ABSTRACT
The aim of this study was to evaluate the effects of flunixin meglumine administration on pregnancy rates and luteal phase characteristics in bovine embryo recipients at the moment of embryo transfer. In experiment 1, in vitro produced embryos were transferred to 184 females divided as control and treated group (recipients treated with 1.1mg/kg flunixin meglumine). In experiment 2, 22 females were divided as control group; group 2 (animals submitted to a reproductive tract manipulation similar to an embryo transfer on the 7th day after estrous); and group 3 (females submitted to a manipulation and treatment with 1.1mg/kg flunixin meglumine). In experiment 1 no difference was observed between control and treated groups (40.2% and 44.6%, respectively) for pregnancy rates. In experiment 2 no difference was observed on the length of luteal phase between groups, however, animals in group 2 presented lower plasma progesterone concentrations than the control group and group 3. Therefore, we concluded that although the administration of flunixin meglumine at the moment of embryo transfer inhibited the reduction plasma progesterone concentrations, it was not effective in increasing pregnancy rates of bovine recipients.(AU)
O objetivo deste estudo foi avaliar os efeitos da administração de flunixina meglumina sobre as taxas de prenhez e características da fase lútea da receptora no momento da transferência de embriões em bovinos. No experimento 1, embriões produzidos in vitro foram transferidos para 184 fêmeas, divididas em grupos controle e tratado (tratados com 1,1mg/kg de flunixina meglumina). No experimento 2, 22 fêmeas foram divididas em grupo controle (n=7); grupo 2 (n=8; animais submetidos à manipulação do trato reprodutivo semelhante à transferência de embriões no sétimo dia pós-cio); e grupo 3 (n=7; fêmeas submetidas à manipulação e ao tratamento com 1,1mg/kg de flunixina meglumina). No experimento 1, não foi observada diferença nos grupos controle e tratado (40,2% e 44,6%, respectivamente) para as taxas de prenhez. No experimento 2, não houve diferença na extensão da fase lútea entre os grupos, entretanto os animais do grupo 2 apresentaram concentrações plasmáticas de progesterona mais baixas que o grupo controle e o grupo 3. Portanto, conclui-se que a administração de flunixina meglumina no momento da transferência de embriões inibiu a redução das concentrações plasmáticas de progesterona, no entanto não foi eficaz para aumentar as taxas de prenhez de receptoras em bovinos.(AU)
Subject(s)
Animals , Female , Pregnancy , Cattle , Pregnancy Rate , Embryo Culture Techniques/veterinary , Luteal Phase/physiology , Meglumine , Progesterone , In Vitro Techniques/veterinaryABSTRACT
The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50µM/mL Cysteamine; T3)2.5µg/mL; T4)5.0µg/mL and T5)10.0µg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.(AU)
O objetivo desse estudo foi avaliar a suplementação de meio de cultura de embriões com antioxidante obtido do extrato oleoso da Lippia origanoides no desenvolvimento e na qualidade de blastocistos produzidos in vitro. Oócitos coletados de ovários de matadouros foram maturados e fertilizados in vitro segundo procedimento laboratorial padrão. Zigotos foram cultivados em meio SOF suplementado de acordo com os seguintes tratamentos: T1) meio de cultivo embrionário sem suplementação antioxidantes; T2) 50µM/mL Cisteamina; T3) 2,5µg/mL; T4) 5,0µg/mL e T5) 10,0µg/mL do antioxidante obtido do extrato oleoso de Lippia origanoides. No sétimo dia de cultivo, os blastocistos foram fixados e avaliados para taxa de apoptose, número total de células e massa celular interna através do teste TUNEL. O uso de antioxidantes durante cultivo não aumentou (P>0,05) a taxa de produção final de blastócitos. Os tratamentos T2, T3, T4 e T5 tiverem menor índice apoptótico (p>0,05 - 4,5±1,1%, 8,4±2,5%, 3,4±1,1% e 5,5±0,9%, respectivamente) quando comparados a T2 (10,0±1,4%). O valor de massa celular interna não diferenciou (p>0,05) entre embriões de diferentes tratamentos. A adição de antioxidante obtido do extrato oleoso de Lippia origanoides reduziu a taxa de apoptose e melhorou a qualidade sem aumentar a produção in vitro de embriões bovinos.(AU)
Subject(s)
Animals , Cattle , Apoptosis , Lippia , Embryo Culture Techniques/veterinary , Embryonic Development , AntioxidantsABSTRACT
No presente estudo, utilizou-se a melatonina e a proteína específica do oviduto (pOSP) nos meios de maturação in vitro. Foram avaliadas a expansão do complexo cumulus-ovócito (CCOs), as concentrações intracelulares de espécies reativas de oxigênio (ROS) e o desenvolvimento embrionário nos diferentes grupos (C = controle; T1 = somente com melatonina; T2 = com melatonina e pOSP e T3 somente com pOSP). No tocante à expansão do CCOs, houve diferença (P<0,05) dos valores obtidos no grupo C em relação aos valores médios dos grupos T1, T2 e T3, porém não houve diferença entre os valores obtidos nos tratamentos (P>0,05). Na dosagem de ROS, não houve diferença entre os valores médios obtidos no grupo C (26,4±10,9) e o valor verificado no grupo T1 (23,4±7,8), porém no grupo T2 (21,3±9,7) o valor médio mostrou-se satisfatório em relação ao valor do grupo C. No entanto, o valor médio do grupo T3 (16,6±10,5) foi o que demonstrou resultado mais satisfatório quando comparado aos demais grupos (P<0,05). A produção de embriões foi avaliada por meio da taxa de clivagem. Não houve diferença (P >0,05) entre os valores obtidos entre o grupo C (48,9 %) e os valores verificados nos grupos T1 (51,5 %), T2 (50 %), T3 (57,7 %), nem destes entre si. Este estudo permitiu concluir que a proteína específica do oviduto recombinante e a melatonina foram eficientes em melhorar a expansão dos CCOs. Além disso, as células tratadas com pOSP mostraram-se com menor quantidade de ROS, podendo a pOSP ser considerada um antioxidante proteico.(AU)
The present study used melatonin and recombinant oviduct specific protein (pOSP) in in vitro maturation medium (IVM). The expansion of the cumulus-oocyte complexes (COCs), the intracellular concentrations of reactive oxygen species (ROS) and embryo development of the different groups were evaluated (C = control; T1 = melatonin; T2 = melatonin and pOSP and T3 = pOSP). Regarding the COCs expansion, the groups T1, T2 and T3 showed satisfactory results compared with group C (P<0.05), but there was no difference between treatments (P>0.05). In the ROS dosage, there was no difference between the mean values obtained in group C (26.4 ± 10.9) and group 1 (23.4 ± 7.8). However, in group 2 (21.3 ± 9.7), the average value was found to be satisfactory in relation group C. Despite that, the average value of treatment 3 (16.6 ± 10.5) was the most satisfactory result found compared to the other groups (P<0.05). The production of embryos was evaluated by cleavage rate, there was no difference between the values obtained in group C and the values recorded in groups T1 (51.5 %), T2 (50 %), T3 (57.7 %), and among them. This study showed that the pOSP and the melatonin were effective in the improvement of the expansion of COCs cells. In addition, the cells that were treated with pOSP presented a lower amount of ROS, allowing the pOSP to be considered a proteic antioxidant.(AU)
Subject(s)
Animals , Female , Embryonic Development , Fallopian Tubes/chemistry , Melatonin/administration & dosage , Swine , Antioxidants , Cleavage Stage, Ovum , Embryo Culture Techniques/veterinary , In Vitro Techniques/veterinaryABSTRACT
In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation...
A produção in vitro (PIV) de embriões de bovinos não é apenas de grande importância econômica para a pecuária, mas é também um importante modelo para estudar o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a modificação de histona, H3R26me2 durante o desenvolvimento pré-implantacional em embriões bovinos produzidos in vitro, cultivados com ou sem suplementação de soro fetal bovino (SFB), bem como comparar essa modificação específica entre mórulas produzidas in vitro e in vivo. Após a maturação in vitro e fertilização, embriões foram cultivados com suplementação de 0 ou 2,5% SFB. O desenvolvimento embrionário foi avaliado e embriões foram coletados e fixados em diferentes fases durante o desenvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). Os embriões fixados foram avaliados por imunofluorescência utilizando um anticorpo para H3R26me2. Imagens de embriões corados foram analisadas baseadas na porcentagem do DNA total. Embriões cultivados com 2,5% SFB tiveram uma taxa de desenvolvimento ao estágio de blastocisto maior que o grupo que não recebeu suplementação com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de H3R26me2 variaram para ambos os grupos ao longo do desenvolvimento. No grupo 0% SFB, a marcação para H3R26me2 foi mais intensa nos estágios de 4 células (P<0,05), 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, apenas os embriões de 4 células tiveram marcação significativamente maior que todas as outras fases (P<0,01). Mórulas produzidas in vivo apresentaram níveis de H3R26me2 semelhantes ao grupo 0% SFB, e ambos foram significativamente maiores que o grupo 2.5% SFB. Estes resultados sugerem que a modificação de histona H3R26me2 é regulada durante o desenvolvimento pré-implantacional de embriões bovinos, e que as condições de cultura alteram de maneira importante esta regulação...
Subject(s)
Animals , Cattle , Cattle/embryology , Embryonic Development , Histones/analysis , Immunohistochemistry/veterinary , Morula , In Vitro Techniques/veterinary , Embryo Culture Techniques/veterinaryABSTRACT
The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
Subject(s)
Animals , Blastocyst/cytology , Cloning, Organism/veterinary , Culture Media/metabolism , Dogs/embryology , Embryo Culture Techniques/veterinary , Embryonic Development , Nuclear Transfer Techniques/veterinaryABSTRACT
A inseminação artificial intrauterina profunda (IIP) é de grande importância para a indústria suinícola, em função do maior número de doses produzidas por reprodutores de alto mérito genético e da possibilidade da utilização de biotecnologias, como sêmen sexado e/ou congelado. Entretanto, necessita-se compreender com maior propriedade os mecanismos pelos quais os espermatozoides colonizam as tubas uterinas. Assim sendo, pretende-se com o presente experimento avaliar a existência ou não de migração intraperitoneal de espermatozoides inseminados profundamente em um dos cornos uterinos, mediante a obtenção de oócitos fertilizados no corno contralateral à inseminação e seccionado na base, na junção com o corpo do útero. Quatorze fêmeas pluríparas foram divididas em dois grupos experimentais, sendo que em um deles as fêmeas foram submetidas à secção da base de um dos cornos uterinos (Grupo Operado, n = 7), enquanto as do Grupo Controle (n = 7) não foram submetidas a nenhuma intervenção cirúrgica. Ambos os grupos foram submetidos à IIP, sendo as fêmeas abatidas 5±1,2 dias após a última inseminação. Os sistemas genitais das fêmeas foram coletados, dissecados e o número de corpos lúteos contados em ambos os ovários. A recuperação dos embriões foi feita por meio de lavagem das tubas e cornos uterinos com solução de PBS (Phosphate Buffered Saline), após o que se avaliou os fluidos coletados em lupa para a identificação de embriões. Em ambos os grupos experimentais, foram encontrados embriões nos segmentos do sistema genital de ambos os lados. Apenas uma fêmea apresentou embriões nos segmentos em somente um dos lados no grupo operado. Diante dos resultados aqui observados, concluiu-se que a migração espermática no suíno pode ocorrer tanto por via retrógrada pelo útero quanto por migração intraperitoneal. Estes achados certamente contribuirão para aumentar a eficiência da técnica de IIP, sendo de grande valia para o aprimoramento da indústria suinícola...
Deep intrauterine insemination (DUI) is of great importance for the swine industry as it can increase the efficiency in the use of boars of high genetic merit, and facilitate the use of biotechnologies such as frozen and sexed semen. However, a better understanding of the mechanisms by which the sperm colonize the uterine tubes is essential. The aim of the present study was to investigate the existence of intrauterine sperm migration after DUI in one uterine horn, through the fertilization of oocytes in the contra lateral uterine horn. Fourteen multiparous sows were divided into two experimental groups: Operated (n = 7), where females had a segment close to the base of the uterine horn surgically removed, and Control (n = 7), females with intact uterus. Both groups were inseminated through DUI and slaughtered 5±1.2 days after the last insemination. The reproductive tracts collected were dissected and the number of corpora lutea counted in both ovaries. Embryo recovery was performed though flushings of uterine tubes and horns with Phosphate Buffered Saline solution and further examination under a dissecting microscope. Embryos were found in the uterine horns of both sides of the reproductive tract in both experimental groups. In the operated group, just one female had embryos in only one side of the reproductive tract. The results presented herein suggest that sperm migration in pigs may occur both in a retrograde way through the uterus and by intraperitoneal migration. These findings will certainly contribute to increase the efficiency of the DUP technique, which is of great importance for the improvement of the swine industry...
Subject(s)
Animals , Insemination, Artificial/veterinary , Sperm Motility , Swine , Oocytes , Semen Preservation/veterinary , Embryo Culture Techniques/veterinaryABSTRACT
Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.
Subject(s)
Animals , Cattle , Female , Blastocyst/physiology , Embryo Culture Techniques/veterinary , Embryonic Development/physiology , Gene Expression Regulation, Developmental/physiology , Nuclear Transfer Techniques/veterinary , Embryo Culture Techniques/methodsABSTRACT
The objective of the present study was to examine the effect of green tea polyphenols (GTPs) supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5°C for 24 h) and fertilized (38.5°C for 15-18 h) and embryos were cultured (38.5°C for 192 h) in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99 percent catechin derivatives, with the major components being 50 percent (-)-epigallocatechin gallate, 22 percent (-)-epicatechin gallate, 18 percent (-)-epigallocatechin, and 10 percent (-)-epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 æM) of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4 percent, P < 0.05). However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 æM) were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 æM) significantly reduced the rate of blastocyst formation (P < 0.05). Supplementation of the culture medium with 15 æM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 æM) significantly reduced embryo development (P < 0.05). In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 æM) during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.
Subject(s)
Animals , Cattle , Female , Embryo Culture Techniques/veterinary , Fertilization in Vitro/veterinary , Flavonoids/pharmacology , Oocytes/drug effects , Phenols/pharmacology , Tea/chemistry , Fertilization in Vitro/drug effects , Flavonoids/chemistry , Oocytes/growth & development , Phenols/chemistryABSTRACT
The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p > 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 microsec (19 +/- 2% vs. 77 +/- 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 microsec, although this was still lower than the rate of fusion in the CCCs (33 +/- 1% vs. 80 +/- 2%). The rates of cleavage (57 +/- 5%) and blastocyst formation (1 +/- 1%) in the DCC-derived embryos did not differ from those (55 +/- 6% and 3 +/- 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 +/- 2%) showed higher levels of blastocyst formation (p > 0.01) than CCC-derived autologous SCNT embryos (1 +/- 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.
Subject(s)
Animals , Female , Animals, Genetically Modified , Cloning, Organism , Cumulus Cells/metabolism , Electric Stimulation , Embryo Culture Techniques/veterinary , Embryonic Development , Fibroblasts/metabolism , Nuclear Transfer Techniques/veterinary , Oocytes/metabolism , Ovarian Follicle/metabolism , Swine/embryologyABSTRACT
This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or beta-mercaptoethanol (beta-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 micrometer CYS or 100 micrometer beta-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. beta-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.