Evaluation of novel SexedULTRA-4M technology for in vitro bovine embryo production

SexedULTRA-4M™ is made using an improved method of sex-sorting sperm in a less damaging environment for better retaining sperm integrity throughout the sorting process. The objective of this research was to compare conventional (CONV) and SexedULTRA-4M™ (ULTRA-4M) semen for bovine IVP using four Angus bulls. Matured slaughterhouse oocytes (n = 4000) were divided into the CONV group and the ULTRA-4M group (2000 COCs for each semen type). The IVF process was implemented with CONV and ULTRA-4M semen from the same bull. The cleavage rates, eight cell embryos and blastocysts on day 7 of culture were evaluated for each semen type and each bull. The statistical analysis was carried out with the ANOVA procedure SAS software. The results were 54.45% ± 1.03 and 58.10% ± 1.07; 35% ± 1.57 and 39.15% ± 1.62; 22.8% ± 1.09 and 27.15% ± 1.12 for CONV and ULTRA-4M, respectively, for cleavage rate, eight cell embryos and blastocysts on day 7 for the average of all bulls, comparing only the semen type. Concerning only the semen type, ULTRA-4M was significantly superior to CONV for cleavage rates (P = 0.01) and blastocysts on day 7 (P = 0.009). There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analyzed for Bull 1 and Bull 4, however, for Bull 2 ULTRA-4M was significantly superior to CONV for cleavage rates and blastocysts on day 7 (P< 0.05). In Bull 3, ULTRA-4M was significantly higher (P< 0.05) for blastocysts on day 7 compared to CONV. In conclusion, under the conditions of this research the ULTRA-4M and CONV semen produced similar bovine IVP results overall.(AU)

Effect of ovarian status on reproductive performance in dairy heifers inseminated with sexed semen

Background: The intensive reproductive management in the dairy farms requires inclusion of a large group of replacement heifers in the breeding program for a shorter period. In this aspect, a creation of effective estrus synchronization protocols with timed artificial insemination (TAI) by sexed semen and optimization of the current ones have a crucial role for obtaining high pregnancy rate. These protocols are beneficial, because they led to reduced interval to first AI, lack of need for estrus detection, and allow obtaining a large group of female calves for on time. Because of limited fertilizing potential of sexed spermatozoa this type of semen is applied mainly for heifers, as fertility is higher compared to lactating cows. The main objective of the present study was to investigate the effect of ovarian status on the reproductive performance in dairy heifers subjected to estrus synchronization and timed artificial insemination with sexed semen. Materials, Methods & Results: Forty-eight Holstein healthy heifers separated in 2 groups were subjected to PGF2α-GnRH pre-treatment and Ovsynch or PRID-5-day estrus synchronization protocol and timed artificial insemination (TAI) with sex-sorted semen, staring 6 days after end of the hormonal pre-treatment. The ovarian status (presence of follicles with or not a corpus luteum) of the heifers at the begin of the treatment, on day of TAI and pregnancy rate in different groups were determined and compared. Additionally, the ovarian status at the begin of the treatment, ovarian status and size of preovulatory follicle (PF) on day of TAI and total values for both groups according to reproductive performance (pregnant or non-pregnant) were also analyzed. On day of TAI the animals with PF and a lack of corpus luteum (CL) in both groups were more that those with PF and CL (39.3% and 30% vs. 60.7 % and 70%), with significant (P < 0.05) difference in PRID-5-day group. The pregnancy rate tended to be higher in PRID-5 day than Ovsynch treatment (65% vs. 35.7%). A higher percentage (100% and 67.9%) of the pregnant animals in both treatments had not CL on day of TAI, and the size of the PF (1.58 ± 0.12 cm and 1.64 ± 0.13 cm) was increased (P < 0.05). Similar effects of the ovarian status on reproductive performance were obtained after a comparison of the total values between pregnant and non-pregnant animals. The percentage of heifers with observed PF without CL on day of TAI was significantly greater (P < 0.05) in pregnant compared to non-pregnant group (91.3% vs. 40%). The opposite dependence was determined for the parameter presence of follicles and corpus luteum (8.7% vs. 60%; P < 0.05). Furthermore, the size of the PF measured immediately before TAI was increased in animals became pregnant (1.60 ± 0.12 cm vs. 1.34 ± 0.17 cm; P < 0.05). Discussion: The analysis of the obtained results showed that the ovarian status on day of TAI affects the reproductive performance in dairy heifers subjected to estrus synchronization and timed artificial insemination with sexed semen. Hormonal pre-treatment with onset of PRID-5-day protocol 6 days later and TAI with sex-sorted semen ensure acceptable pregnancy rate. The greater preovulatory follicle and a lack of corpus luteum before insemination provide significantly (P < 0.05) more pregnant animals, compared to the cases when CL is presented. Ultrasound determination of the ovarian function before insemination can be used in selection of heifers for TAI with sex-sorted semen.

Evolução da utilização de espermatozoides sexados / Evolution of sexed spermatozoa utilization

A sexagem de espermatozoides tem relevante aplicação na produção animal. Várias publicações demonstram a eficiência do processo de sexagem por citometria de fluxo que separa os espermatozoides X e Y em função da mensuração do conteúdo de DNA. Importantes modificações no processo de sexagem por citometria de fluxo, tais como pressão, intensidade do laser, diluidores tem levado à diminuição dos danos nos espermatozoides e aumentado a viabilidade dos mesmos.(AU)

Spermatozoa sexing has an important application for livestock production. Results have been published worldwide that demonstrate effectiveness of the flow cytometer sexing process based on sorting sperm with differential DNA content as the X and Y sperm marker. Major improvements in the sexing sorting process as pressure, laser power, extenders had led to the decrease of sperm damage and increase of sperm viability(AU)

Evolução da utilização de espermatozoides sexados / Evolution of sexed spermatozoa utilization

A sexagem de espermatozoides tem relevante aplicação na produção animal. Várias publicações demonstram a eficiência do processo de sexagem por citometria de fluxo que separa os espermatozoides X e Y em função da mensuração do conteúdo de DNA. Importantes modificações no processo de sexagem por citometria de fluxo, tais como pressão, intensidade do laser, diluidores tem levado à diminuição dos danos nos espermatozoides e aumentado a viabilidade dos mesmos.

Spermatozoa sexing has an important application for livestock production. Results have been published worldwide that demonstrate effectiveness of the flow cytometer sexing process based on sorting sperm with differential DNA content as the X and Y sperm marker. Major improvements in the sexing sorting process as pressure, laser power, extenders had led to the decrease of sperm damage and increase of sperm viability

Centrifugación en gradiente de densidad: alternativa para sexar espermatozoides caninos? / Density gradient centrifugation: alternative for sexing sperm in dogs? / Centrifugação em gradiente de densidade: alternativa para sexagem espermática em cães?

La evolución de técnicas para la separación del sexo de esperma en los últimos años ha permitido avances significativos, pero todavía no hay método que ha sido utilizado con éxito en perros. Apear de la citometría de flujo ser un método eficaz para el sexado de esperma, en perros puede ser considerado difícil de implementar en vista del alto costo de la aplicación de técnica y la recuperación baja de espermatozoides después de la separación. En contraste, la centrifugación en gradiente de densidad también puede ser utilizado para la separación de espermatozoides en los perros. Esta técnica también se basa en la diferencia de contenido en DNA entre los espermatozoides portadores de los cromosomas X e Y. Separación de esperma puede ser confirmada por diversos métodos tales como citometría de flujo, hibridación in situ fluorescente (FISH), coloración con quinacrina mostaza y PCR. El objetivo de esta revisión es abordar el centrifugacion en gradiente de densidad como alternativa a la separación del sexo de espermatozoides en los perros.(AU)

The technical progress concerning sperm sexing in recent years has enabled significant advances, but still none method was used successfully in dogs. Despite the flow cytometry to be an effective method for sperm sexing, in dogs it can be considered difficult to implement in view of high cost of application of the technique and low sperm recovery after separation. In contrast, density gradient centrifugation can also be used for separating sperm in dogs. This technique is also based on the DNA content difference between sperm with chromosomes X and Y. Sperm separation can be confirmed by various methods such as flow cytometry, fluorescence in situ hybridization (FISH) staining with quinacrine mustard and PCR. The objective of this review to address the density gradient centrifugation as an alternative to sperm sexing in dogs.(AU)

A evolução das técnicas de sexagem espermática nos últimos anos permitiu avanços significativos, mas ainda nenhum método foi empregado com sucesso na espécie canina. Apesar da citometria de fluxo ser um método eficiente para sexagem espermática, nos cães pode ser considerada de difícil aplicação, em vista do alto custo da aplicação da técnica e da baixa recuperação espermática após a separação. Em contrapartida, a centrifugação em gradiente de densidade também pode ser utilizada para separação espermática em cães. Essa técnica também se baseia na diferença do conteúdo de DNA entre os espermatozoides portadores dos cromossomos X e Y. A separação espermática pode ser confirmada por diferentes métodos, como citometria de fluxo, hibridização in situ fluorescente (FISH), coloração com quinacrina mostarda e PCR. Objetivou-se, com esta revisão abordar a centrifugação em gradiente de densidade como uma alternativa a sexagem espermática em cães.(AU)

Centrifugación en gradiente de densidad: alternativa para sexar espermatozoides caninos? / Density gradient centrifugation: alternative for sexing sperm in dogs? / Centrifugação em gradiente de densidade: alternativa para sexagem espermática em cães?

La evolución de técnicas para la separación del sexo de esperma en los últimos años ha permitido avances significativos, pero todavía no hay método que ha sido utilizado con éxito en perros. Apear de la citometría de flujo ser un método eficaz para el sexado de esperma, en perros puede ser considerado difícil de implementar en vista del alto costo de la aplicación de técnica y la recuperación baja de espermatozoides después de la separación. En contraste, la centrifugación en gradiente de densidad también puede ser utilizado para la separación de espermatozoides en los perros. Esta técnica también se basa en la diferencia de contenido en DNA entre los espermatozoides portadores de los cromosomas X e Y. Separación de esperma puede ser confirmada por diversos métodos tales como citometría de flujo, hibridación in situ fluorescente (FISH), coloración con quinacrina mostaza y PCR. El objetivo de esta revisión es abordar el centrifugacion en gradiente de densidad como alternativa a la separación del sexo de espermatozoides en los perros.

The technical progress concerning sperm sexing in recent years has enabled significant advances, but still none method was used successfully in dogs. Despite the flow cytometry to be an effective method for sperm sexing, in dogs it can be considered difficult to implement in view of high cost of application of the technique and low sperm recovery after separation. In contrast, density gradient centrifugation can also be used for separating sperm in dogs. This technique is also based on the DNA content difference between sperm with chromosomes X and Y. Sperm separation can be confirmed by various methods such as flow cytometry, fluorescence in situ hybridization (FISH) staining with quinacrine mustard and PCR. The objective of this review to address the density gradient centrifugation as an alternative to sperm sexing in dogs.

A evolução das técnicas de sexagem espermática nos últimos anos permitiu avanços significativos, mas ainda nenhum método foi empregado com sucesso na espécie canina. Apesar da citometria de fluxo ser um método eficiente para sexagem espermática, nos cães pode ser considerada de difícil aplicação, em vista do alto custo da aplicação da técnica e da baixa recuperação espermática após a separação. Em contrapartida, a centrifugação em gradiente de densidade também pode ser utilizada para separação espermática em cães. Essa técnica também se baseia na diferença do conteúdo de DNA entre os espermatozoides portadores dos cromossomos X e Y. A separação espermática pode ser confirmada por diferentes métodos, como citometria de fluxo, hibridização in situ fluorescente (FISH), coloração com quinacrina mostarda e PCR. Objetivou-se, com esta revisão abordar a centrifugação em gradiente de densidade como uma alternativa a sexagem espermática em cães.

Postnatal consequences of assisted reproductive technologies in cattle

Experiments in mice and clinical observations in humans are indicative that adult phenotype can be altered in offspring derived from embryos that were subjected to culture or that were produced by the combination of in vitro oocyte maturation, fertilization and embryonic development (in vitro production; IVP). The most commonly observed changes are in body and organ size, growth rate, cardiovascular function and regulation of glucose homeostasis. In cattle, IVP is associated with increased birth weight and neonatal death loss but little is known about the long-term consequences of embryo technologies. Recently, postnatal characteristics were compared between females born as a result of artificial insemination, IVP using conventional semen, IVP using reverse X-sorted semen, and multiple ovulation – embryo transfer. Females born following IVP using reverse X-sorted semen produced less milk, milk fat, and milk protein than females in the other three groups, which were similar to each other. These results point out the importance of 1) examining other data sets to examine long-term impact of assisted reproductive technologies on adult physiology and performance and 2) evaluating whether use of sexed semen alters the adult phenotype when used for artificial insemination.

Postnatal consequences of assisted reproductive technologies in cattle

Experiments in mice and clinical observations in humans are indicative that adult phenotype can be altered in offspring derived from embryos that were subjected to culture or that were produced by the combination of in vitro oocyte maturation, fertilization and embryonic development (in vitro production; IVP). The most commonly observed changes are in body and organ size, growth rate, cardiovascular function and regulation of glucose homeostasis. In cattle, IVP is associated with increased birth weight and neonatal death loss but little is known about the long-term consequences of embryo technologies. Recently, postnatal characteristics were compared between females born as a result of artificial insemination, IVP using conventional semen, IVP using reverse X-sorted semen, and multiple ovulation embryo transfer. Females born following IVP using reverse X-sorted semen produced less milk, milk fat, and milk protein than females in the other three groups, which were similar to each other. These results point out the importance of 1) examining other data sets to examine long-term impact of assisted reproductive technologies on adult physiology and performance and 2) evaluating whether use of sexed semen alters the adult phenotype when used for artificial insemination.(AU)

Sêmen Convencional e Sexado: Características In Vitro e Fertilidade de Vacas Nelore com Ou Sem a Aplicação de Prostaglandina no Momento da Iatf.

O presente estudo teve por objetivo avaliar a fertilidade do sêmen convencional e sexado utilizado em vacas Nelore multíparas que receberam tratamento com PGF2 no momento da inseminação artificial (IA) e expressaram diferentes intensidades de comportamento de estro (com ou sem expressão de estro) em um protocolo de IATF. Adicionalmente o estudo avaliou laboratorialmente as amostras de sêmen sexado e convencional a fim de comparar as características seminais in vitro com os resultados de fertilidade obtidos a campo. No experimento a campo, foram avaliadas as taxas de concepção (TC) de vacas multíparas lactantes da raça Nelore locadas em duas fazendas comerciais de gado de corte (fazenda 1: n=348 e fazenda 2: n=355). Em ambas as fazendas, as vacas foram submetidas ao mesmo protocolo de IATF. Quarenta e oito hs após a remoção do dispositivo de Progesterona (dia 11; D11), a IA foi realizada com sêmen convencional ou sexado de dois touros diferentes, de forma aleatória. Adicionalmente, no momento da IA, as vacas receberam, aleatoriamente, 12,5 mg im de dinoprost trometamina (Dinoprost; Lutalyse®; tratamento PGF) ou 2,5 mL im de soro fisiológico estéril (Controle). O comportamento estral foi aferido no D11 (momento da IATF) pela ativação do dispositivo estrotect®. Os dados foram analisados por regressão logística a um grau de significância de 5%. No experimento laboratorial, foram avaliadas amostras de sêmen sexado e convencional dos dois touros (touros 1 e 2), utilizados na fazenda 1 deste estudo. Foram analisados parâmetros convencionais de qualidade espermática, avaliação da integridade de membrana plasmática (pelo teste hiposmótico), análise computadorizada do sêmen (CASA) e avaliação de morfometria e parâmetros da cromatina espermática pelo Azul de Toluidina. Os dados laboratoriais foram analisados por ANOVA considerando 5% de significância. De acordo com os resultados do estudo a campo, a TC geral foi de 32,8% na Fazenda 1 e 42,3% na Fazenda 2 (P = 0,01). Escore de condição corporal (P = 0,02), comportamento estral (P=0,01) e tipo de sêmen (P <0,001) foram fatores que afetaram a TC. Vacas inseminadas com sêmen convencional tiveram 2,73 vezes mais probabilidade de tornar-se gestantes do que vacas inseminadas com sêmen sexado e vacas exibindo estro tiveram 2,5 vezes mais chance de gestação do que vacas que não exibiram estro. Nenhum efeito do tratamento com PGF2 (P=0,67) foi detectado em vacas que receberam sêmen convencional ou sexado. Ainda assim, foi detectada tendência estatística de interação (P=0,08) entre o tratamento (PGF ou controle) e o comportamento estral (estro ou não). O tratamento com PGF2 no momento da IA aumentou em 70% a TC das vacas que não expressaram estro. Nas análises laboratoriais, o sêmen convencional de ambos os touros apresentou maior (P<0,05) motilidade visual, concentração espermática, espermatozoides móveis na palheta e integridade de membrana plasmática em relação ao sêmen sexado. Nas avaliações pelo sistema CASA, motilidade total (MT) e progressiva (MP) foram significativamente menores nas amostras de sêmen sexado em relação ao convencional no touro 1 (P<0.05). A amplitude de deslocamento lateral de cabeça (ALH) foi maior (P<0.05) no sêmen sexado comparado ao sêmen convencional nos dois animais avaliados. Os resultados de análise morfométrica demonstraram que o touro 2 apresentou menor elipsidade nos espermatozoides sexados em relação ao sêmen convencional (P<0,05), enquanto no touro 1 foi observado menor Fourier1 no sêmen sexado em relação ao convencional. Não foram observadas diferenças na descompactação e heterogeneidade do DNA, quando comparados sêmen convencional e sexado em ambos os touros. Concluiu-se que a fertilidade do sêmen sexado foi menor do que o sêmen convencional, independente do tratamento com PGF2. Porém, vacas que não apresentaram estro e receberam o tratamento com PGF2 no momento da IA demonstraram TC semelhante às vacas em estro, independentemente do tipo de sêmen. Além disso, o processo de sexagem prejudicou diversas características de viabilidade espermática, bem como parâmetros cinéticos e morfométricos dos espermatozoides que estão relacionados com menor capacidade de fertilização deste tipo de sêmen.

The present study aimed to evaluate the fertility of conventional and sexed semen used in multiparous Nelore cows that received PGF2 treatment at the moment of AI and expressed different intensities of estrus behavior (estrus or no estrus expression) in timed-AI protocol. Additionally, laboratory assessment of sexed and conventional semen was performed in order to compare in vitro sperm characteristics with field fertility results. In the field experiment, conception rates (CR) of lactating multiparous Nelore cows from two commercial beef farms (farm 1: n=348 and farm 2: n=355) were evaluated. The animals were submitted to the same timed-AI protocol and 48 hs after the progesterone device removal (day 11; D11), AI was randomly performed with conventional or sexed semen from two different bulls. Additionally, at the time of AI, cows randomly received 12.5 mg im of dinoprost tromethamine (Dinoprost; Lutalyse®; PGF treatment) or 2.5 mL im of sterile saline (Control). Estrus behavior was measured at D11 (moment of timed-AI) by estrotect® device. The data were analyzed by logistic regression at 5% of significance level. In the laboratory experiment, sexed and conventional semen samples from the bulls (bulls 1 and 2) used in farm 1 were evaluated. Conventional sperm quality parameters, plasma membrane integrity (hyposmotic test), computerized semen analysis (CASA) and sperm morphometry and chromatin (Toluidine Blue) assessment were analyzed. The laboratory data were analyzed by ANOVA at 5% of significance. The overall CR was 32.8% in Farm 1 and 42.3% in Farm 2 (P = 0.01). Body condition score (P = 0.02), estrous behavior (P = 0.01) and semen type (P <0.001) were factors affecting CR. Cows inseminated with conventional semen were 2.73 times more likely to become pregnant than cows inseminated with sexed semen and cows exhibiting estrus were 2.5 times more likely to become pregnant than cows not exhibiting estrus. No effect of PGF2 treatment (P=0.67) was detected in cows that received conventional or sexed semen. Still, a statistical tendency of interaction (P=0.08) was detected between treatment (PGF or control) and estrous behavior (estrus or no estrus). Treatment with PGF2 at the time of AI increased by 82% the CR of cows with no estrus behavior. In laboratory analyses, conventional semen from both bulls demonstrated higher (P<0.05) visual motility, sperm concentration, number of motile sperm in the straw and plasma membrane integrity compared to sexed semen. In the CASA assessment, total

Status of embryo production in the world

In the last century, agriculture has seen the introduction of innovating reproductive biotechnologies that have permitted this field to grow significantly. In the early 20th century, introduction of semen cryopreservation and artificial insemination has propelled animal agriculture worldwide with the possibility to import and export in a biosecure way genetics from different species. Then, with the development of embryo transfer, it was possible to import and export not only half of the genetic component by disseminating frozen embryos in biosecure manners. Later, the introduction of ultrasonography (which gave us transvaginal ovum pick-up) and in vitro fertilization (IVF) revolutionized the speed at which generations of embryos could be produced thus shortening the generation gaps between these important genetics for farmers. Finally, the introduction of genomics again revolutionized the precision and speed at which farmers could identify the desired genetics. The bovine industry is an example of a niche that profited by the development of these technologies. In the last 15 years, IVF embryo production has increased significantly year after year with an all-time high of 42% of the total embryos produced in 2013 were of IVF origin. There are several reasons why IVF is being used more and more in the embryo transfer business: in vitro culture media have improved significantly; Introduction of sexed semen for IVF permits farmers to get over 90% of embryos of the desired sex; The interval between generations has reduced significantly with the identification of the next elite male and female genetics using genomic technology. The international agricultural community will benefit by integrating new technologies such as IVF in their operations. It is important that international societies such as SBTE and IETS continue to support scientists and players in this field to develop these technologies.(AU)

Status of embryo production in the world

In the last century, agriculture has seen the introduction of innovating reproductive biotechnologies that have permitted this field to grow significantly. In the early 20th century, introduction of semen cryopreservation and artificial insemination has propelled animal agriculture worldwide with the possibility to import and export in a biosecure way genetics from different species. Then, with the development of embryo transfer, it was possible to import and export not only half of the genetic component by disseminating frozen embryos in biosecure manners. Later, the introduction of ultrasonography (which gave us transvaginal ovum pick-up) and in vitro fertilization (IVF) revolutionized the speed at which generations of embryos could be produced thus shortening the generation gaps between these important genetics for farmers. Finally, the introduction of genomics again revolutionized the precision and speed at which farmers could identify the desired genetics. The bovine industry is an example of a niche that profited by the development of these technologies. In the last 15 years, IVF embryo production has increased significantly year after year with an all-time high of 42% of the total embryos produced in 2013 were of IVF origin. There are several reasons why IVF is being used more and more in the embryo transfer business: in vitro culture media have improved significantly; Introduction of sexed semen for IVF permits farmers to get over 90% of embryos of the desired sex; The interval between generations has reduced significantly with the identification of the next elite male and female genetics using genomic technology. The international agricultural community will benefit by integrating new technologies such as IVF in their operations. It is important that international societies such as SBTE and IETS continue to support scientists and players in this field to develop these technologies.

Sex-sorted sperm for artificial in semination and embryo transfer programs in cattle

The selection of offspring from the desired sex can be one of the determining factors to increase the genetic progress and farmer ́s profitability in either beef or dairy cattle. In fact, the use of sex-sorted sperm has been applied worldwide combined with artificial insemination (AI) upon estrus detection in heifers. Additionally, several researches have been performed aiming to investigate the use of sex-sorted sperm during timed AI (TAI) programs and for insemination of superstimulated donors for in vivo embryo programs. Pregnancy per AI (P/AI) of cyclic heifers inseminated in estrus with sex-sorted sperm has been reported to be approximately 75 to 80% of the P/AI of heifers inseminated with non-sorted sperm. Insemination of superstimulated cows with sex-sorted sperm has been reported to reduce the production of viable embryos. Recently, however, it has been demonstrated that P/AI and embryo production per flushing resulting from AI with sex-sorted sperm may be improved when the time of AI is postponed in relation to the time of AI with non-sorted sperm. The P/AI of non stimulated females and fertilization rates and number of embryos recovered from superstimulated females were increased when AI occurred between 16 and 24 h after the onset of estrus (i.e. 6 to 14 h before ovulation). Nonetheless, despite the improvements achieved in the last decade, there is still a significant individual variability in fertility among bulls that have their sperm se x-sorted. It is critical that the pre-determination of the sire fertility is a paramount when sex-sorted sperm is utilized in commercial AI and ET programs. Thus, the aim of this review is to discuss the main concepts related to the use of sex-sorted sperm in TAI and ET programs, addressing some strategies to increase the efficiency of the technique.

Sex-sorted sperm for artificial in semination and embryo transfer programs in cattle

The selection of offspring from the desired sex can be one of the determining factors to increase the genetic progress and farmer ́s profitability in either beef or dairy cattle. In fact, the use of sex-sorted sperm has been applied worldwide combined with artificial insemination (AI) upon estrus detection in heifers. Additionally, several researches have been performed aiming to investigate the use of sex-sorted sperm during timed AI (TAI) programs and for insemination of superstimulated donors for in vivo embryo programs. Pregnancy per AI (P/AI) of cyclic heifers inseminated in estrus with sex-sorted sperm has been reported to be approximately 75 to 80% of the P/AI of heifers inseminated with non-sorted sperm. Insemination of superstimulated cows with sex-sorted sperm has been reported to reduce the production of viable embryos. Recently, however, it has been demonstrated that P/AI and embryo production per flushing resulting from AI with sex-sorted sperm may be improved when the time of AI is postponed in relation to the time of AI with non-sorted sperm. The P/AI of non stimulated females and fertilization rates and number of embryos recovered from superstimulated females were increased when AI occurred between 16 and 24 h after the onset of estrus (i.e. 6 to 14 h before ovulation). Nonetheless, despite the improvements achieved in the last decade, there is still a significant individual variability in fertility among bulls that have their sperm se x-sorted. It is critical that the pre-determination of the sire fertility is a paramount when sex-sorted sperm is utilized in commercial AI and ET programs. Thus, the aim of this review is to discuss the main concepts related to the use of sex-sorted sperm in TAI and ET programs, addressing some strategies to increase the efficiency of the technique. (AU)

Validação de um programa de produção in vitro de embriões bovinos com transporte de oócitos e de embriões por longas distâncias / Validation of a program production in vitro embryo cattle with transport of oocytes and embryo for long distance

This study aimed to evaluate the program of in vitro embryo production (IVEP) feasibility whose cattle oocyte maturation and embryo development occurred partly during the transport, fertilization (IVF) was performed with conventional or sexed semen and the embryos were transferred at different periods of development. Therefore, oocytes aspirated in the state of Bahia were transported to a commercial laboratory in São Paulo city, where the steps of in vitro production continued, the IVF was performed with sperm derived from conventional semen (CONV group) or sexed group (SEX group). On the sixth day of cultivation, the embryos produced were sent to Bahia and transferred to recipients at different periods of development (6.5, 7.0 and 7.5 days). The females had a mean of 46.18 ± 32.7 oocytes recovered, and 30.74 ± 24.3 viable oocytes by aspiration. The rates of embryos produced and pregnancy were 32.85% and 33.12%, respectively. The type of semen did not affect neither the number of cleaved oocytes nor the pregnancy rates, however, it affected the number of embryos produced, and the age of the embryo interfered in the pregnancy rates. The results demonstrated that the transport of oocytes and embryos was effective in cattle IVEP program, thus, it can be used in large scale.

Objetivou-se avaliar a viabilidade de um programa de produção in vitro de embriões (PIVE) bovinos cuja maturação oocitária e cultivo embrionário ocorreram parcialmente durante o transporte, a fertilização (FIV) foi realizada com sêmen convencional ou sexado e os embriões foram transferidos com diferentes idades de desenvolvimento. Para tanto, oócitos aspirados no estado da Bahia foram transportados para um laboratório comercial em São Paulo, onde realizaram-se as etapas de produção in vitro, sendo a FIV realizada com espermatozoides oriundos de sêmen convencional (grupo CONV) ou sexado (grupo SEX). No dia seis de cultivo, os embriões produzidos foram remetidos à Bahia e transferidos às receptoras com diferentes idades de desenvolvimento (6,5; 7,0 e 7,5 dias). As fêmeas tiveram média de 46,18±32,7 oócitos recuperados e 30,74±24,3 oócitos viáveis por aspiração. As taxas de embriões produzidos e de gestação foram de 32,85% e 33,12%, respectivamente. O tipo de sêmen não influenciou no número de oócitos clivados e nas taxas de gestação, entretanto, influenciou no número de embriões produzidos e a idade do embrião interferiu nas taxas de gestação. Os resultados encontrados demonstraram que o transporte de oócitos e de embriões foi eficiente em um programa de PIVE bovinos, podendo ser utilizado em larga escala.

Validação de um programa de produção in vitro de embriões bovinos com transporte de oócitos e de embriões por longas distâncias

This study aimed to evaluate the program of in vitro embryo production (IVEP) feasibility whose cattle oocyte maturation and embryo development occurred partly during the transport, fertilization (IVF) was performed with conventional or sexed semen and the embryos were transferred at different periods of development. Therefore, oocytes aspirated in the state of Bahia were transported to a commercial laboratory in São Paulo city, where the steps of in vitro production continued, the IVF was performed with sperm derived from conventional semen (CONV group) or sexed group (SEX group). On the sixth day of cultivation, the embryos produced were sent to Bahia and transferred to recipients at different periods of development (6.5, 7.0 and 7.5 days). The females had a mean of 46.18 ± 32.7 oocytes recovered, and 30.74 ± 24.3 viable oocytes by aspiration. The rates of embryos produced and pregnancy were 32.85% and 33.12%, respectively. The type of semen did not affect neither the number of cleaved oocytes nor the pregnancy rates, however, it affected the number of embryos produced, and the age of the embryo interfered in the pregnancy rates. The results demonstrated that the transport of oocytes and embryos was effective in cattle IVEP program, thus, it can be used in large scale.

Objetivou-se avaliar a viabilidade de um programa de produção in vitro de embriões (PIVE) bovinos cuja maturação oocitária e cultivo embrionário ocorreram parcialmente durante o transporte, a fertilização (FIV) foi realizada com sêmen convencional ou sexado e os embriões foram transferidos com diferentes idades de desenvolvimento. Para tanto, oócitos aspirados no estado da Bahia foram transportados para um laboratório comercial em São Paulo, onde realizaram-se as etapas de produção in vitro, sendo a FIV realizada com espermatozoides oriundos de sêmen convencional (grupo CONV) ou sexado (grupo SEX). No dia seis de cultivo, os embriões produzidos foram remetidos à Bahia e transferidos às receptoras com diferentes idades de desenvolvimento (6,5; 7,0 e 7,5 dias). As fêmeas tiveram média de 46,18±32,7 oócitos recuperados e 30,74±24,3 oócitos viáveis por aspiração. As taxas de embriões produzidos e de gestação foram de 32,85% e 33,12%, respectivamente. O tipo de sêmen não influenciou no número de oócitos clivados e nas taxas de gestação, entretanto, influenciou no número de embriões produzidos e a idade do embrião interferiu nas taxas de gestação. Os resultados encontrados demonstraram que o transporte de oócitos e de embriões foi eficiente em um programa de PIVE bovinos, podendo ser utilizado em larga escala.

Validação de um programa de produção in vitro de embriões bovinos com transporte de oócitos e de embriões por longas distâncias / Validation of a program production in vitro embryo cattle with transport of oocytes and embryo for long distance

This study aimed to evaluate the program of in vitro embryo production (IVEP) feasibility whose cattle oocyte maturation and embryo development occurred partly during the transport, fertilization (IVF) was performed with conventional or sexed semen and the embryos were transferred at different periods of development. Therefore, oocytes aspirated in the state of Bahia were transported to a commercial laboratory in São Paulo city, where the steps of in vitro production continued, the IVF was performed with sperm derived from conventional semen (CONV group) or sexed group (SEX group). On the sixth day of cultivation, the embryos produced were sent to Bahia and transferred to recipients at different periods of development (6.5, 7.0 and 7.5 days). The females had a mean of 46.18 ± 32.7 oocytes recovered, and 30.74 ± 24.3 viable oocytes by aspiration. The rates of embryos produced and pregnancy were 32.85% and 33.12%, respectively. The type of semen did not affect neither the number of cleaved oocytes nor the pregnancy rates, however, it affected the number of embryos produced, and the age of the embryo interfered in the pregnancy rates. The results demonstrated that the transport of oocytes and embryos was effective in cattle IVEP program, thus, it can be used in large scale.(AU)

Objetivou-se avaliar a viabilidade de um programa de produção in vitro de embriões (PIVE) bovinos cuja maturação oocitária e cultivo embrionário ocorreram parcialmente durante o transporte, a fertilização (FIV) foi realizada com sêmen convencional ou sexado e os embriões foram transferidos com diferentes idades de desenvolvimento. Para tanto, oócitos aspirados no estado da Bahia foram transportados para um laboratório comercial em São Paulo, onde realizaram-se as etapas de produção in vitro, sendo a FIV realizada com espermatozoides oriundos de sêmen convencional (grupo CONV) ou sexado (grupo SEX). No dia seis de cultivo, os embriões produzidos foram remetidos à Bahia e transferidos às receptoras com diferentes idades de desenvolvimento (6,5; 7,0 e 7,5 dias). As fêmeas tiveram média de 46,18±32,7 oócitos recuperados e 30,74±24,3 oócitos viáveis por aspiração. As taxas de embriões produzidos e de gestação foram de 32,85% e 33,12%, respectivamente. O tipo de sêmen não influenciou no número de oócitos clivados e nas taxas de gestação, entretanto, influenciou no número de embriões produzidos e a idade do embrião interferiu nas taxas de gestação. Os resultados encontrados demonstraram que o transporte de oócitos e de embriões foi eficiente em um programa de PIVE bovinos, podendo ser utilizado em larga escala.(AU)

Produção de embriões bovinos in vitro com sêmen sexado / In vitro production of bovine embryos with sex-sorted semen

Com o advento da sexagem espermática a escolha do sexo da prole se tornou uma grande vantagem, principalmente para os criadores de gado leiteiro, que necessitam de novas matrizes para a manutenção do seu rebanho e para os criadores de gado de corte, que almejam mais machos para a produção de carne. Atualmente, a única técnica conhecida capaz de separar as populações de espermatozoides X e Y de mamíferos, com eficiência e pureza acima de 90%, é a citometria de fluxo. A melhor forma de uso do sêmen sexado é na fertilização in vitro, devido ao reduzido número de espermatozoides disponíveis após a sexagem. Vários fatores podem influenciar os resultados da produção in vitro de embriões na espécie bovina utilizando sêmen sexado; dentre eles destacam-se as baixas taxas de fertilização, de clivagem, de blastocistos, de gestações; capacitação espermática parcial; amostras seminais diluídas e a variação entre touros. Alguns estudos têm sido realizados com o intuito de avaliar as alterações genéticas e epigenéticas decorrentes do uso do sêmen sexado na produção de embriões bovinos in vitro. Portanto, este trabalho tem como objetivo realizar uma revisão de literatura sobre as limitações e as vantagens do uso do sêmen sexado, com ênfase na técnica de citometria de fluxo, durante a produção embrionária in vitro na espécie bovina.

The advent of sex-sorting-sperm sex selection of offspring became a great advantage, especially for dairy farms, which need new heifers for maintenance of his herds, and for beef cattle breeders, who crave more males for meat production. Currently, the only known technique capable of separating the populations of X and Y mammals sperm cells with efficiency and purity above 90% is flow cytometry. The best way of using sexed semen is in vitro fertilization, due to the small number of available sperm after sexing. Several factors can influence the results of in vitro embryos production using bovine sexed semen; among them there are the low rates of fertilization, cleavage, blastocyst, pregnancies, partially sperm capacitation, diluted semen samples and variation among bulls. Several studies have been conducted in order to assess the genetic and epigenetic alterations resulting from the use of sexed semen in the production of bovine embryos in vitro. Therefore, this paper aims to conduct a literature review on the limitations and advantages of using sexed semen, with emphasis on the technique of flow cytometry, during in vitro embryo production in cattle.

Produção de embriões bovinos in vitro com sêmen sexado / In vitro production of bovine embryos with sex-sorted semen

Com o advento da sexagem espermática a escolha do sexo da prole se tornou uma grande vantagem, principalmente para os criadores de gado leiteiro, que necessitam de novas matrizes para a manutenção do seu rebanho e para os criadores de gado de corte, que almejam mais machos para a produção de carne. Atualmente, a única técnica conhecida capaz de separar as populações de espermatozoides X e Y de mamíferos, com eficiência e pureza acima de 90%, é a citometria de fluxo. A melhor forma de uso do sêmen sexado é na fertilização in vitro, devido ao reduzido número de espermatozoides disponíveis após a sexagem. Vários fatores podem influenciar os resultados da produção in vitro de embriões na espécie bovina utilizando sêmen sexado; dentre eles destacam-se as baixas taxas de fertilização, de clivagem, de blastocistos, de gestações; capacitação espermática parcial; amostras seminais diluídas e a variação entre touros. Alguns estudos têm sido realizados com o intuito de avaliar as alterações genéticas e epigenéticas decorrentes do uso do sêmen sexado na produção de embriões bovinos in vitro. Portanto, este trabalho tem como objetivo realizar uma revisão de literatura sobre as limitações e as vantagens do uso do sêmen sexado, com ênfase na técnica de citometria de fluxo, durante a produção embrionária in vitro na espécie bovina.(AU)

The advent of sex-sorting-sperm sex selection of offspring became a great advantage, especially for dairy farms, which need new heifers for maintenance of his herds, and for beef cattle breeders, who crave more males for meat production. Currently, the only known technique capable of separating the populations of X and Y mammals sperm cells with efficiency and purity above 90% is flow cytometry. The best way of using sexed semen is in vitro fertilization, due to the small number of available sperm after sexing. Several factors can influence the results of in vitro embryos production using bovine sexed semen; among them there are the low rates of fertilization, cleavage, blastocyst, pregnancies, partially sperm capacitation, diluted semen samples and variation among bulls. Several studies have been conducted in order to assess the genetic and epigenetic alterations resulting from the use of sexed semen in the production of bovine embryos in vitro. Therefore, this paper aims to conduct a literature review on the limitations and advantages of using sexed semen, with emphasis on the technique of flow cytometry, during in vitro embryo production in cattle.(AU)

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.(AU)