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)

Use of new recombinant proteins for ovarian stimulation in ruminants

Currently, gonadotropin products (follicle stimulating hormone, FSH, and luteinizing hormone, LH) used in animal reproduction are produced by extraction and purification from abattoir-derived pituitary glands. This method, relying on animal-derived materials, carries the potential risk of hormone contamination and pathogen transmission. Additionally, chorionic gonadotropins are extracted from the blood of pregnant mares (equine chorionic gonadotropin; eCG) or the urine of pregnant women (human chorionic gonadotropin; hCG). However, recent advancements have introduced recombinant gonadotropins for assisted animal reproduction therapies. The traditional use of FSH for superovulation has limitations, including labor requirements and variability in superovulation response, affecting the success of in vivo (SOV) and in vitro (OPU/IVEP) embryo production. FSH treatment for superstimulation before OPU can promote the growth of a homogenous follicular population and the recovery of competent oocytes suitable for IVEP procedures. At present, a single injection of a preparation of long-acting bovine recombinant FSH (rFSH) produced similar superovulation responses resulting in the production of good-quality in vivo and in vitro embryos. Furthermore, the treatment with eCG at FTAI protocol has demonstrated its efficacy in promoting follicular growth, ovulation, and P/AI, mainly in heifers and anestrous cows. Currently, treatment with recombinant glycoproteins with eCG-like activity (r-eCG) have shown promising results in increasing follicular growth, ovulation, and P/AI in cows submitted to P4/E2 -based protocols. Bovine somatotropin (bST) is a naturally occurring hormone found in cows. Recombinant bovine somatotropin (rbST), produced through genetic engineering techniques, has shown potential in enhancing reproductive outcomes in ruminants. Treatment with rbST has been found to improve P/IA, increase donor embryo production, and enhance P/ET in recipients. The use of recombinant hormones allows to produce non-animal-derived products, offering several advantages in assisted reproductive technologies for ruminants. This advancement opens up new possibilities for improving reproductive efficiency and success rates in the field of animal reproduction.(AU)

Infuence of heat stress on in vitro oocyte and embryo production in high-yielding Holstein cows / Influência do estresse calórico na produção in vitro de oócitos e embriões de vacas Holandesas de alta produtividade

The objective of this study was to evaluate the influence of thermal shock on oocytes used in the production of in vitro embryos (IVP) of high productivity Holstein cows on the day of follicular aspiration (OPU; 0), 30, 60 and 90 days before the OPU. From the mean temperature on day 0 and on the previous 30, 60 and 90 days, they were classifed into comfort group (TC; up to 15°C) and heat stress (HS; above 15°C) groups.Anegative influence was observed on oocytes and viable embryos (total and grade I). The heat stress in the periods of 30 and 60 days prior to OPU resulted in lower production of viable oocytes (P=0.0028; P=0.0092, respectively). Under stress, on the day of OPU (HS-OPU), cows showed no reduction in the amount of viable oocytes (P=0.5497) and there was no influence of temperature for the group stressed 90 days before OPU (P=0.8287). For total embryos, the difference occurred only in the HS-30 group (P=0.0317), where the groups HS-OPU, HS-60, HS-90 presented, respectively, P=0. 1987, P=0.0596 and P=0.4580. Regarding the production of embryos of grade 1, there was no difference for the groups HS-OPU (P=0.2291) and HS-90 (P=0.2868), but there was a reduction for HS-30 (P=0.0143) and HS-60 (P=0.0253). In summary, heat stress had a negative impact when it occurred 30 or 60 days before follicular aspiration. In addition, 30 days seems to be the period of more susceptibility and that causes the greatest deleterious effects on oocyte viability and IVP.(AU)

Objetivou-se avaliar a infuência do estresse térmico em oócitos utilizados na produção in vitro de embriões (PIV) bovinos da raça Holandesa de alta produtividade no dia da aspiração folicular (OPU; 0), 30, 60 e 90 dias antes da OPU. A partir da temperatura média no dia 0 e aos 30, 60 e 90 dias anteriores, foram classificados nos grupos conforto (CT; até 15°C) e estresse por calor (ET -acima de 15°C). Observou-se infuência negativa em oócitos e embriões viáveis (total e grau I). A submissão ao estresse térmico nos períodos de 30 e 60 dias anteriores à OPU resultou em menor produção de oócitos viáveis (P=0,0028; P=0,0092, respectivamente). Sob estresse, no dia da OPU (ET-OPU), as vacas não apresentaram redução na quantidade de oócitos viáveis (P=0,5497) e não houve infuência da temperatura para o grupo estressado 90 dias antes da OPU (P=0,8287). Para embriões totais, a diferença ocorreu apenas no grupo ET-30 (P=0,0317), onde os grupos ET-OPU, ET-60, ET-90 apresentaram, respectivamente, P=0,1987, P=0,0596 e P=0,4580. Em relação à produção de embriões grau 1, não houve diferença para os grupos ET-OPU (P=0,2291) e ET-90 (P=0,2868), porém houve redução para ET-30 (P=0,0143) e ET- 60 (P=0,0253). Em resumo, o estresse por calor teve impacto negativo quando ocorreu 30 ou 60 dias antes da aspiração folicular. Além disso, 30 dias parece ser o período de maior suscetibilidade e que causa os maiores efeitos deletérios na viabilidade oocitária e na PIV.(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.

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.

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)

Effect of the antral follicle count of Bos taurus × Bos indicus dairy cows on in vitro embryo production / Efeito da contagem de folículos antrais de vacas Bos taurus x Bos indicus com aptidão leiteira na produção in vitro de embriões

This study aimed to evaluate the potential of Girolando (Bos taurus × Bos indicus) cows with high and low antral follicle counts (AFC) for the in vitro production of bovine embryos (IVEP), as well as the pregnancy rates of the recipients of these embryos. Girolando cows (Bos taurus × Bos indicus) were classified as high and low AFC when they had 35-52 (n = 13) and 11-17 follicles (n = 15), respectively. All animals were subjected to repeated follicular aspiration [Ovum pick-up (OPU)] and subsequent IVEP sessions. The synchronization protocol of the recipients was performed on a random day of the estrous cycle (Day 0) with the implantation of progesterone, estradiol benzoate, and prostaglandin. The high AFC group had higher aspirated oocyte/OPU (42.6 ± 5.2 vs. 14.6 ± 1.9; p < 0.01) and cultured oocyte/OPU (38.1 ± 6.6 vs. 12.3 ± 2.8; p < 0.01) averages as well as a higher blastocyst percentage on D7 (23.0 ± 1.0% vs. 18.4 ± 1.5%; p < 0.05) and higher pregnancy rate (42.7 ± 2.7% vs. 39.7 ± 4.6%; p< 0.05) than the low AFC group. Thus, we can conclude that animals with high AFC had better IVEP success rates than animals with low AFC.

Este estudo teve como objetivo avaliar o potencial de produção in vitro de embriões (PIVE) bovinos de vacas da raça Girolando (Bos taurus x Bos indicus) com alta e baixa contagem de folículos antrais (CFA), bem como a taxa de prenhez nas receptoras destes embriões. As vacas da raça Girolando (Bos taurus x Bos indicus) foram classificadas como de alta CFA quando apresentavam de 35 a 52 folículos(n = 13) e baixa CFA, quando apresentavam de 11 a 17 folículos (n = 15). Todos os animais foram submetidos a repetidas sessões de aspiração folicular (OPU) e subsequente PIVE. O protocolo de sincronização das receptoras foi realizado em um dia aleatório do ciclo estral (Dia 0) com implante de progesterona, benzoato de estradiol e prostaglandina. O grupo de alta CFA obteve uma maior média de oócitos aspirados / OPU (42,6 ± 5,2 vs. 14,6 ± 1,9; p < 0,01); oócitos cultivados / OPU (38,1 ± 6,6 vs.12,3 ± 2,8; p < 0,01); porcentagem de blastocistos em D7 (23,0 ± 1,0% vs. 18,4 ± 1,5%; p < 0,05) e taxa de prenhez (42,7 ± 2,7% vs. 39,7 ± 4,6%; p < 0,05) maiores quando comparado ao grupo de baixa CFA. Desta maneira, podemos concluir que os animais de alta CFA apresentaram melhores taxas de PIVE que os animais de baixa CFA.

Effect of the antral follicle count of Bos taurus × Bos indicus dairy cows on in vitro embryo production / Efeito da contagem de folículos antrais de vacas Bos taurus x Bos indicus com aptidão leiteira na produção in vitro de embriões

This study aimed to evaluate the potential of Girolando (Bos taurus × Bos indicus) cows with high and low antral follicle counts (AFC) for the in vitro production of bovine embryos (IVEP), as well as the pregnancy rates of the recipients of these embryos. Girolando cows (Bos taurus × Bos indicus) were classified as high and low AFC when they had 35-52 (n = 13) and 11-17 follicles (n = 15), respectively. All animals were subjected to repeated follicular aspiration [Ovum pick-up (OPU)] and subsequent IVEP sessions. The synchronization protocol of the recipients was performed on a random day of the estrous cycle (Day 0) with the implantation of progesterone, estradiol benzoate, and prostaglandin. The high AFC group had higher aspirated oocyte/OPU (42.6 ± 5.2 vs. 14.6 ± 1.9; p < 0.01) and cultured oocyte/OPU (38.1 ± 6.6 vs. 12.3 ± 2.8; p < 0.01) averages as well as a higher blastocyst percentage on D7 (23.0 ± 1.0% vs. 18.4 ± 1.5%; p < 0.05) and higher pregnancy rate (42.7 ± 2.7% vs. 39.7 ± 4.6%; p< 0.05) than the low AFC group. Thus, we can conclude that animals with high AFC had better IVEP success rates than animals with low AFC.(AU)

Este estudo teve como objetivo avaliar o potencial de produção in vitro de embriões (PIVE) bovinos de vacas da raça Girolando (Bos taurus x Bos indicus) com alta e baixa contagem de folículos antrais (CFA), bem como a taxa de prenhez nas receptoras destes embriões. As vacas da raça Girolando (Bos taurus x Bos indicus) foram classificadas como de alta CFA quando apresentavam de 35 a 52 folículos(n = 13) e baixa CFA, quando apresentavam de 11 a 17 folículos (n = 15). Todos os animais foram submetidos a repetidas sessões de aspiração folicular (OPU) e subsequente PIVE. O protocolo de sincronização das receptoras foi realizado em um dia aleatório do ciclo estral (Dia 0) com implante de progesterona, benzoato de estradiol e prostaglandina. O grupo de alta CFA obteve uma maior média de oócitos aspirados / OPU (42,6 ± 5,2 vs. 14,6 ± 1,9; p < 0,01); oócitos cultivados / OPU (38,1 ± 6,6 vs.12,3 ± 2,8; p < 0,01); porcentagem de blastocistos em D7 (23,0 ± 1,0% vs. 18,4 ± 1,5%; p < 0,05) e taxa de prenhez (42,7 ± 2,7% vs. 39,7 ± 4,6%; p < 0,05) maiores quando comparado ao grupo de baixa CFA. Desta maneira, podemos concluir que os animais de alta CFA apresentaram melhores taxas de PIVE que os animais de baixa CFA.

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)

Efeito da somatotropina bovina recombinante (rbST) na produção in vivo e na maturação in vitro de oócitos bovinos da raça Sindi / Effect of recombinant bovine somatotropin (rbST) on the in vivo production and in vitro maturation of bovine oocytes of the Sindi breed

A fim de avaliar o efeito de diferentes doses da rbST sobre a dinâmica folicular, a produção e a maturação in vitro de oócitos, 20 vacas Sindi, divididas em três grupos, receberam um dispositivo de progesterona intravaginal, estradiol e PGF2α, além de 2mL de solução salina (grupo controle), 250 (grupo rbST 250) ou 500mg de rbST (grupo rbST 500). Cinco dias depois, realizou-se a ovum pick up, e os complexos cumulus-oócitos (CCOs) recuperados foram selecionados, classificados e maturados in vitro. Os dados de contagem foram comparados pelo procedimento glht (General Linear Hypothesis Test), e os dados em porcentagem foram submetidos ao qui-quadrado, no programa estatístico R, onde as diferenças foram consideradas significativas quando P<0,05. Não houve diferença (P>0,05) entre os grupos quanto à quantidade de folículos e à taxa de maturação. Os grupos rbST 250 e rbST 500 foram significativamente superiores (P<0,05) ao grupo controle em relação ao número de folículos grandes (0,42±0,20 vs. 0). O grupo rbST 500 apresentou maior (P<0,05) porcentagem de oócitos viáveis (91,52%) do que os grupos controle (67,85%) e rbST 250 (53,33%). A rbST aumenta o número de folículos grandes, e 500mg de rbST aumentam a porcentagem de oócitos viáveis em vacas Sindi.(AU)

In order to evaluate the effect of different doses of rbST on the follicular dynamics, production, and in vitro maturation of oocytes, 20 Sindhi cows were divided into three groups, receiving an intravaginal progesterone device, estradiol and PGF2α, and 2mL of solution saline (Control Group), 250 (rbST 250 Group) or 500mg rbST (rbST 500 Group). Five days later, the ovum pick up was performed, and the cumulus-oocyte (CCO) complexes recovered were selected, classified, and matured in vitro. The counting data were compared by the glht (General Linear Hypothesis Test) procedure, and the percentage data were submitted to Qui- square, in the statistical program R, where differences were considered significant when P< 0.05. There was no difference (P> 0.05) between the groups regarding follicle quantity and maturation rate. The rbST 250 and rbST 500 groups were significantly higher (P< 0.05) than the Control group in relation to the number of large follicles (0.42±0.20 versus 0). The rbST 500 group presented higher (P< 0.05) percentage of viable oocytes (91.52%) than the Control (67.85%) and rbST 250 (53.33%) groups. rbST increases the number of large follicles and 500mg rbST increases the percentage of viable oocytes in Sindhi cows.(AU)

Efeito da somatotropina bovina recombinante (rbST) na produção in vivo e na maturação in vitro de oócitos bovinos da raça Sindi / Effect of recombinant bovine somatotropin (rbST) on the in vivo production and in vitro maturation of bovine oocytes of the Sindi breed

A fim de avaliar o efeito de diferentes doses da rbST sobre a dinâmica folicular, a produção e a maturação in vitro de oócitos, 20 vacas Sindi, divididas em três grupos, receberam um dispositivo de progesterona intravaginal, estradiol e PGF2α, além de 2mL de solução salina (grupo controle), 250 (grupo rbST 250) ou 500mg de rbST (grupo rbST 500). Cinco dias depois, realizou-se a ovum pick up, e os complexos cumulus-oócitos (CCOs) recuperados foram selecionados, classificados e maturados in vitro. Os dados de contagem foram comparados pelo procedimento glht (General Linear Hypothesis Test), e os dados em porcentagem foram submetidos ao qui-quadrado, no programa estatístico R, onde as diferenças foram consideradas significativas quando P<0,05. Não houve diferença (P>0,05) entre os grupos quanto à quantidade de folículos e à taxa de maturação. Os grupos rbST 250 e rbST 500 foram significativamente superiores (P<0,05) ao grupo controle em relação ao número de folículos grandes (0,42±0,20 vs. 0). O grupo rbST 500 apresentou maior (P<0,05) porcentagem de oócitos viáveis (91,52%) do que os grupos controle (67,85%) e rbST 250 (53,33%). A rbST aumenta o número de folículos grandes, e 500mg de rbST aumentam a porcentagem de oócitos viáveis em vacas Sindi.(AU)

In order to evaluate the effect of different doses of rbST on the follicular dynamics, production, and in vitro maturation of oocytes, 20 Sindhi cows were divided into three groups, receiving an intravaginal progesterone device, estradiol and PGF2α, and 2mL of solution saline (Control Group), 250 (rbST 250 Group) or 500mg rbST (rbST 500 Group). Five days later, the ovum pick up was performed, and the cumulus-oocyte (CCO) complexes recovered were selected, classified, and matured in vitro. The counting data were compared by the glht (General Linear Hypothesis Test) procedure, and the percentage data were submitted to Qui- square, in the statistical program R, where differences were considered significant when P< 0.05. There was no difference (P> 0.05) between the groups regarding follicle quantity and maturation rate. The rbST 250 and rbST 500 groups were significantly higher (P< 0.05) than the Control group in relation to the number of large follicles (0.42±0.20 versus 0). The rbST 500 group presented higher (P< 0.05) percentage of viable oocytes (91.52%) than the Control (67.85%) and rbST 250 (53.33%) groups. rbST increases the number of large follicles and 500mg rbST increases the percentage of viable oocytes in Sindhi cows.(AU)

Supplementation of cows with linseed: effects on antral follicle count, oocyte quality, and nutritional and plasma metabolite parameters / Suplementação de fêmeas girolando com linhaça: efeitos sobre contagem de folículos antrais, qualidade oocitária, parâmetros nutricionais e metabólitos plasmáticos

Linseed is distinguished by its composition of fatty acids, which are rich in unsaturated acids, and by the slow release of oil in the rumen, consequently providing a greater amount of lipids of interest for intestinal absorption. The aim of this study was to evaluate the effect of linseed supplementation on the antral follicle count (AFC) and oocyte quality of Girolando cows, as well as their digestive parameters and plasma metabolite. Twelve Girolando cows were used in the study and were randomly distributed in two experimental groups: control (CTL, n = 6) and linseed (LINS, n = 6). During the 126 days of supplementation, seven transvaginal ultrasound-guided ovum pick-up (OPU) sessions were performed at intervals of 21 days. AFC was performed in each OPU session. The cows fed with linseed showed no significant difference in the dry matter coefficient of digestibility (63.11 and 62.88), crude protein (62.7 and 55.26), neutral detergent fiber (NDF; 75.36 and 77.15), or acid detergent fiber (ADF; 72.45 and 74.77) for LINS and CTL, respectively. On the contrary, a higher level of ethereal extract was observed for LINS (69.31 vs. 40.7). There was no effect (p > 0.05) of lipid supplementation on plasma metabolite between groups and OPU sessions. There was no difference (p > 0.05) in the mean number of antral follicles (CTL: 31.14 ± 1.97; LINS: 25.52 ± 2.68), cumulus oocyte complexes recovered (CTL: 6.45 ± 1.66; LINS: 5.28 ± 1.18), or oocyte quality (CTL: 60.48% ± 8.46; LINS: 64.54% ± 7.77). The supplementation of 800g of linseed in the diet of Girolando cows did not alter the apparent digestibility of nutrients, AFC, or quality of oocytes obtained by OPU.

A linhaça destaca-se pela composição de ácidos graxos insaturados e por apresentar lenta liberação do óleo no rúmen e consequentemente fornecendo maior quantidade de lipídios de interesse para absorção intestinal. O objetivo deste estudo foi avaliar o efeito da suplementação com linhaça sobre a contagem de folículos antrais e a qualidade oocitária de vacas Girolando, bem como parâmetros digestivos e metabólitos plasmáticos. Foram utilizadas doze fêmeas bovinas da raça Girolando distribuídas aleatoriamente em 2 grupos experimentais: controle (CTL n = 6) e linhaça (LINS, n = 6). Durante os 126 dias de suplementação foram realizadas 7 sessões de aspiração folicular guiada por ultrassonografia (OPU) com intervalo de 21 dias entre si. A cada sessão de OPU a CFA era realizada. Os animais alimentados com linhaça não apresentaram diferença no coeficiente de digestibilidade de matéria seca (63,11 vs. 62,88), proteína bruta (62,7 vs. 55,26), fibra em detergente neutro (75,36 vs. 77,15), fibra em detergente ácido (72,45 vs. 74,77) para LINS e CTL respectivamente. Por outro lado, os níveis de extrato etéreo foram superiores para LINS (69,31 vs. 40,7). Não houve efeito (P > 0,05) da suplementação lipídica sobre os metabólitos plasmáticos entre os grupos e as sessões de OPU. Não houve diferença (P > 0,05) no número médio de folículos antrais (31,14±1,97 vs. 25,52±2,68), COCs recuperados (6,45±1,66 vs. 5,28±1,18) e na qualidade dos oócitos (60,48%±8,46 vs. 64,54%±7,77) para LINS e CTL respectivamente. A suplementação de 800 g de linhaça na dieta de vacas Girolando não alterou a digestibilidade aparente dos nutrientes, bem como o número e a qualidade dos oócitos obtidos por OPU.

Supplementation of cows with linseed: effects on antral follicle count, oocyte quality, and nutritional and plasma metabolite parameters / Suplementação de fêmeas girolando com linhaça: efeitos sobre contagem de folículos antrais, qualidade oocitária, parâmetros nutricionais e metabólitos plasmáticos

Linseed is distinguished by its composition of fatty acids, which are rich in unsaturated acids, and by the slow release of oil in the rumen, consequently providing a greater amount of lipids of interest for intestinal absorption. The aim of this study was to evaluate the effect of linseed supplementation on the antral follicle count (AFC) and oocyte quality of Girolando cows, as well as their digestive parameters and plasma metabolite. Twelve Girolando cows were used in the study and were randomly distributed in two experimental groups: control (CTL, n = 6) and linseed (LINS, n = 6). During the 126 days of supplementation, seven transvaginal ultrasound-guided ovum pick-up (OPU) sessions were performed at intervals of 21 days. AFC was performed in each OPU session. The cows fed with linseed showed no significant difference in the dry matter coefficient of digestibility (63.11 and 62.88), crude protein (62.7 and 55.26), neutral detergent fiber (NDF; 75.36 and 77.15), or acid detergent fiber (ADF; 72.45 and 74.77) for LINS and CTL, respectively. On the contrary, a higher level of ethereal extract was observed for LINS (69.31 vs. 40.7). There was no effect (p > 0.05) of lipid supplementation on plasma metabolite between groups and OPU sessions. There was no difference (p > 0.05) in the mean number of antral follicles (CTL: 31.14 ± 1.97; LINS: 25.52 ± 2.68), cumulus oocyte complexes recovered (CTL: 6.45 ± 1.66; LINS: 5.28 ± 1.18), or oocyte quality (CTL: 60.48% ± 8.46; LINS: 64.54% ± 7.77). The supplementation of 800g of linseed in the diet of Girolando cows did not alter the apparent digestibility of nutrients, AFC, or quality of oocytes obtained by OPU.(AU)

A linhaça destaca-se pela composição de ácidos graxos insaturados e por apresentar lenta liberação do óleo no rúmen e consequentemente fornecendo maior quantidade de lipídios de interesse para absorção intestinal. O objetivo deste estudo foi avaliar o efeito da suplementação com linhaça sobre a contagem de folículos antrais e a qualidade oocitária de vacas Girolando, bem como parâmetros digestivos e metabólitos plasmáticos. Foram utilizadas doze fêmeas bovinas da raça Girolando distribuídas aleatoriamente em 2 grupos experimentais: controle (CTL n = 6) e linhaça (LINS, n = 6). Durante os 126 dias de suplementação foram realizadas 7 sessões de aspiração folicular guiada por ultrassonografia (OPU) com intervalo de 21 dias entre si. A cada sessão de OPU a CFA era realizada. Os animais alimentados com linhaça não apresentaram diferença no coeficiente de digestibilidade de matéria seca (63,11 vs. 62,88), proteína bruta (62,7 vs. 55,26), fibra em detergente neutro (75,36 vs. 77,15), fibra em detergente ácido (72,45 vs. 74,77) para LINS e CTL respectivamente. Por outro lado, os níveis de extrato etéreo foram superiores para LINS (69,31 vs. 40,7). Não houve efeito (P > 0,05) da suplementação lipídica sobre os metabólitos plasmáticos entre os grupos e as sessões de OPU. Não houve diferença (P > 0,05) no número médio de folículos antrais (31,14±1,97 vs. 25,52±2,68), COCs recuperados (6,45±1,66 vs. 5,28±1,18) e na qualidade dos oócitos (60,48%±8,46 vs. 64,54%±7,77) para LINS e CTL respectivamente. A suplementação de 800 g de linhaça na dieta de vacas Girolando não alterou a digestibilidade aparente dos nutrientes, bem como o número e a qualidade dos oócitos obtidos por OPU.(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)

Estratégias para aumentar a produção de embriões em bovinos / Strategies to increase embryo production in cattle

As biotecnologias para produção de embriões in vivo e in vitro são de grande importância para acelerar o ganho genético dos rebanhos de corte e de leite. No entanto, o sucesso desses processos depende diretamente da quantidade e da qualidade dos oócitos da doadora de embrião. Diversos fatores influenciam a produção de embriões como a população folicular, a idade da doadora, o estresse térmico, o status metabólico e o status reprodutivo. Na atualidade, vários estudos têm sido desenvolvidos para elaborar estratégias que contornem estes desafios e aumentem a eficiência das técnicas de produção de embriões. Dentre os tratamentos estabelecidos para melhorar a produção de embriões das doadoras destacam-se a utilização de FSH, rBST, propilenoglicol e células tronco mesenquimais.(AU)

Biotechnologies for producing in vivo and in vitro embryos are very important to accelerate the genetic gain of beef and dairy herds. However, the success of these processes depend directly on the quantity and quality of the donors oocytes. Many factors influence the embryo production, such as follicle population, age, heat stress, metabolic status and reproductive status. Recently, many studies have been conducted to elaborate strategies that can overcome these issues and increase the efficiency of the embryo production techniques. Among the treatments developed to improve the embryo production, the use of FSH, rBST, propylene glycol and mesenchymal stem cells stand out.(AU)

Estratégias para aumentar a produção de embriões em bovinos / Strategies to increase embryo production in cattle

As biotecnologias para produção de embriões in vivo e in vitro são de grande importância para acelerar o ganho genético dos rebanhos de corte e de leite. No entanto, o sucesso desses processos depende diretamente da quantidade e da qualidade dos oócitos da doadora de embrião. Diversos fatores influenciam a produção de embriões como a população folicular, a idade da doadora, o estresse térmico, o status metabólico e o status reprodutivo. Na atualidade, vários estudos têm sido desenvolvidos para elaborar estratégias que contornem estes desafios e aumentem a eficiência das técnicas de produção de embriões. Dentre os tratamentos estabelecidos para melhorar a produção de embriões das doadoras destacam-se a utilização de FSH, rBST, propilenoglicol e células tronco mesenquimais.

Biotechnologies for producing in vivo and in vitro embryos are very important to accelerate the genetic gain of beef and dairy herds. However, the success of these processes depend directly on the quantity and quality of the donors oocytes. Many factors influence the embryo production, such as follicle population, age, heat stress, metabolic status and reproductive status. Recently, many studies have been conducted to elaborate strategies that can overcome these issues and increase the efficiency of the embryo production techniques. Among the treatments developed to improve the embryo production, the use of FSH, rBST, propylene glycol and mesenchymal stem cells stand out.

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.