RESUMO

The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil.

RESUMO

A presente revisão tem por objetivo abordar aspectos relacionados ao bem-estar animal na produção in vivo de embriões ovinos. A cobrança da sociedade tem impulsionado o uso de práticas que atendam os preceitos de bem-estar na produção animal. Nesse contexto, destaca-se a necessidade de aprimoramento de procedimentos menos invasivos, como a coleta não cirúrgica de embriões. Avanços recentes nos protocolos de dilatação cervical melhoraram os resultados e tornaram essa técnica uma alternativa viável na espécie ovina. No entanto, a avaliação do estado de bem-estar das doadoras submetidas à coleta transcervical mostra a necessidade de melhor controle da dor durante a sua realização. Assim, acreditamos que a associação dos esforços de diferentes pesquisas pode proporcionar a resolução desses entraves e possibilitar maior aplicabilidade comercial da biotécnica.(AU)

This review aims to address aspects related to animal welfare in the in vivo production of ovine embryos. The use of practices that meet the precepts of welfare in animal production has been driven by demands from society. In this context, there is a need to improve less invasive procedures, such as nonsurgical embryo collection. Recent advances in cervical dilation protocols have improved results and made this technique a viable alternative in sheep. However, the assessment of the state of welfare of donors undergoing transcervical collection shows the need for better pain control during its performance. Thus, the association of different research efforts can provide the resolution of these obstacles and enable greater commercial applicability of the biotechnique.(AU)

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In the past two decades, average litter size (ALS) in Entlebucher Mountain dogs decreased by approximately 0.8 puppies. We conducted a GWAS for ALS using the single-step methodology to take advantage of 1632 pedigree records, 892 phenotypes and 372 genotypes (173 662 markers) for which only 12% of the dogs had both phenotypes and genotypes available. Our analysis revealed associations towards the growth differentiation factor 9 gene (GDF9), which is known to regulate oocyte maturation. The trait heritability was estimated at 43.1%, from which approximately 15% was accountable by the GDF9 locus alone. Therefore, markers flanking GDF9 explained approximately 6.5% of the variance in ALS. Analysis of WGSs revealed two missense substitutions in GDF9, one of which (g.11:21147009G>A) affected a highly conserved nucleotide in vertebrates. The derived allele A was validated in 111 dogs and shown to be associated with decreased ALS (-0.75 ± 0.22 puppies per litter). The variant was further predicted to cause a proline to serine substitution. The affected residue was immediately followed by a six-residue deletion that is fixed in the canine species but absent in non-canids. We further confirmed that the deletion is prevalent in the Canidae family by sequencing three species of wild canids. Since canids uniquely ovulate oocytes at the prophase stage of the first meiotic division, requiring maturation in the oviduct, we conjecture that the amino acid substitution and the six-residue deletion of GDF9 may serve as a model for insights into the dynamics of oocyte maturation in canids.

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This study investigated the feasibility of applying fixed-time (cryopreserved) embryo transfer in ewes. Embryos (n = 106) were non-surgically recovered from superovulated donors (n = 39) on day 6-7 after oestrus. Straws containing one or two embryos (morulae and/or blastocysts) subjected to either slow freezing (SF, n = 62) or vitrification (VT, n = 44) were randomly used within fixed-time embryo transfer on Day 8.5. Recipient ewes were nulliparous (n = 58) bearing corpora lutea after synchronous oestrous induction protocol. The pregnancy rate was higher (p = .03) in SF (39.4%) than VT (16.9%) and survival rate tended (p = .08) to be higher in SF than in VT (25.8% vs. 15.9%). Lambing rates were similar (p = .13) between SF (20.9%) and VT (15.9%). Embryos recovered by non-surgical route after cervical dilation treatment and later cryopreserved by either slow freezing or vitrification produced reasonable pregnancy rates after FTET.

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In the present study, 2.228 cycles of 180 Polo Argentino donor mares from an embryo transfer program in Argentina were examined to evaluate the effects of: (1) Interval from Prostaglandin F2alpha analog treatment to ovulation (ITO) on embryo recovery rate (ERR); (2) ITO on number of embryos per flushing (EPF); (3) ITO on multiple ovulation (MO) rate; (4) ITO from donor mare on pregnancy rate (PR) in recipient mares. Mares were inseminated with fresh semen from 31 fertile stallions in the induced estrus. Embryo flushing was performed 7-8 days postovulation. Following embryo flushing, donor mares were treated with prostaglandin F2alpha analog (cloprostenol 250 µg). The ERR increased along with the ITO (P = .01), with the lowest ERR (30.7%) for mares with an ITO of <4 days, and the highest (78.3%) in mares with an ITO of 10 days. The ITO from the donor mare in which the embryo was recovered did not have a significant effect on PR: ITO <6, 6 to 10, and >10 days were 74.6, 81.4, and 77.3%, respectively. The number of EPF and MO rate increased gradually along with the ITO (P < .05). In conclusion, the results of this study indicated that the ITO is positively correlated with the embryo recovery and the multiple ovulation rate.

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This study investigated the influence of feed intake on superovulatory response and embryo production of Nelore heifers. Pubertal heifers were kept in a feedlot and were submitted to the same diets, but with different levels of feed consumption: High (1.7 M; n = 20) or Low (0.7 M; n = 19) feed intake. Heifers in the 1.7 M treatment consumed 170% (2.6% of body weight [BW] in dry matter) and the 0.7 M heifers ate 70% (1.1% of BW in dry matter) of a maintenance diet. After 7 wk on these diets, heifers were treated with eight decreasing doses of follicle-stimulating hormone (FSH) given every 12 h, totaling 133 mg Folltropin (Folltropin-V; Bioniche Animal Health, Canada) per heifer. Seven d after AI, heifers had their uteri flushed and embryos were recovered and graded according to the International Embryo Technology Society standards. Data were analyzed using the GLIMMIX procedure of SAS and results are presented as least-squares means ± SEM (P < 0.05). At the onset of the FSH treatment (Day 0 of the protocol), 1.7 M heifers had greater body condition score (BCS), BW and serum insulin concentrations than 0.7 M heifers (4.1 ± 0.1 vs. 3.0 ± 0.1; 462.5 ± 10.1 vs. 382.7 ± 10.4 kg; and 14.3 ± 1.7 vs. 3.5 ± 0.8 µIU/mL, respectively). The 0.7 M heifers had more follicles ≥6 mm at the time of the last FSH (Day 7; 47.9 ± 6.4 vs. 23.5 ± 4.3 follicles), related to a better follicle superstimulatory response to FSH. Similarly, 0.7 M heifers had more corpora lutea at the time of embryo collection (33.6 ± 1.4 vs. 15.7 ± 0.9) than the 1.7 M heifers, which resulted in greater number of recovered embryos and ova (9.9 ± 0.7 vs. 6.7 ± 0.6) and viable embryos (5.3 ± 0.5 vs. 3.8 ± 0.4), despite having similar proportions of viable embryos (∼62%). A negative correlation between circulating insulin and follicle superstimulatory response to FSH was observed (r = -0.68). Therefore, we conclude that high feed intake, for a long period of time, compromised the superovulatory response and embryo production potential of Bos indicus heifers possibly related to the elevation in circulating insulin.

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This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced in vivo. Forty Dorper ewes were selected and superovulated. Immediately prior to the embryo collection by laparotomy, a laparoscopy was performed to verify the superovulatory response. The recovered flushing was followed by embryo evaluation and embryos were divided in two experimental groups where embryos from Control group were submitted to a traditional vitrification protocol and embryos from Sucrose group to a modified vitrification protocol with sucrose. After warming, embryos were again divided regarding cryoprotectant removal (Indirect) or not (Direct). The embryo quality was classified as embryos of degrees I (excellent or good), II (regular), III (poor) and IV (dead or degenerate). It was also verified the homogeneity of mass, occurrence of embryonic mass retraction and rupture of pellucid zone. The results were expressed as percentages and were subjected to Chi-square test with P 0.05. The embryos vitrified in the presence of sucrose had lower proportions of lower-quality embryos after warming (22.20 vs. 44.50%), higher percentages of homogeneous embryos after warming (63.89 vs. 38.89 %) while concerning other parameters there was no difference between these groups. It can be concluded that the addition of 0.4 M sucrose during vitrification improves the embryo quality.

Este estudo objetivou avaliar a eficácia da adição de sacarose na solução de vitrificação de embriões ovinos produzidos in vivo. Foram selecionadas 40 ovelhas da raça Dorper as quais foram superovuladas. Imediatamente antes da colheita de embriões por laparotomia, uma laparoscopia foi realizada para verificar a resposta superovulatória. O lavado recuperado foi submetido à procura e avaliação de embriões e estes foram divididos em dois grupos experimentais, onde os embriões do Grupo Controle foram submetidos ao protocolo tradicional de vitrificação e os embriões do Grupo Sacarose a um protocolo modificado de vitrificação com sacarose. Após a descongelação, os embriões foram novamente divididos considerando a remoção (Indireto) ou não (Direto) do crioprotetor. A qualidade embrionária foi classificada como embriões de graus I (excelente ou bom), II (regular), III (pobre) e IV (morto ou degenerado). Foi também verificada a homogeneidade da massa, ocorrência de retração da massa e ruptura de zona pelúcida. Os resultados foram expressos em porcentagem e foram submetidos ao teste do Qui-quadrado com P < 0.05. Os embriões vitrificados na presença de sacarose apresentaram menores proporções de embriões de menor qualidade após a descongelação (22,20 vs. 44,50%), e maiores percentuais de embriões homogêneos após a descongelação (63,89 vs. 38,89%) enquanto com relação aos demais parâmetros não existiram diferenças entre grupos. Pode-se concluir que a adição de 0,4 M de sacarose durante os procedimentos de vitrificação e descongelação beneficia a qualidade embrionária.

Assuntos

RESUMO

This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced in vivo. Forty Dorper ewes were selected and superovulated. Immediately prior to the embryo collection by laparotomy, a laparoscopy was performed to verify the superovulatory response. The recovered flushing was followed by embryo evaluation and embryos were divided in two experimental groups where embryos from Control group were submitted to a traditional vitrification protocol and embryos from Sucrose group to a modified vitrification protocol with sucrose. After warming, embryos were again divided regarding cryoprotectant removal (Indirect) or not (Direct). The embryo quality was classified as embryos of degrees I (excellent or good), II (regular), III (poor) and IV (dead or degenerate). It was also verified the homogeneity of mass, occurrence of embryonic mass retraction and rupture of pellucid zone. The results were expressed as percentages and were subjected to Chi-square test with P 0.05. The embryos vitrified in the presence of sucrose had lower proportions of lower-quality embryos after warming (22.20 vs. 44.50%), higher percentages of homogeneous embryos after warming (63.89 vs. 38.89 %) while concerning other parameters there was no difference between these groups. It can be concluded that the addition of 0.4 M sucrose during vitrification improves the embryo quality.(AU)

Este estudo objetivou avaliar a eficácia da adição de sacarose na solução de vitrificação de embriões ovinos produzidos in vivo. Foram selecionadas 40 ovelhas da raça Dorper as quais foram superovuladas. Imediatamente antes da colheita de embriões por laparotomia, uma laparoscopia foi realizada para verificar a resposta superovulatória. O lavado recuperado foi submetido à procura e avaliação de embriões e estes foram divididos em dois grupos experimentais, onde os embriões do Grupo Controle foram submetidos ao protocolo tradicional de vitrificação e os embriões do Grupo Sacarose a um protocolo modificado de vitrificação com sacarose. Após a descongelação, os embriões foram novamente divididos considerando a remoção (Indireto) ou não (Direto) do crioprotetor. A qualidade embrionária foi classificada como embriões de graus I (excelente ou bom), II (regular), III (pobre) e IV (morto ou degenerado). Foi também verificada a homogeneidade da massa, ocorrência de retração da massa e ruptura de zona pelúcida. Os resultados foram expressos em porcentagem e foram submetidos ao teste do Qui-quadrado com P < 0.05. Os embriões vitrificados na presença de sacarose apresentaram menores proporções de embriões de menor qualidade após a descongelação (22,20 vs. 44,50%), e maiores percentuais de embriões homogêneos após a descongelação (63,89 vs. 38,89%) enquanto com relação aos demais parâmetros não existiram diferenças entre grupos. Pode-se concluir que a adição de 0,4 M de sacarose durante os procedimentos de vitrificação e descongelação beneficia a qualidade embrionária.(AU)

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RESUMO

Realizaram-se estudos para aferir as prováveis causas da baixa taxa de recuperação de estruturas embrionárias em búfalas superovuladas. No primeiro estudo (Experimento 1), foram utilizados sistemas genitais de búfalas e de bovinas tratadas para a indução de ovulações únicas ou múltiplas, os quais foram submetidos à morfometria, seguidos de lavagem dos ovidutos para a recuperação dos oócitos. Posteriormente, os ovidutos foram encaminhados à histologia. No Experimento 2, foram utilizados ovidutos de búfalas e de bovinas, tratadas para a indução de ovulação única. O lúmen do oviduto foi exposto e, após isso, os ovidutos foram incubados em meio de cultura com ou sem E2, com posterior colocação de microesferas na sua superfície para a aferição do movimento ciliar. No Experimento 3, foram utilizados ovidutos de búfalas e de bovinas tratadas para a indução de ovulação única. Os ovidutos foram incubados em meio de cultura com ou sem E2, com a inserção de oócitos bubalinos ou bovinos em seu lúmen, sendo posteriormente lavados para a recuperação e contagem dos oócitos. No Experimento 4, búfalas e bovinas foram tratadas para a indução de ovulações únicas ou múltiplas. Após a ovulação, os animais foram submetidos à laparotomia para a inserção de oócitos bubalinos ou bovinos no oviduto.

Studies were performed to assess the probable causes of the low embryonic structures recovery rate in superovulated buffaloes. In the first study (Experiment 1) were used buffaloes and bovines genital systems treated to induce single or multiple ovulations, which were submitted to morphometry followed by oviducts flushing for the oocytes recovery. Subsequently, the oviducts were sent to histology. In Experiment 2, were used buffaloes and bovines oviducts treated for single ovulation. The oviduct lumen was exposed and, thereafter, incubated in culture medium with or without E2, with subsequent placement of microspheres on its surface for the ciliary movement measure. In Experiment 3, were used buffaloes and bovines oviducts treated for a single ovulation. The oviducts were incubated in culture medium with or without E2, with the inclusion of bovine or buffalo oocytes in the lumen, and subsequently flushed for the oocytes recovery and counting. In Experiment 4, buffaloes and bovines were treated to induce single or multiple ovulations. After ovulation, the animals underwent laparotomy for the insertion of bovine or buffalo oocytes in the oviduct. Later (five and six days after buffalo and bovine oocytes insertion, respectively), the genital systems were flushed in vivo for the embryonic structures recovery.

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A Transferência de Embrião e Múltipla Ovulação (MOET) possibilitam a produção de mais de uma cria por fêmea por ano e pode ser impulsionada com o uso de sêmen sexado, já que a tolerância a criopreservação dos embriões produzidos in vitro ainda é baixa, limitando o uso desta biotécnica. Todavia, pesquisas têm procurado estabelecer protocolos de sincronização/superovulação para elevar a taxa de fertilização na MOET com sêmen sexado. No entanto, os resultados com sêmen sexado, quando utilizado em animais superovulados, ainda são inferiores àqueles obtidos com o sêmen convencional. Desta forma, nesta revisão objetivou- -se abordar alguns aspectos da produção in vivo de embriões bovinos e o processo de sexagem espermática, bem como os resultados relatados com a utilização de sêmen sexado em programas de MOET.(AU)

The Multiple Ovulation and Embryo Transfer (MOET) enable the production of more than one calf per female per year and may be stimulated with the use of sorted semen, as the tolerance of the in vitro produced embryo is still low, limiting the use of this biotechnique. However, researches have aimed to establish synchronization/ superovulation protocols to increase the fertilization rate in MOET with sorted semen. Still, the results with sexed semen, when utilized in superovulated cows still are inferior than unsorted semen. Thus, the objective of this review is to approach some aspects of the in vivo embryo production from cow and the process of sorting of semen, as well as the results reported with use of sorted semen in MOET programs. (AU)

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RESUMO

Realizaram-se estudos para aferir as prováveis causas da baixa taxa de recuperação de estruturas embrionárias em búfalas superovuladas. No primeiro estudo (Experimento 1), foram utilizados sistemas genitais de búfalas e de bovinas tratadas para a indução de ovulações únicas ou múltiplas, os quais foram submetidos à morfometria, seguidos de lavagem dos ovidutos para a recuperação dos oócitos. Posteriormente, os ovidutos foram encaminhados à histologia. No Experimento 2, foram utilizados ovidutos de búfalas e de bovinas, tratadas para a indução de ovulação única. O lúmen do oviduto foi exposto e, após isso, os ovidutos foram incubados em meio de cultura com ou sem E2, com posterior colocação de microesferas na sua superfície para a aferição do movimento ciliar. No Experimento 3, foram utilizados ovidutos de búfalas e de bovinas tratadas para a indução de ovulação única. Os ovidutos foram incubados em meio de cultura com ou sem E2, com a inserção de oócitos bubalinos ou bovinos em seu lúmen, sendo posteriormente lavados para a recuperação e contagem dos oócitos. No Experimento 4, búfalas e bovinas foram tratadas para a indução de ovulações únicas ou múltiplas. Após a ovulação, os animais foram submetidos à laparotomia para a inserção de oócitos bubalinos ou bovinos no oviduto.(AU)

Studies were performed to assess the probable causes of the low embryonic structures recovery rate in superovulated buffaloes. In the first study (Experiment 1) were used buffaloes and bovines genital systems treated to induce single or multiple ovulations, which were submitted to morphometry followed by oviducts flushing for the oocytes recovery. Subsequently, the oviducts were sent to histology. In Experiment 2, were used buffaloes and bovines oviducts treated for single ovulation. The oviduct lumen was exposed and, thereafter, incubated in culture medium with or without E2, with subsequent placement of microspheres on its surface for the ciliary movement measure. In Experiment 3, were used buffaloes and bovines oviducts treated for a single ovulation. The oviducts were incubated in culture medium with or without E2, with the inclusion of bovine or buffalo oocytes in the lumen, and subsequently flushed for the oocytes recovery and counting. In Experiment 4, buffaloes and bovines were treated to induce single or multiple ovulations. After ovulation, the animals underwent laparotomy for the insertion of bovine or buffalo oocytes in the oviduct. Later (five and six days after buffalo and bovine oocytes insertion, respectively), the genital systems were flushed in vivo for the embryonic structures recovery.(AU)

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Data on 1390 Zebu donors multiple ovulation followed by embryo transfer results were analyzed. The donors came from different regions of Brazil and belonged to Brahman (31), Gir (22), Guzerá (68) and Nelore (411) breeds. The age of donors ranged from 1.5 to 19.5 years old. The first recovery data and consecutive recovery data were submitted to multivariate analysis. The estimated Pearson correlation coefficients were: 0.72 between total recovered structures (ER) and number of viable embryos (EV); 0.66 between ER and total of positive pregnant receptors (PP); 0.53 between ER and number of palpated corpora luteum; 0.89 between EV and PP; 0.33 between EV and CL and 0.30 between PP and CL. Relationships among inbreeding coefficient, age of donors to superovulation, stimulating drug, drug dosage, recovery number and number of executed insemination were also observed. Factor analysis indicated the following factors for consecutive recoveries: factor 1 - factors that reduce superovulation response, factor 2 - superovulation scheme according to characteristics of donors, factor 3 - success of superovulation scheme. For first recovery analysis the following factors were designated: factor 1 - optimum age for superovulation, factor 2 - superovulation scheme according to characteristics of donors, and factor 3 - alternative superovulation scheme. The analysis also indicated the results of superovulation are quite variable and that the age of donors to superovulation, inbreeding coefficient of donors, recovery number, drug dosage and number of inseminations have high effect on the results. The number of viable embryos and the correlation between number of viable embryos and total of positive pregnant receptors can be indicators of MOET success.

Resultados de 1390 ovulações múltiplas seguidas de transferência de embriões foram submetidas à análise multivariada para estudo da relação entre algumas variáveis envolvidas no processo. As superovulações foram realizadas no período de 1989 a 1999 em várias doadoras zebuínas das raças Brahman (31), Gir (22), Guzerá (68) e Nelore (411), provenientes de 61 rebanhos de diversas regiões do Brasil. As doadoras tinham idades entre 1,5 e 19,5 anos. Analisaram-se apenas dados de primeira colheita e dados de colheitas consecutivas. As principais correlações encontradas com a análise de ambos os arquivos pelo método de Pearson foram de: 0,72 entre o total de estruturas recuperadas (ER) e o número de embriões viáveis (EV); 0,66 entre ER e o total de prenhezes positivas (PP); 0,53 entre ER e o número de corpos lúteos palpados (CL); 0,89 entre EV e PP; 0,33 entre EV e CL; e 0,30 entre PP e CL. Observou-se associação entre coeficiente de endogamia, idade das doadoras à superovulação, droga estimuladora, dose da droga estimuladora, ordem de colheita e número de inseminações. Foram estimados os seguintes fatores nas análises de colheitas consecutivas: fator 1 - que reduz a resposta à superovulação, fator 2 - que define os esquemas de superovulação de doadoras e fator 3 - que define o sucesso do esquema de superovulação. Com os dados de primeira colheita foram obtidos: fator 1 - que define a idade ótima à superovulação, fator 2 - que define os esquemas de superovulação de doadoras e fator 3 - que define esquema alternativo de superovulação. As análises indicaram que os resultados de superovulação são muito variáveis e que há grande efeito da idade da doadora à superovulação, do coeficiente de endogamia da doadora, da ordem de colheita, da dose da droga e do número de inseminações sobre esses resultados. O número de embriões viáveis por superovulação e a correlação entre número de embriões viáveis e total de prenhezes positivas podem ser indicadores da eficiência do MOET.