estrus was correlated with the test result immediately before embryo collection, the latter was preceded by the cervical dilation protocol which consisted of: estradiol benzoate (100 µg, iv) and cloprostenol (0.12 mg, im) 12 h before embryo collection, plus oxytocin (100 IU, iv) 15 min before collection. In the second experiment, heart rate, rectal temperature, serum concentrations of total proteins, albumin, globulin, cortisol and glucose were measured in animals submitted to TC or LP. The rates of fluid recovery and structures were also measured. In the third and fourth experiments, the groups were formed by ewes treated with the cervix dilation protocol (described above) or ewes control (saline). In the third experiment, luteal function was analyzed using Doppler ultrasound and measurement of progesterone concentrations. The embryos were evaluated for morphology and gene expression. A proteomic analysis of the recovered uterine lavage was also performed. In the fourth experiment, the recovered embryos were fixed for the TUNEL test and another part was cultured in vitro and submitted to qRT-PCR. In the first experiment, the transposition test showed an accuracy of 80.0% and a kappa index of 0.52. In the second experiment, TC collection was more efficient than LP (fluid recovery rates, P = 0.0002; and structures, P = 0.0180). During and immediately after procedures, the rectal temperature was greater in the TC collection, while the heart rate was greater in the late postoperative period of the LP collection (P < 0.05). There was a peak during and after the procedures in the measurements of glucose (greater in the TC collection, P < 0.05) and cortisol (greater in the LP collection, P < 0.05). The collection technique did not affect the concentrations of total proteins, albumin and globulin. In the last two experiments, the morphological quality and apoptotic index were not affected by the protocol, but the corpora lutea of control animals had greater blood perfusion compared to treated animals (P = 0.002). Progesterone concentrations were reduced in the treated group at 6, 9 and 12 h after treatment administration (P < 0.0001). The expression of the BAX, BCL2, PRDX1, and HSP90 genes was not affected by the protocol. However, embryos in the treated group had fewer NANOG and OCT4 transcripts than embryos in the control group (P = 0.008; P = 0.006, respectively). After culture, there were no differences between groups in any gene. In the uterine lavage, the treated group obtained 436 exclusive proteins, which 16 were significantly more expressed than in the control group (P < 0.05). In control group, 126 exclusive proteins and five differential proteins were found (P < 0.05). In conclusion, the cervical transposition test is a screening option for embryo donors by TC technique, which is more efficient and, possibly less stressful than the LP technique. However, the hormonal protocol for cervical dilation promotes luteolysis and a temporary alteration in the gene expression of the embryos, as well as a modified uterine fluid protein profile.

Uso de procedimentos não cirúrgicos para a produção, recuperação e inovulação de embriões em pequenos ruminantes / In vivo embryo production, recovery and transfer in small ruminants by non surgical techniques

O objetivo deste artigo foi descrever técnicas não cirúrgicas associadas à recuperação e inovulação de embriões em caprinos e ovinos. Os procedimentos relativos a estas etapas da produção in vivo de embriões destes animais foram descritos de forma sequencial e detalhada, a fim de permitir sua execução por veterinários especialistas na área.(AU)

The objective of this article was to describe non surgical techniques related to recovery and transfer of sheep and goat embryos. The main procedures for these steps in the in vivo embryo production from these animals were described sequentially and with the requested details to support their performance by veterinarian experts.(AU)

Uso de procedimentos não cirúrgicos para a produção, recuperação e inovulação de embriões em pequenos ruminantes / In vivo embryo production, recovery and transfer in small ruminants by non surgical techniques

O objetivo deste artigo foi descrever técnicas não cirúrgicas associadas à recuperação e inovulação de embriões em caprinos e ovinos. Os procedimentos relativos a estas etapas da produção in vivo de embriões destes animais foram descritos de forma sequencial e detalhada, a fim de permitir sua execução por veterinários especialistas na área.

The objective of this article was to describe non surgical techniques related to recovery and transfer of sheep and goat embryos. The main procedures for these steps in the in vivo embryo production from these animals were described sequentially and with the requested details to support their performance by veterinarian experts.

Sincronização do Estro e Coleta de Embriões em Suínos Nacionais

Objetivou-se avaliar coletas de embriões consecutivas e protocolos de sincronização estral, em porcas de raças localmente adaptadas, na qualidade embrionária e recuperação de embriões. No Capítulo 2, os animais (n=30), das raças Piau (n=23) e Moura (n=7), foram submetidos a coletas de embriões, 14 foram coletados três vezes, 12 foram coletados duas vezes, e 4 foram coletadas apenas uma vez. No Capítulo 3, as doadoras de embriões Piau (n=9) foram submetidas a protocolos de sincronização de estro, todos os animais passaram nos três protocolos: GP4 = 20 mg da progesterona oral (Altrenogest), 18 dias; GGnRH = 20 mg de Altrenogest, 18 dias, após 104 horas aplicação intramuscular (IM) de 25 µg de GnRH; GeCG+GnRH = 20 mg de Altrenogest (18 dias), 24 horas após, aplicação IM de 1000 UI de eCG, após 80 horas, aplicação IM de GnRH. A partir de 24 horas após a última administração da P4, foi utilizado um cachaço para detecção de estro. A coleta de embriões nos dois experimentos foram realizadas seis dias após a primeira inseminação e todas as estruturas coletas foram analisadas. No capítulo 2, a taxa de recuperação foi similar entre os tratamentos (P>0,05). O número de CL, estruturas totais, embriões viáveis e congeláveis e grau de aderência teve diferença significativa entre os tratamentos (P<0,05). No Capítulo 3 o tempo para a manifestação do estro foi semelhantes entre os tratamentos(P>0,05) O número de CL, folículos anovulatórios, estruturas totais, embriões viáveis, embriões congeláveis e a taxa de recuperação apresentaram diferença significativa entre o GeCG+GnRH e os demais tratamentos (P<0,05). A técnica de coleta de embriões suínos por laparotomia mostra-se eficiente para recuperar embriões de qualidade até a segunda coleta. Todos os protocolos foram eficazes para sincronizar o estro até sete dias em marrãs. No entanto, o protocolo do GeCG+GnRH, não foi eficaz para recuperar embriões e não teve efeitos benéficos na qualidade embrionária.

This study aimed to evaluate consecutive embryos collections and estrus synchronization protocols in locally adapted breed swines, in the embryos quality and embryos recovery. In Chapter 2, animals (n = 30), of Piau race (n = 23) and Moura race (n = 7) underwent embryos collections, 14 of these animals were collected three times, 12 were collected twice and 4 were collected only once time. In Chapter 3, the Piau embryos donnors (n = 9) were subjected to estrus synchronization protocols. All animals passed in three protocols: GP4 = 20 mg of oral progesterone (Altrenogest), 18 days; GGnRH = 20 mg of Altrenogest, 18 days, after 104 hours intramuscular application (IM) of 25 µg GnRH; GeCG+GnRH = 20 mg of Altrenogest (18 days), after 24 hours, IM administration of 1000 IU eCG, after 80 hours, GnRH IM application. From 24 hours after the last administration of P4, was used a boar to estrus detection. In two experiments, the embryos collection was realized six days after the first insemination and all the collections structures were analyzed. In Chapter 2, the recovery rate was similar between treatments (P>0.05). The number of CL, total structures, viable and freezable embryos and adherence degree, was significant difference between treatments (P<0,05). In Chapter 3, the time for the estrus manifestation was similar between treatments (P>0.05). The number of CL, anovulatory follicles, totalstructures, viable embryos, freezable embryos and the recovery rate showed a significant difference between the GeCG+GnRH and the other treatments (P<0.05). The pig embryos collection technique by laparotomy appear to be efficient to recover quality embryos until to the twice collection. All protocols were effective for synchronizing the estrus until seven days in gilts. However, the protocol GeCG+GnRH wasnt effective to recover embryos and hadnt beneficial effect in embryonic quality.