Differentially methylated regions identified in bovine embryos are not observed in adulthood

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.(AU)

The waveless model: clinical and research applications of follicular wave suppression in cattle / Modelo waveless: aplicações clínicas e na pesquisa da supressão das ondas foliculares em bovinos

The follicular growth waves are directly linked to the fluctuations in plasma gonadotrophins, which are controlled by the hypothalamic GnRH release pattern. Therefore, if the actions of the GnRH are inhibited or blocked, the final stages of the antral follicle growth are suppressed, resulting in an induced anestrus (a.k.a. waveless model). In the human medicine, GnRH agonists or antagonists are broadly used in the control of ovarian disfunctions, as well as in the preparation of women for assisted reproductive cycles. In cattle, a similar effect can be obtained by active immunization against GnRH. This was shown to be a viable strategy, for example, for the control of chronic cases of cystic ovarian disease in oocyte donors. However, on shall take into account the substantial individual variation on the immune response and, consequently, the lack of control of the duration of the anestrus induced. The waveless model is also very useful as a research model, once it controls the potential interference of the endogenous FSH and LH, improving the sensitivity of essays with exogenous hormones and consequently reducing the required number of replicas within studies.(AU)

O padrão de crescimento folicular em ondas está diretamente associado às flutuações nas concentrações plasmáticas de gonadotrofinas, controladas por sua vez pelo padrão de liberação de GnRH hipotalâmico. Desta forma, a inibição ou bloqueio da ação do GnRH suprime as etapas finais do crescimento folicular, resultando em anestro induzido (também chamado modelo waveless). Na medicina humana, agonistas ou antagonistas de GnRH são utilizados tanto no controle de disfunções ovarianas quanto na preparação de pacientes para procedimentos de reprodução assistida. Em bovinos, este efeito pode ser obtido pela imunização ativa contra GnRH, e mostrou-se estratégia viável, por exemplo, no controle de casos crônicos de doença ovariana cística em doadoras de oócitos. Contudo, é importante considerar a grande variação individual na resposta à imunização e consequente impossibilidade de controlar a duração do anestro induzido. O modelo waveless também é de grande utilidade na pesquisa, uma vez que elimina a potencial interferência do FSH e LH endógenos, aumentando a sensibilidade nos ensaios com hormônios exógenos e consequentemente reduzindo o número de réplicas necessárias nos estudos.(AU)

Genetic evaluation of oocyte and embryo production in dairy Gir cattle using repeatability and random regression models

The objective of this work is to estimate genetic parameters and breeding values to improve embryo and oocyte production, using repeatability and random regression models (RRM) for Gir dairy cattle. We used 11,398 records of ovum pick-up from 1,747 dairy Gir donors and evaluated sixteen different models: the traditional repeatability model and fifteen RRM, each of which considered a different combination of Legendre polynomial regressors to describe the additive genetic and permanent environment effects. The 4G1P model (four regressors for the genetic effect and one regressor for the permanent environment effect) is the most suitable model to analyze the number of viable and total oocytes, while the 3G1P is the best model to analyze the number of cleaved and viable embryos, according to the values of the Akaike information criterion (AIC) and the Bayesian information criterion (BIC). The heritability estimated with the RRM was higher than that estimated with the repeatability model. The high repeatability reported for oocyte and embryo count traits indicates that donors, which had high oocyte and embryo counts in the first ovum pick-up, should maintain this result in the next ovum pick-up. Genetic correlations between adjacent ages were high and positive, while genetic correlations between extreme ages were weak. We observed a reranking of the top sires and females (heifers and cows) over the period evaluated. The reliability of the estimated breeding values by RRM showed changes across age, and the expected genetic gains by RRM are larger. This shows that RRM is most suitable alternative for the evaluation and selection of oocyte and embryo count traits.(AU)

Synchronization with estradiol benzoate in the presence of the corpus luteum in Wagyu cows increases the number of medium follicles but does not interfere with in vitro production of embryos / Sincronização com benzoato de estradiol na presença do corpo lúteo em vacas de Wagyu aumenta o número de folículos médios, mas não interfere com a produção in vitro de embriões

Reproductive biotechnologies are emerging as an important element for livestock; however, some strategies must be modified to adapt to different breeding systems, such as the use of follicular synchronization protocols. This study aimed to evaluate follicular synchronization using estradiol benzoate (EB), in the presence of the corpus luteum (CL) from Wagyu oocyte donors in in vitro embryo production (IVEP). Rounds of IVEP were performed in heifers and cows (n=19) that were classified into three groups: G1/CL - animals with CL, G2/WCL - animals without CL, and G3/CL + EB - animals with CL that were subjected to follicular synchronization with EB at D0. The groups G1/CL and G2/WCL were considered the control and undertook the natural process of follicular dynamics. The results showed that the synchronization of the follicular wave with the application of EB in the presence of CL, presented a smaller number of small (6.05 ± 0.55) and large follicles (0.45 ± 0.15), but increased (P < 0.05) the number of medium-sized follicles (16.20 ± 0.90). However, the results of ovum pick up showed that regardless of whether or not EB was applied, and regardless of the presence or absence of CL in the Wagyu donor, there was no difference among the groups (P > 0.05) concerning the number of viable oocytes and the viability rate. It was concluded that follicular synchronization using EB in Wagyu oocyte donors that presented a CL, increased the number of medium-sized follicles. However, there was no improvement in the efficiency of ovum pick up, in vitro embryo production, and pregnancy rate.(AU)

As biotecnologias reprodutivas estão emergindo como um elemento importante para a pecuária, entretanto, algumas estratégias devem ser modificadas para se adaptar a diferentes sistemas de criação, como o uso de protocolos de sincronização folicular. Este estudo teve como objetivo avaliar a sincronização folicular utilizando benzoato de estradiol (BE), na presença do corpo lúteo (CL) de doadoras de oócitos Wagyu na produção in vitro de embriões (PIVE). Sessões de PIVE foram realizadas em novilhas e vacas (n=19) que foram classificadas em três grupos: G1/CL - animais com CL, G2/SCL - animais sem CL e G3/CL+BE - animais com CL que foram submetidos para sincronização folicular com BE em D0. Os grupos G1/CL e G2/ SCL foram considerados o controle e realizaram o processo natural da dinâmica folicular. Os resultados mostraram que a sincronização da onda folicular com a aplicação de BE na presença de CL, apresentou um menor número de folículos pequenos (6,05 ± 0,55) e grandes (0,45 ± 0,15), mas aumentou (P < 0,05) o número de folículos de tamanho médio (16,20 ± 0,90). No entanto, os resultados da ovum pick up mostraram que independente da aplicação ou não de BE, e independente da presença ou ausência de CL na doadora Wagyu, não houve diferença entre os grupos (P > 0,05) quanto ao número de oócitos viáveis e a taxa de viabilidade. Concluiu-se que a sincronização folicular por meio de BE em doadoras de oócitos Wagyu que apresentaram CL, aumentou o número de folículos de tamanho médio. No entanto, não houve melhora na eficiência da ovum pick up, na produção in vitro de embriões e na taxa de prenhez.(AU)

In vitro performance of Zebu (Bos indicus) and Taurus (Bos taurus) donor cow embryos / Desempenho de vacas doadoras zebuínas (Bos indicus) e taurinas (Bos taurus) na produção de embrião in vitro

In this study, the in vitro production of bovine embryos from zebu and taurine donors was compared. Cumulus-oocyte complexes (COCs) were obtained from 167 Bos taurus and 161 Bos indicus donors by ovum pick-up. COCs were classified based on their morphological quality, matured in incubators for 22 to 24 h in maturation medium, and then fertilized for 18 to 22 h. The zygotes were transferred to the culture medium for seven days. The embryos were classified as morula (OM), initial blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), before being transferred to synchronized recipient cows. Pregnancy was diagnosed 30-45 days post-transfer. The Bos indicus donors had a higher oocyte yield (n = 2556) than Bos taurus donors (n = 1903) (P = 0.008). The COCs from zebu donors had a better morphological quality than those from taurine donors (n = 689 vs. 444 for grade 1 COC, P < 0.0001; n = 681 vs. 509 for grade 2 COC, P = 0.010, for zebu and taurine donors, respectively). There were differences in embryo production percentages obtained from OM (0.44% from zebu and 6.42% from taurine, P = 0.017), BL (14.18% from zebu and 3.74% from taurine, P < 0.0001), and BX (81.43% from zebu and 75.13% from taurine, P < 0.0001). No significant difference was observed for embryo production from BI and pregnancy rate (P > 0.05). The Bos indicus cows showed greater oocyte recovery, number of viable oocytes, and production of viable embryos than the Bos taurus cows.(AU)

O objetivo foi avaliar a produção in vitro de embriões bovinos a partir de doadoras zebuína e taurina. Os complexos cumulus oócitos (COCs) foram obtidos de 167 doadoras Bos taurus e 161 Bos indicus, por meio de Ovum pick-up. Os COCs foram classificados quanto à qualidade morfológica, maturados em incubadora por 22 a 24h em meio de maturação, e encaminhados à fertilização entre 18 e 22h. Os zigotos foram transferidos para meio de cultivo, onde permaneceram durante sete dias. Os embriões foram classificados em mórula (MO), blastocisto inicial (BI), blastocisto (BL) e blastocisto expandido (BX), envasados em palhetas contendo meio de cultivo, e inovulados em receptoras sincronizadas. Após 30 e 45 dias da inovulação, realizou-se o diagnóstico de gestação. Os animais Bos indicus apresentaram maior produção oocitária (n = 2556) em comparação aos Bos taurus (n = 1903) (P = 0,008). Os COCs das doadoras zebu apresentaram melhor qualidade morfológica do que das doadoras taurina (n = 689 vs. 444 para COCs grau I, P < 0,0001; n = 681 vs. 509 para COCs grau II, P = 0,010, para doadoras zebu e taurina, respectivamente). Quanto à produção de embriões, houve diferença nas porcentagens obtidas de MO (0,44% zebuínas e 6,42% taurinas, P = 0,017), BL (14,18% zebuínas e 3,74% taurinas, P < 0,0001) e BX (81,43% zebuínas e 75,13% taurinas, P < 0,0001). Não foi observado diferença para BI e taxa de prenhez (P > 0,05). Os animais Bos indicus apresentaram maior recuperação oocitária e maior número de oócitos viáveis em comparação às vacas Bos taurus, como também, maior produção de embriões viáveis.(AU)

In vitro performance of Zebu (Bos indicus) and Taurus (Bos taurus) donor cow embryos / Desempenho de vacas doadoras zebuínas (Bos indicus) e taurinas (Bos taurus) na produção de embrião in vitro

In this study, the in vitro production of bovine embryos from zebu and taurine donors was compared. Cumulus-oocyte complexes (COCs) were obtained from 167 Bos taurus and 161 Bos indicus donors by ovum pick-up. COCs were classified based on their morphological quality, matured in incubators for 22 to 24 h in maturation medium, and then fertilized for 18 to 22 h. The zygotes were transferred to the culture medium for seven days. The embryos were classified as morula (OM), initial blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), before being transferred to synchronized recipient cows. Pregnancy was diagnosed 30-45 days post-transfer. The Bos indicus donors had a higher oocyte yield (n = 2556) than Bos taurus donors (n = 1903) (P = 0.008). The COCs from zebu donors had a better morphological quality than those from taurine donors (n = 689 vs. 444 for grade 1 COC, P 0.05). The Bos indicus cows showed greater oocyte recovery, number of viable oocytes, and production of viable embryos than the Bos taurus cows.

O objetivo foi avaliar a produção in vitro de embriões bovinos a partir de doadoras zebuína e taurina. Os complexos cumulus oócitos (COC’s) foram obtidos de 167 doadoras Bos taurus e 161 Bos indicus, por meio de Ovum pick-up. Os COC’s foram classificados quanto à qualidade morfológica, maturados em incubadora por 22 a 24h em meio de maturação, e encaminhados à fertilização entre 18 e 22h. Os zigotos foram transferidos para meio de cultivo, onde permaneceram durante sete dias. Os embriões foram classificados em mórula (MO), blastocisto inicial (BI), blastocisto (BL) e blastocisto expandido (BX), envasados em palhetas contendo meio de cultivo, e inovulados em receptoras sincronizadas. Após 30 e 45 dias da inovulação, realizou-se o diagnóstico de gestação. Os animais Bos indicus apresentaram maior produção oocitária (n = 2556) em comparação aos Bos taurus (n = 1903) (P = 0,008). Os COCs das doadoras zebu apresentaram melhor qualidade morfológica do que das doadoras taurina (n = 689 vs. 444 para COCs grau I, P 0,05). Os animais Bos indicus apresentaram maior recuperação oocitária e maior número de oócitos viáveis em comparação às vacas Bos taurus, como também, maior produção de embriões viáveis.

Estacionalidade e a produção de oócitos e embriões in vitro em vacas Gir leiteiro na região sudeste do Brasil / Seasonality and the production of oocytes and in vitro embryos in Gir dairy cows in the southeast region of Brazil

A eficiência reprodutiva de doadoras de embriões Bos taurus é afetada pelas estações devido ao estresse calórico em países de clima tropical e temperado. O objetivo do estudo foi avaliar o efeito das condições climáticas de verão e inverno na produção de oócitos e embriões in vitro na raça Gir Leiteiro (Bos indicus) em fazendas da região Sudeste do Brasil. Os parâmetros avaliados foram: total de oócitos recuperados (OT), oócitos viáveis (OV) e percentual de oócitos viáveis sobre o total (POV); embriões produzidos (EP) e percentual de embriões produzidos (PEP) sobre o total de estruturas recuperadas pela aspiração folicular guiada por ultrassonografia (OPU). Os oócitos foram obtidos de 153 doadoras pluríparas de diferentes idades e ordens de parição de 10 rebanhos. As médias obtidas em 380 aspirações foliculares (OPU) nos meses de verão foram: OT=14,8; OV=12,6; EP= 3,4; PEP=22,3%, e no inverno de OT=13,4; OV=10,8; EP=3,5; PEP=26,1%, apresentando coeficiente de variação de até 84%, porém sem diferença estatísticas (p<0,05) entre estes períodos, utilizando-se o teste de Kruscal Wallis s (p<0,05). Apenas a percentagem de oócitos viáveis foi maior (p<0,05) no período de verão (POV=84,4 vs. 80,0%), contudo, o total de embriões PIVE foi semelhante nos dois períodos. Conclui-se que a produção de embriões da raça Gir Leiteiro, criadas na região Sudeste do Brasil, não foi afetada pela sazonalidade climática.

The reproductive performance of B.taurus embryo donors is affected in many tropical and temperate countries. The objective of this study was to evaluate the effects of the climatic conditions of summer and winter climatic conditions on the production of oocytes and in vitro embryos in dairy cows of the breed Gir Leiteiro (Bos indicus) at farms in Southeast of the Brazil. The variable answers evaluated were the total oocytes recovered (TO) and percentage of viable oocytes over the total (PVO); embryo production (EP) and percentage of embryos in viable oocytes (PE) over the total of structures recovered by follicular aspiration guided by ultrasonography (OPU). The oocytes analyzed were obtained from 10 herds and 153 pubertal donors of different ages and orders of parturition. The average from 380 ovum pick up (OPU) during summer were: TO=14.8; PVO=12.6; EP=3.4; PE=22.3%, and TO=13.4; PVO=10.8; EP=3.5; PE=26.1%, with variation coefficient of up to 84%, and no differences evaluated through the Kruscal Wallis (p<0.05). Only the percentage of viable oocytes was higher (p<0.05) in the summer period (PVO=84.4 vs. 80.0%). However, the embryo production (IVEP) was similar in both seasons (summer and winter). Therefore, we could conclude that the embryo production of Dairy Gir cows in Southeast of the Brazil was not affected by weather seasonality.

Estacionalidade e a produção de oócitos e embriões in vitro em vacas Gir leiteiro na região sudeste do Brasil / Seasonality and the production of oocytes and in vitro embryos in Gir dairy cows in the southeast region of Brazil

A eficiência reprodutiva de doadoras de embriões Bos taurus é afetada pelas estações devido ao estresse calórico em países de clima tropical e temperado. O objetivo do estudo foi avaliar o efeito das condições climáticas de verão e inverno na produção de oócitos e embriões in vitro na raça Gir Leiteiro (Bos indicus) em fazendas da região Sudeste do Brasil. Os parâmetros avaliados foram: total de oócitos recuperados (OT), oócitos viáveis (OV) e percentual de oócitos viáveis sobre o total (POV); embriões produzidos (EP) e percentual de embriões produzidos (PEP) sobre o total de estruturas recuperadas pela aspiração folicular guiada por ultrassonografia (OPU). Os oócitos foram obtidos de 153 doadoras pluríparas de diferentes idades e ordens de parição de 10 rebanhos. As médias obtidas em 380 aspirações foliculares (OPU) nos meses de verão foram: OT=14,8; OV=12,6; EP= 3,4; PEP=22,3%, e no inverno de OT=13,4; OV=10,8; EP=3,5; PEP=26,1%, apresentando coeficiente de variação de até 84%, porém sem diferença estatísticas (p<0,05) entre estes períodos, utilizando-se o teste de Kruscal Wallis s (p<0,05). Apenas a percentagem de oócitos viáveis foi maior (p<0,05) no período de verão (POV=84,4 vs. 80,0%), contudo, o total de embriões PIVE foi semelhante nos dois períodos. Conclui-se que a produção de embriões da raça Gir Leiteiro, criadas na região Sudeste do Brasil, não foi afetada pela sazonalidade climática.(AU)

The reproductive performance of B.taurus embryo donors is affected in many tropical and temperate countries. The objective of this study was to evaluate the effects of the climatic conditions of summer and winter climatic conditions on the production of oocytes and in vitro embryos in dairy cows of the breed Gir Leiteiro (Bos indicus) at farms in Southeast of the Brazil. The variable answers evaluated were the total oocytes recovered (TO) and percentage of viable oocytes over the total (PVO); embryo production (EP) and percentage of embryos in viable oocytes (PE) over the total of structures recovered by follicular aspiration guided by ultrasonography (OPU). The oocytes analyzed were obtained from 10 herds and 153 pubertal donors of different ages and orders of parturition. The average from 380 ovum pick up (OPU) during summer were: TO=14.8; PVO=12.6; EP=3.4; PE=22.3%, and TO=13.4; PVO=10.8; EP=3.5; PE=26.1%, with variation coefficient of up to 84%, and no differences evaluated through the Kruscal Wallis (p<0.05). Only the percentage of viable oocytes was higher (p<0.05) in the summer period (PVO=84.4 vs. 80.0%). However, the embryo production (IVEP) was similar in both seasons (summer and winter). Therefore, we could conclude that the embryo production of Dairy Gir cows in Southeast of the Brazil was not affected by weather seasonality.(AU)

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P 0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P 0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.(AU)

How can we improve embryo production and pregnancy outcomes of Holstein embryos produced in vitro? (12 years of practical results at a California dairy farm)

Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.

How can we improve embryo production and pregnancy outcomes of Holstein embryos produced in vitro? (12 years of practical results at a California dairy farm)

Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.(AU)

Strategies to increment in vivo and in vitro embryo production and transfer in cattle

Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and resynchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.

Strategies to increment in vivo and in vitro embryo production and transfer in cattle

Knowledge of follicular wave dynamics obtained through the use of real-time ultrasonography and the development of the means by which follicular wave dynamics can be controlled have provided practical approaches for the in vivo and in vitro production and transfer of embryos in cattle. The elective control of follicular wave emergence and ovulation has had a great impact on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. Although estradiol and progestins have been used for many years, practitioners in countries where estradiol cannot be used have turned to alternative treatments, such as mechanical follicle ablation or the administration of GnRH for the synchronization of follicle wave emergence. In vitro embryo production also benefits from the synchronization of follicle wave emergence prior to Cumulus Oocyte Complexes (COCs) recovery. As Bos indicus cattle have high antral follicle population, large numbers of oocytes can be obtained by ovum pick-up (OPU) without superstimulation. However, synchronization of follicular wave emergence and superstimulation is necessary to obtain high numbers of COCs by OPU and blastocysts following in vitro fertilization in Bos taurus donors. Finally, embryos can now be transferred in commercial beef or dairy herds using efficacious synchronization and resynchronization protocols that are easily implemented by farm personnel. These technologies can also be used to resolve reproductive problems such as the reduced fertility observed during summer heat stress and/or in repeat-breeder cows in commercial dairy herds.(AU)

In vitro embryo production in buffaloes: from the laboratory to the farm

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.

In vitro embryo production in buffaloes: from the laboratory to the farm

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.(AU)

Breed composition does not influence the performance of Holstein-Gyr crossbred as oocyte donors for OPU/IVP

Holstein-Gyr crossbred cattle are strategic for dairy systems in tropical countries, since they combine milk yield genetics with adaptability to tropical climate. However, Holstein (Bos taurus) and Gyr (Bos indicus) breeds present remarkable differences regarding reproductive physiology. Brazil stands out as the world’s largest user of embryo in vitro production (IVP) in bovine, and the use of this technique is increasing in dairy systems. As Holstein-Gyr crossbreds are important oocyte donors for IVP, the present work aimed at investigating whether increased Gyr or Holstein breed composition influences donor’s performance. Sixteen Holstein-Gyr crossbred females presenting increased (HG, 71.4 to 87.5% Holstein; n = 9) or decreased (GH, 40.2 to 46.6% Holstein; n = 7) Holstein composition were submitted to three ovum pick up (OPU) sessions. We observed similar (P = 0.2946) antral follicle count between HG and GH donors (24.8 ± 3.2 vs 29.4 ± 2.8 respectively; mean ± SEM). Groups also display similar morphological oocyte grading (Grade I: 0.1 ± 0.1 vs 0.1 ± 0.1 – P = 0.9680; Grade II: 0.9 ± 0.5 vs 1.9 ± 0.5 – P = 0.1942; Grade III, 4.0 ± 1.2 vs 7.2 ± 1.4 – P = 0.1047, HG vs GH respectively; mean ± SEM). Additionally, the proportion of viable oocyte was similar between HG and GH groups (27.8% vs 31.9%, respectively, P = 0.3500) and oocyte lipid area fraction (6.8% vs 9.5%, respectively; P = 0.1539). Our results indicate that the individual variation has more influence than breed composition of crossbred oocyte donors.

Breed composition does not influence the performance of Holstein-Gyr crossbred as oocyte donors for OPU/IVP

Holstein-Gyr crossbred cattle are strategic for dairy systems in tropical countries, since they combine milk yield genetics with adaptability to tropical climate. However, Holstein (Bos taurus) and Gyr (Bos indicus) breeds present remarkable differences regarding reproductive physiology. Brazil stands out as the worlds largest user of embryo in vitro production (IVP) in bovine, and the use of this technique is increasing in dairy systems. As Holstein-Gyr crossbreds are important oocyte donors for IVP, the present work aimed at investigating whether increased Gyr or Holstein breed composition influences donors performance. Sixteen Holstein-Gyr crossbred females presenting increased (HG, 71.4 to 87.5% Holstein; n = 9) or decreased (GH, 40.2 to 46.6% Holstein; n = 7) Holstein composition were submitted to three ovum pick up (OPU) sessions. We observed similar (P = 0.2946) antral follicle count between HG and GH donors (24.8 ± 3.2 vs 29.4 ± 2.8 respectively; mean ± SEM). Groups also display similar morphological oocyte grading (Grade I: 0.1 ± 0.1 vs 0.1 ± 0.1 P = 0.9680; Grade II: 0.9 ± 0.5 vs 1.9 ± 0.5 P = 0.1942; Grade III, 4.0 ± 1.2 vs 7.2 ± 1.4 P = 0.1047, HG vs GH respectively; mean ± SEM). Additionally, the proportion of viable oocyte was similar between HG and GH groups (27.8% vs 31.9%, respectively, P = 0.3500) and oocyte lipid area fraction (6.8% vs 9.5%, respectively; P = 0.1539). Our results indicate that the individual variation has more influence than breed composition of crossbred oocyte donors.(AU)

Laparoscopic ovum pick-up for in vitro embryo production from dairy bovine andbuffalo calves

Laparoscopic ovum pick-up (LOPU) conducted on bovine and buffalo calves of 2-6-month of age, followed by in vitro embryo production and transfer into synchronous adult recipients, is a powerful tool for accelerated genetic gain and early dissemination of top genetics. In its current state, the technology is characterized by higher oocyte recovery rates, lower oocyte-to-embryo yields, and similar pregnancy and term development rates compared with adult counterparts. Improvements in oocyte competence have been made in recent years mainly through gonadotropin stimulation protocols tailored for prepubertal donors. These advances have brought the technology to the point of been apt for commercial application. However, future research must focus on increasing the proportion of fully competent oocytes recovered from calves thereby further empowering the role this technology platform can play in programs for accelerated dissemination of superior genetics.

Laparoscopic ovum pick-up for in vitro embryo production from dairy bovine andbuffalo calves

Laparoscopic ovum pick-up (LOPU) conducted on bovine and buffalo calves of 2-6-month of age, followed by in vitro embryo production and transfer into synchronous adult recipients, is a powerful tool for accelerated genetic gain and early dissemination of top genetics. In its current state, the technology is characterized by higher oocyte recovery rates, lower oocyte-to-embryo yields, and similar pregnancy and term development rates compared with adult counterparts. Improvements in oocyte competence have been made in recent years mainly through gonadotropin stimulation protocols tailored for prepubertal donors. These advances have brought the technology to the point of been apt for commercial application. However, future research must focus on increasing the proportion of fully competent oocytes recovered from calves thereby further empowering the role this technology platform can play in programs for accelerated dissemination of superior genetics.(AU)