Bem-estar animal nos procedimentos de produção in vivo de embriões na espécie ovina / Animal welfare in the in vivo production of sheep embryos

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)

Desafios e perspectivas na produção comercial de embriões in vivo e in vitro de raças taurinas e sintéticas / Current challenges and perspectives of in vivo and in vitro commercial embryo production of taurine and synthetic breeds

As biotécnicas de produção in vivo e in vitro de embriões bovinos permitem aumentar significativamente o número de descendentes de fêmeas genética e/ou zootecnicamente importantes. Porém, antes de se optar por um dos métodos, deve-se avaliar suas peculiaridades. A produção in vivo pode ser empregada de forma satisfatória tanto em zebuínos quanto em raças sintéticas e taurinas, permitindo a obtenção de, em média, seis a sete embriões viáveis por coleta, com boa tolerância à criopreservação. Já a produção in vitro é mais eficiente em raças zebuínas e sintéticas, visto que possuem maior número de folículos antrais aspiráveis. Além disso, esta técnica permite a produção de embriões sem estímulos hormonais exógenos, porém com menor criotolerância. Desse modo, a presente revisão discute os desafios atuais e perspectivas futuras na produção de embriões in vivo e in vitro com base nos dados da rotina de uma central de doadoras e laboratório de produção de embriões que desenvolve simultaneamente ambas as técnicas de produção de embriões na região Sul do Brasil.(AU)

Biotechniques for in vivo and in vitro production of bovine embryos allow to significantly increase the number of descendants from cows genetically and/or zootechnically superior. However, before opting for one of the methods, one should evaluate its peculiarities. In vivo production can be used satisfactorily both in zebu cattle and in synthetic and taurine breeds, allowing to obtain, on average, six to seven viable embryos per procedure, with good tolerance to cryopreservation. In vitro production is more efficient in Zebu and synthetic breeds, since they have a greater number of aspirable antral follicles. In addition, this technique allows the production of embryos without exogenous hormonal stimuli, but with lower cryotolerance. This review discusses current challenges and future perspectives in in vivo and in vitro embryo production based on routine data from an embryo production center and a laboratory that develop, simultaneously, the two embryo production techniques in southern Brazil.(AU)

Processos que afetam a taxa de gestação pós-inovulação do embrião equino / Processes that affect pregnancy rate after embryo transfer

A transferência de embriões equinos é uma biotecnologia que traz inúmeros benefícios reprodutivos para um haras. Entretanto, os índices reprodutivos obtidos numa estação de monta podem ser afetados por diversos fatores que interferem tanto na recuperação embrionária que normalmente são relacionados à doadora de embriões, quanto na taxa de gestação na receptora de embriões. O objetivo do presente artigo foi revisar os fatores relacionados à receptora de embriões em programas de transferência de embriões equinos.(AU)

Equine embryo transfer is a biotechnology that brings reproductive benefits to a stud farm. However, the reproductive rates obtained in a breeding season can be affected by several factors that interfere both in the embryonic recovery that are normally related to the embryo donor, and in the pregnancy rate in the embryo recipient. The aim of this article was to review the factors related to the recipient of embryos in equine embryo transfer programs.(AU)

Taxa de recuperação embrionária em éguas Mangalarga Marchador inseminadas com sêmen refrigerado / Embryonic recovery rate in Mangalarga Marchador mares inseminated with refrigerated semen

O acompanhamento reprodutivo da espécie equina tem sido cada vez mais aplicado na aplicade de diversos países, resultando em melhores índices reprodutivos para a espécie e em animais de linhagens selecionadas. Nos programas de transferência de embrião, a taxa de recuperação embrionária consiste em um parâmetro essencial a fim de verificar o sucesso da técnica. Por conseguinte, este trabalho propõe-se a avaliar as taxas de recuperação embrionária de éguas doadoras submetidas à inseminação artificial com sêmen refrigerado de maneira a complementar os dados relacionados ao assunto. Conforme os dados coletados durante a estação de monta de agosto de 2018 a fevereiro de 2019, obteve-se 129 lavados uterinos provenientes de 20 éguas e 83 embriões recuperados. Desse modo, a taxa de recuperação embrionária obtida foi de 64,34%. Diante dos dados apresentados neste estudo, conclui-se que a aplicabilidade da técnica de recuperação embrionária visando o uso do sêmen refrigerado na inseminação artificial de éguas se mostra promissora, pois permite o transporte de material genético de alto valor entre propriedades sem interferir drasticamente nos índices dos programas de reprodução.

The reproductive monitoring of the equine species has been increasingly applied in several countries, resulting in better reproductive rates for the species and in animals from selected strains. In embryo transfer programs, the embryo recovery rate is an essential parameter to verify the technique success. Therefore, the present work proposes to evaluate the embryo recovery rates of donor mares submitted to artificial insemination with refrigerated semen to complement the data related to the subject. According to the data collected during the breeding season for the period from August 2018 to February 2019, 129 uterine washes were obtained from 20 mares and 83 recovered embryos. Thus, the embryonic recovery rate obtained was 64.34%. Given the data presented in this study, it is concluded that the applicability of the embryonic recovery technique aiming at the use of refrigerated semen in artificial insemination of mares is promising, as it allows the transport of high-value genetic material between properties without interfering dramatically in breeding program indexes.

Pontos críticos na produção de embriões equinos com enfoque na doadora / Critical points in the production of equine embryos with a donor approach

Inúmeras são as vantagens da utilização da técnica de transferência de embriões (TE) em equinos. No entanto, alguns fatores podem afetar a taxa de recuperação embrionária: dia da colheita do embrião; condição uterina e idade da doadora; manejos reprodutivo, sanitário e nutricional; garanhão/qualidade do sêmen; habilidade do técnico; condições climáticas, número de ovulações e adaptação da doadora. O objetivo deste artigo é revisar os fatores que podem exercer influência sobre a taxa de recuperação embrionária tendo impacto direto nos índices reprodutivos de um programa de TE.(AU)

There are countless advantages of using the embryo transfer (ET) technique in horses. However, some factors may affect the embryonic recovery rate: day of embryo collection; uterine condition and donor age; reproductive, health and nutritional management; stallion/semen quality; skill of the technician; climatic conditions, number of ovulations and adaptation of the donor. The objective of this article is to review those factors that can influence the embryonic recovery rate, having a direct impact on the reproductive rates of an ET program.(AU)

Estudos para o incremento da produção de embriões in vivo e in vitro em vacas de corte taurinas e sintéticas / Studies to improve in vivo and in vitro embryo production in beef cows from taurine and synthetic breeds

O Brasil destaca-se por ser um dos líderes mundiais na produção in vitro de embriões, o que é decorrente do rebanho nacional ser predominantemente composto por raças zebuínas, que possuem maior número de folículos antrais aspiráveis. Por outro lado, vacas taurinas apresentam menor população folicular antral, o que limita o número de ovócitos obtidos por seção de aspiração folicular (ovum pick-up; OPU). Portanto, na região Sul do Brasil há demanda para produção tanto in vivo como in vitro de embriões de doadoras de raças taurinas e sintéticas. Este texto discute estudos recentes que buscam estratégias para aperfeiçoar as etapas envolvidas na produção de embriões bovinos, bem como para aumentar o aproveitamento de receptoras utilizadas nos programas de transferência de embriões (TE), com foco em vacas taurinas e sintéticas.(AU)

Brazil stands among the leaders on in vitro embryo production, as the Brazilian herd is predominantly composed by Zebu breeds, which have a greater number of antral follicles available to ovum pick-up (OPU). On the other hand, taurine cows have a lower antral follicle population, which limits the number of oocytes obtained by each OPU section. Therefore, in the Brazilian Southern region there is a demand for both in vivo and in vitro production of embryos from donors of taurine and synthetic breeds. This text discusses recent studies that seek strategies to improve the steps involved in the production of bovine embryos, as well as to increase the use of recipients in embryo transfer (ET) programs, focusing on taurine and synthetic cows.(AU)

Impacto da qualidade seminal e dos fatores relativos ao macho na produção in vitro de embriões bovinos / Impact of semen quality and other male-related factors on in vitro embryo production in cattle

A importância da qualidade do sêmen no processo comercial de produção in vitro de embriões (PIVE) é bem conhecida, ainda que não devidamente relatada na literatura. Existe não apenas uma significativa diferença entre touros nas taxas de clivagem e de blastocistos, mas também nas taxas de prenhezes subsequentes. Adicionalmente, há evidências de interação entre touro e tecnologia de processamento do sêmen (particularmente na separação de espermatozoides por sexo), entre touro e protocolo de preparação do sêmen para fertilização in vitro, e ainda entre touro e doadora. Controlar estes efeitos em uma rotina comercial tem sido um desafio crescente para os laboratórios, particularmente com a alta oferta de novos touros decorrente da recente adoção da seleção genômica. O presente trabalho aborda algumas destas questões, com base na experiência da Bio Biotecnologia da Reprodução Animal nesta área.(AU)

The importance of semen quality in a commercial in vitro embryo production (IVEP) routine is well-known, although underreported in the literature. There is not only a significant difference among sires on cleavage and blastocyst rates, but also on subsequent pregnancy rates. Moreover, there are evidences of interaction between sire and sperm processing technology (particularly in the case of sex-sorted semen), between sire and the protocol for sperm preparation for in vitro fertilization, and between sire and donor. Controlling such effects in a commercial routine has been a growing challenge for the laboratories, especially due to the high turnover of sires caused by the recent adoption of genomic selection in most breeds. The current study discusses some of these aspects, from the perspective of the experience of Bio Biotecnologia da Reprodução Animal in this field.(AU)

Maternal contributions to pregnancy success: from gamete quality to uterine environment

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.(AU)

The embryo non-invasive pre-implantation diagnosis era: how far are we?

Advancements in assisted reproduction (AR) methodologies have allowed significant improvements in live birth rates of women who otherwise would not be able to conceive. One of the tools that allowed this improvement is the possibility of embryo selection based on genetic status, performed via preimplantation genetic testing (PGT). Even though the widespread use of PGT from TE biopsy helped to decrease the interval from the beginning of the AR intervention to pregnancy, especially in older patients, in AR, there are still many concerns about the application of this invasive methodology in all cycles. Therefore, recently, researchers started to study the use of cell free DNA (cfDNA) released by the blastocyst in its culture medium to perform PGT, in a method called non-invasive PGT (niPGT). The development of a niPGT would bring the diagnostics power of conventional PGT, but with the advantage of being potentially less harmful to the embryo. Its implementation in clinical practice, however, is under heavy discussion since there are many unknowns about the technique, such as the origin of the cfDNA or if this genetic material is a true representative of the actual ploidy status of the embryo. Available data indicates that there is high correspondence between results observed in TE biopsies and the ones observed from cfDNA, but these results are still contradictory and highly debatable. In the present review, the advantages and disadvantages of niPGT are presented and discussed in relation to tradition TE biopsy-based PGT. Furthermore, there are also presented some other possible non-invasive tools that could be applied in the selection of the best embryo, such as quantification of other molecules as quality biomarkers, or the use artificial intelligence (AI) to identify the best embryos based on morphological and/or morphokitetic parameters.(AU)

Insulin-like growth factor-1 improves in vitro meiotic resumption of dromedary camel (Camelus Dromedarius) oocytes

Despite relatively high maturation rate of in vitro matured oocytes in the dromedary camel, however, blastocyst production is very low after in vitro fertilization (IVF). Herein, the influences of oocyte collection method (follicular aspiration vs slicing; Experiment I), the addition of Insulin-like growth factor I (IGF-I) to the maturation medium (Experiment II) on in vitro maturation (IVM) of oocyte were investigated. Although the nuclear maturation did not differ regardless of collecting method, follicular aspiration led to lower degeneration rates than those in controls (P < 0.05). The percentages of oocytes at MII were greater in the presence of IGF-1 than in its absence (71.9% vs 48.4%, respectively, P<0.05). Additionally, the percentages of degenerated oocytes were higher in the control group compared to oocytes cultured in the presence of IGF-I (23.6% vs 10.4%, respectively, P<0.05). IGF-I treatment improved the quality of MII matured oocytes as evidenced by the decrease of cathepsin B (CTSB) activity, a marker of poor quality oocytes, when compared to control ones (P < 0.05). In conclusion, follicular aspiration decreased the degeneration rate; however, it had no effect on completion of maturation. IGF-I enhanced the IVM of oocyte and decreased degeneration rate.(AU)

Prostaglandin F2α treatment concurrent with artificial insemination does not affect bovine embryo production / Administração de prostaglandina F2 alfa no momento da inseminação artificial não afeta a produção de embriões bovinos

Treatment with prostaglandin F2α (PGF) induces ovulation and increases conception rates in cows, while improving embryo production in buffalos. However, its effect on superovulated cows is unknown. This study verified whether single PGF administration concurrent with artificial insemination (AI) improves fertilization and embryo production rates in superovulated cows. In each replicate, embryo donor cows were equally allocated to two groups: the untreated control and PGF groups. The latter of which received 482 µg of cloprostenol concurrent with the first AI. Each cow (n = 35) was subjected to two superovulations (SOV) in a crossover design (total = 70 embryo collections). In the control and PGF groups, respectively, the observed responses were [median (95% CI)]: 12 (10-18) and 15 (12-18) total structures, 9 (7-11) and 7 (6-10) viable embryos, 1 (0-1) and 1 (1-3) degenerated embryos, and 1 (0-3) and 2 (0-5) oocytes (P > 0.05). In conclusion, single PGF treatment concurrent with the first AI did not affect embryo production in superovulated cows.

A prostaglandina F2α (PGF) pode induzir a ovulação e melhorar tanto a concepção em vacas, como a produção de embriões em búfalas, mas o efeito em vacas superovuladas é desconhecido. Esse estudo teve como objetivo verificar se a administração de uma dose de PGF na inseminação artificial (IA) após a superovulação (SOV) melhora as taxas de fecundação e produção embrionária em vacas. Em cada replicação, vacas doadoras de embriões foram equilibradamente alocadas em dois grupos: controle, não tratado, ou PGF, que recebeu 482 µg de cloprostenol no momento da primeira IA. Cada doadora (n = 35) foi submetida a duas SOV em um delineamento crossover (total = 70 coletas de embriões). Nos grupos controle e PGF, respectivamente, foram observados [medianas (IC 95%)]: 12 (10-18) e 15 (12-18) estruturas totais; 9 (7-11) e 7 (6-10) embriões viáveis; 1 (0-1) e 1 (1-3) embriões degenerados; e 1 (0-3) e 2 (0-5) ovócitos (P > 0,05). Conclui-se que uma única administração de PGF no momento da primeira IA não afeta a produção embrionária de vacas superovuladas.

The effect of adding L-ergothioneine in culture medium on blastocyst development rates

Background: Oocytes and embryos produce energy through mitochondrial oxidative phosphorylation by using oxygen. The membrane structure of the embryo is mostly composed of unsaturated fatty acids, for this reason DNA fragmentation, apoptosis, and abnormal gene expression are shaped as a result of the lipid peroxidation during culture. Oxidative stress (OS) is one of the most important problems affecting the in vitro embryo development. Antioxidant supplementation to the culture medium has been an alternative way to reduce cell damage caused by oxidative stress in in vitro embryo production systems. In this study, it was aimed to determine the effect of L-ergothioneine on blastocyst development when added to the culture medium. Materials, Methods & Results: The material of the study consisted of oocytes aspirated from the ovaries of Holstein cows which were collected from the local slaughterhouse. The ovaries were delivered to the laboratory within 2-3 h in a thermos which provided a constant temperature of 25-30o C with physiological saline (0.9%) containing antibiotics. All follicles in the 3-8 mm range on the ovaries were aspirated using 20 G needle. The collected follicle fluid was filtered through filters with a pore diameter of 70 micrometers. Cells remaining in the filter were washed with OPU medium and transferred to the petri dishes. Fluids were examined under a stereomicroscope. The cumulus-oocyte complexes were classified, and A and B quality oocytes were included to the study (A, B, C, and D quality COC). Oocytes aspirated from the ovaries and collected later on were incubated in IVM medium for 22 h. After maturation, it was taken into IVF medium, semen was added and incubated for 20-22 h. Possible zygotes to be taken to the culture stage were transferred to culture (IVC) drops with (L-ergothioneine 100 µL/mL (n:121) added and without antioxidant (control (n:124)), and kept in the incubator for 6-7 days. Evaluated on the 7th day differences in in vitro embryo production stages were evaluated with the Chi-square test. The study was run in 5 replicates each time, with at least 20 possible zygotes for per group being cultured. It was determined that 262 (87.33%) of a total of 300 oocytes undergoing in vitro maturation were matured. It was determined that 245 of the mature oocytes were fertilized (93.51%). The cleavage rates of the groups were determined as 87.60% and 86.29%, respectively. Eighty-two (33.47%) blastocysts were obtained from 245 zygotes taken into the culture stage, and the blastocyst rates in the groups were found to be 40.50% and 26.61%, respectively. After the study, it was determined that the statistical difference between L-ergothioneine and control in cleavage rates was insignificant (P > 0.05) and blastocyst rates was significant (P < 0.05) Discussion: Oxygen content above normal ratios can increase the formation of reactive oxygen species (ROS), particularly hydrogen peroxide (H2 O2 ), hydroxyl radical (HO·), and peroxyl radicals (ROO·). The increased rate of ROS negatively affects the success of IVP in mammalian embryos. It was observed that L-ergothioneine, which has high antioxidant activity, improved blastocyst development rates, and higher blastocyst rates could be achieved compared to the control group. By investigating the use of L-ergothioneine in different doses, it was thought that the dose with the highest antioxidant activity could be added to the culture medium in in vitro embryo production and more blastocysts could be produced.

Manual collapse of blastocoels in not effective in increasing the viability of vitrified equine embryos / Colabamento manual da blastocele não foi efetivo para aumentar a viabilidade de embriões equinos vitrificados

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 µm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 µm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.(AU)

Métodos de criopreservação de embriões têm sido utilizados com diversos objetivos. Maiores taxas de coleta embrio-nária em éguas com lavagem uterina realizada 7 a 8 dias pós ovulação. A criopreservação de embriões equinos com diâmetro <300 µm (6-6,5 dias após a ovulação) permite a obtenção de taxas de prenhez satisfatórias. Embriões com diâmetro >300 µm (7º dia pós-ovulação) somente são adequadamente criopreservados quando submetidos a colabamento da blastocele. Objetivando avaliar a viabilidade da punção da blastocele com equipamento de baixa sofisticação e custo, 22 embriões coletados no 8º. dia pós-ovulação (D8) foram alocados aos seguintes grupos: (1) micropunção com uma agulha 30 G assistida por micromanipula-dor antes da vitrificação (n=4); (2) microssecção manual por lâmina antes da vitrificação (n=6); (3) sem manipulação anterior à vitrificação (n=8); e (4) transferidos a fresco (n=4). Apesar de altas taxas de reexpansão após a criopreservação, os embriões manipulados previamente a vitrificação não resultaram em prenhez aos 25 dias. Tanto os embriões não micromanipulados, quanto os transferidos a fresco resultaram em prenhezes aos 25 dias. A microssecção manual não se mostrou eficiente como método para aumento da criotolerância de embriões grandes, necessitando um aprimoramento visando a obtenção de prenhezes.(AU)

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.(AU)

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus­oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL+) and without (CCL- ) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL+), contralateral (CL−), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL− cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL+ group; however, a higher proportion of embryos were obtained from the CCL− group. Besides, COCs from the CL+ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL− group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.(AU)

Effects of heat-stress on oocyte number and quality and in vitro embryo production in Holstein heifers

Background: This study aimed to determine the effects of environmental temperature on the number and quality of oocytes and embryo production rates obtained by performing ovum pick up (OPU). Heat stress leads to long-term, short-term, visible, and invisible effects in dairy cows. Its effects on reproduction are evident in all stages, from oocyte development to birth. Disturbance in ovarian follicle development, follicular dominance deficiency, anoestrus, polyspermia, embryonic losses, decreased fetal growth, and abortion are some examples of responses to these effects. The aim of the present study was aimed to determine the effects of ambient temperature on oocyte quality and number and embryo production rates. Materials, Methods & Results: The animal material used in this study comprised 10 Holstein heifers. At the beginning of the study, the heifers were 13-15 months old. OPU was performed at different times of the year, and weather conditions were recorded. Grouping according to ambient temperature was done as < 10°C (group 1), 10-25°C (group 2), and > 25°C (group 3). The veterinary ultrasonography device and a set of compatible intravaginal OPU probe, catheter, and aspiration device were used for OPU application. All antral follicles with diameters of 2-8 mm in the ovaries were aspirated. The aspirated follicle fluids were examined under a stereo microscope, and the cumulus-oocyte complexes (COC) were collected and classified according to their structure. A, B, and C-quality oocytes were included in the in vitro embryo production process. After performing 69 OPUs on random days of the cycle, the number of oocytes per OPU was found to be 8.72, 6.32, and 6.85 in groups 1, 2, and 3, respectively (P < 0.05). The number of viable oocytes per OPU was 6.83, 4.64, and 4.65 in groups 1, 2, and 3, respectively (P < 0.05). The statistical difference between the first group and the other groups was significant for cleavage and blastocyst counts (P < 0.05). Discussion: All the negative effects of heat stress on animals resulted from the increased body temperature. Reproductive performance is adversely affected by high temperatures and humidity during periods of high ambient temperatures. Metabolic heat is released, and the heat load increases due to the metabolism of nutrients in cattle. Internal body temperature is regulated via the dissipation of metabolic heat to the environment. The amount of heat dissipated via conduction and convection depends on the unit body weight, surface area, skin and coat color, difference in temperature gradient of the animal and ambient temperature, and humidity. In the present study, it was determined that the blastocyst development rates of the oocytes obtained in the warm season (>25°C [group 3]) were lower than those of the other groups. It was concluded that this may be because the oocytes developed under chronic heat stress in the animals, and several cycles were required to enhance oocyte quality and developmental potential. Additional studies are needed to investigate the response of oocytes obtained with OPU to heat stress during embryonic developmental stages and to determine the sensitivity and effects of embryonic tissue damage according to developmental stages. Based on the results of the present study, it was concluded that performing OPU and in vitro embryo production (IVEP) when the ambient temperature is close to the thermoneutral limits may increase the blastocyst development rates.

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using timelapse imaging

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm2, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.(AU)

In vitro toxicity of Niedenzuella (Tetrapterys) multiglandulosa on bovine embryos / Toxicidade in vitro da Niedenzuella (Tetrapterys) multiglandulosa em embriões bovinos

Niedenzuella (Tetrapterys) multiglandulosa, a vine plant found in Brazil, has been correlated to outbreaks of poisoning in cattle and buffaloes, generating economic losses related to the death due to heart failure, miscarriage, abortion, stillbirth, and neonatal mortality. The aim of this study was to examine the embryotoxic potential of the aqueous plant extract on in vitro bovine embryos. In vitro study was performed in five replicates of bovine embryo culture assigned in two groups: control, in vitro embryo culture medium without the aqueous plant extract; treated group, with addition of 2.7mg/mL of aqueous plant extract (10%) to the embryo culture on the sixth day of culture. Cleavage rate was evaluated at day 2 of the cell culture. Viability, hatchability and underdevelopment of blastocysts on the seventh, eighth, and ninth days (D7, D8, and D9, respectively) of culture were assessed under stereoscopic microscope. On day 7, blastocysts were submitted to TUNEL assay to determine apoptotic index. In vitro exposure of bovine embryos to of N. multiglandulosa resulted in reduced embryo development and survival, evaluated by dark cytoplasm indicating poor morphology and poor quality with marked reduction of hatchability. We observed a significant reduction of blastocyst production/number of cleaved embryos (60.6% vs 41.5%); reduction of blastocysts production/total number of matured bovine oocytes (35.1% vs 21.3%); and embryonic hatching rates (38.0% vs 10.0%). However, no effects were observed on the apoptotic rate. In conclusion, aqueous extract of N. multiglandulosa leaves reduces bovine embryo viability in vitro, suggesting possible detrimental effects on embryo development.(AU)

Niedenzuella (Tetrapterys) multiglandulosa, uma videira encontrada no Brasil, tem sido correlacionada a surtos de intoxicações em bovinos e búfalos, gerando perdas econômicas relacionadas à morte por insuficiência cardíaca, aborto, natimorto e mortalidade neonatal. O objetivo deste estudo foi examinar o potencial embriotóxico do extrato vegetal aquoso em embriões bovinos in vitro. O estudo in vitro foi realizado em cinco repetições de cultura de embriões bovinos distribuídos em dois grupos: controle, meio de cultura de embriões in vitro sem o extrato aquoso da planta; grupo tratado, com adição de 2,7mg / mL de extrato vegetal aquoso (10%) à cultura do embrião no sexto dia de cultivo. A taxa de clivagem foi avaliada no dia 2 da cultura de células. Viabilidade, eclodibilidade e subdesenvolvimento de blastocistos no sétimo, oitavo e nono dia (D7, D8 e D9, respectivamente) de cultura foram avaliados em microscópio estereoscópico. No dia 7, os blastocistos foram submetidos ao ensaio TUNEL para determinar o índice apoptótico. Observamos redução significativa da produção de blastocisto / número de embriões clivados (60,6% vs 41,5%); redução da produção de blastocistos / número total de oócitos bovinos maturados (35,1% vs 21,3%); e taxas de eclosão embrionária (38,0% vs 10,0%). No entanto, nenhum efeito foi observado na taxa de apoptose. Em conclusão, o extrato aquoso das folhas de N. multiglandulosa reduz a viabilidade do embrião bovino in vitro, sugerindo possíveis efeitos prejudiciais no desenvolvimento embrionário.(AU)

Does combining estradiol cypionate and GnRH for ovulation induction in recipient cows increase pregnancy rate after timed embryo transfer?

Estradiol cypionate (EC) or GnRH have been widely used for ovulation induction in timed embryo transfer (TET). EC administration increases the proportion of cows that show estrus, whereas GnRH promotes more synchronized ovulations. The aim of the present study was to evaluate the potential beneficial effects of combining EC and GnRH in TET. In experiment 1, no difference was observed on serum progesterone concentrations on Day 6 and 13 after GnRH treatment between GnRH and EC+GnRH groups. In experiment 2, pregnancy per embryo transfer (P/ET) did not differ (p = 0.69) between GnRH (62.8%) and EC+GnRH (58.7%) groups. In conclusion, combining EC and GnRH for ovulation induction does not increase progesterone secretion and pregnancy rate after TET in cattle.(AU)

Uso de éguas receptoras acíclicas em programas de transferência de embrião / Anovulatory recipient mares in embryo transfer programs

O maior entrave para o êxito de um programa de transferência de embrião (TE) está relacionado às receptoras, tanto a qualidade de características reprodutivas, quanto a sincronização entre doadoras e receptoras. Éguas doadoras, geralmente, apresentam melhor escore corporal e disponibilidade alimentar nos períodos de seca, fazendo com que iniciem o seu ciclo estral precocemente enquanto éguas receptoras se encontram ainda em anestro ou período transicional, o que dificulta a TE. Vários estudos foram realizados com o objetivo de utilizar receptoras acíclicas como receptoras de embrião. Assim, podem ser utilizadas nos programas de TE quando submetidas a protocolos hormonais que mimetizam o ciclo estral natural. Uma diversidade de protocolos é encontrada na literatura, usando diferentes doses, posologias e intervalos do uso de estradiol associado a progestágenos. Portanto, objetivou-se analisar resultados de pesquisas com protocolos hormonais que poderiam ajudar a solucionar a escassez de receptoras cíclicas e maximizar os resultados da biotécnica de transferência de embriões.(AU)

There are obstacles to the success of equine embryo transfer programs, concomitantly there has been an increase in the popularity of the technique. One of the most urgent issues to overcome is related to the recipient mare availability and the donor-recipient synchrony. Usually, the donor mares have a higher body condition score and are fed a higher quality diet than recipient mares during the non-breeding season. Therefore, the donor mares start cycling earlier than recipient mares, which makes embryo transfer challenging. Several studies have been carried on finding hormonal protocols that mimic the natural oestrous cycle to allow the use of noncycling recipient mares for embryo transfer programs. Many protocols can be found in the literature, using different doses and intervals. Among the main difference is the use of progestogens, which can be used alone or in combination with oestradiol. The aim of this review was to analyse results that could help solve the lack of recipient mares and improve the results of the embryo transfer technique.(AU)