Cultivo in vitro de folículos pré-antrais bovinos: revisão, desafios, conquistas e perspectivas futuras / In vitro culture of bovine preantral follicles: review, challenges, achievements and future perspectives

A reprodução animal incide diretamente na eficiência e rentabilidade econômica de um rebanho, sendo considerada um dos fatores de maior importância para os sistemas de produção. Consequentemente, o desenvolvimento e a utilização de biotecnologia são indispensáveis para o aumento da eficiência reprodutiva, do melhoramento genético, da produção de alimentos e geração de riquezas. Nas últimas décadas várias foram as biotécnicas associadas à reprodução animal que apresentaram um grande potencial para a geração de genética, especialmente, ao que se referem aos avanços da reprodução assistida. Estes progressos possibilitaram a utilização de folículos pré-antrais como fonte de oócitos para biotecnologias reprodutivas. Deste modo, a técnica de cultivo in vitro proporciona a retomada do desenvolvimento dos folículos presente na reserva ovariana pela ativação in vitro forçada simulando a ocorrência de distúrbios na foliculogênesse. Até então, apenas a especie murina obteve descendentes vivos através dos folículos primordiais cultivados in vitro. Infelizmente, não há relatos de sucesso em outras espécies, tornando o desenvolvimento de sistemas ideias de cultivo in vitro um desafio. Portanto, o objetivo desta revisão é descrever e discutir os principais avanços da situação atual da pesquisa com folículos pré-antrais inclusos em folículos ovarianos.(AU)

Animal reproduction directly affects the efficiency and economic profitability of a herd, being considered one of the most important factors for production systems. Consequently, the development and use of biotechnology are indispensable for increasing reproductive efficiency, genetic improvement, food production and wealth generation. In the last few decades, biotechnologies associated with animal reproduction have shown great potential for the generation of genetics, especially with regard to advances in assisted reproduction. These advances have enabled the use of pre-antral follicles as a source of oocytes for reproductive biotechnologies. Thus, the in vitro culture technique provides the resumption of the development of follicles present in the ovarian reserve by forced in vitro activation, simulating the occurrence of disturbances in folliculogenesis. Until then, only the murine species obtained living descendants through primordial follicles cultivated in vitro. Unfortunately, there are no reports of success in other species, making the development of ideal in vitro culture systems a challenge.Therefore, the purpose of this review is to describe and discuss the main advances in the current research situation with preantral follicles included in ovarian follicles.(AU)

Cultivo in vitro de folículos pré-antrais bovinos: revisão, desafios, conquistas e perspectivas futuras / In vitro culture of bovine preantral follicles: review, challenges, achievements and future perspectives

A reprodução animal incide diretamente na eficiência e rentabilidade econômica de um rebanho, sendo considerada um dos fatores de maior importância para os sistemas de produção. Consequentemente, o desenvolvimento e a utilização de biotecnologia são indispensáveis para o aumento da eficiência reprodutiva, do melhoramento genético, da produção de alimentos e geração de riquezas. Nas últimas décadas várias foram as biotécnicas associadas à reprodução animal que apresentaram um grande potencial para a geração de genética, especialmente, ao que se referem aos avanços da reprodução assistida. Estes progressos possibilitaram a utilização de folículos pré-antrais como fonte de oócitos para biotecnologias reprodutivas. Deste modo, a técnica de cultivo in vitro proporciona a retomada do desenvolvimento dos folículos presente na reserva ovariana pela ativação in vitro forçada simulando a ocorrência de distúrbios na foliculogênesse. Até então, apenas a especie murina obteve descendentes vivos através dos folículos primordiais cultivados in vitro. Infelizmente, não há relatos de sucesso em outras espécies, tornando o desenvolvimento de sistemas ideias de cultivo in vitro um desafio. Portanto, o objetivo desta revisão é descrever e discutir os principais avanços da situação atual da pesquisa com folículos pré-antrais inclusos em folículos ovarianos.

Animal reproduction directly affects the efficiency and economic profitability of a herd, being considered one of the most important factors for production systems. Consequently, the development and use of biotechnology are indispensable for increasing reproductive efficiency, genetic improvement, food production and wealth generation. In the last few decades, biotechnologies associated with animal reproduction have shown great potential for the generation of genetics, especially with regard to advances in assisted reproduction. These advances have enabled the use of pre-antral follicles as a source of oocytes for reproductive biotechnologies. Thus, the in vitro culture technique provides the resumption of the development of follicles present in the ovarian reserve by forced in vitro activation, simulating the occurrence of disturbances in folliculogenesis. Until then, only the murine species obtained living descendants through primordial follicles cultivated in vitro. Unfortunately, there are no reports of success in other species, making the development of ideal in vitro culture systems a challenge.Therefore, the purpose of this review is to describe and discuss the main advances in the current research situation with preantral follicles included in ovarian follicles.

Ocorrência de alterações das glândulas sexuais acessórias de touros de rodeio através do exame ultrassonográfico / Occurrence of alterations in the accessory sex glands of rodeo bulls accessed by ultrasound examination

Objetivou-se com o presente estudo determinar a ocorrência de alterações das glândulas sexuais acessórias de touros de rodeio através da ultrassonografia transretal. Touros mestiços (n = 118) foramavaliados através da palpação e ultrassonografia transretal. As glândulas sexuais acessórias (próstata, ampolas dos ductos deferentes e vesículas seminais) foram palpadas e delimitadas quanto ao tamanho e higidez. Após, tais glândulas foram avaliadas e mensuradas de ponta a ponta com ultrassom (Sonoscape®, China), probe linear, modo-B, com frequência de 7,5 Hz, por via transretal. A ocorrência de alterações das vesículas seminais direita e esquerda foi de 44,91 %. A assimetria vesicular leve obteve a prevalência de 47,17 %, a moderada de 5,66 % e grave cerca de 47,17 %. A medida média da ampola foi de 9 mm. A ocorrência de alterações das ampolas direita e esquerda foi de 6,78 %, nos quais as alterações na próstata foi de 17,8 %. Assim, cerca de 10,17 % dos touros apresentaram redução da sensibilidade da próstata e no que diz respeito a ausência de sensibilidade dessa glândula, cerca de 7,63%. Conclui-se que o exame ultrassonográfico pode auxiliar em melhor acuidade no exame das glândulas sexuais acessórias em touros de rodeio.(AU)

The objective of this study was to determine the occurrence of alterations of the rodeo bull accessory sex glands by transrectal ultrasound. Crossbred bulls (n = 118) were evaluated by palpation and transrectal ultrasound. The accessory sex glands (prostate, ampoules and seminal vesicles) were palpated and delimited for size and higidity. Afterwards, these glands were scanned and measured from end to end with ultrasound (Sonoscape®, China), linear probe, B-mode, with frequency of 7.5 Hz, transrectally. The occurrence of right and left seminal vesicle alterations was 44.91%. Mild vesicularasymmetry was prevalent at 47.17%, moderate at 5.66% and severe at 47.17%. The mean ampoule measurement was 9 mm. Changes in right and left ampoules were 6.78%, in which prostate changes were 17.8%. Besides, about 10.17% of bulls had reduced prostate sensitivity and about 7.63% lack of sensitivity of this gland. It is concluded that the ultrasound examination may help in better accuracy in the examination of the accessory sex glands in rodeo bulls.(AU)

Ocorrência de alterações das glândulas sexuais acessórias de touros de rodeio através do exame ultrassonográfico / Occurrence of alterations in the accessory sex glands of rodeo bulls accessed by ultrasound examination

Objetivou-se com o presente estudo determinar a ocorrência de alterações das glândulas sexuais acessórias de touros de rodeio através da ultrassonografia transretal. Touros mestiços (n = 118) foramavaliados através da palpação e ultrassonografia transretal. As glândulas sexuais acessórias (próstata, ampolas dos ductos deferentes e vesículas seminais) foram palpadas e delimitadas quanto ao tamanho e higidez. Após, tais glândulas foram avaliadas e mensuradas de ponta a ponta com ultrassom (Sonoscape®, China), probe linear, modo-B, com frequência de 7,5 Hz, por via transretal. A ocorrência de alterações das vesículas seminais direita e esquerda foi de 44,91 %. A assimetria vesicular leve obteve a prevalência de 47,17 %, a moderada de 5,66 % e grave cerca de 47,17 %. A medida média da ampola foi de 9 mm. A ocorrência de alterações das ampolas direita e esquerda foi de 6,78 %, nos quais as alterações na próstata foi de 17,8 %. Assim, cerca de 10,17 % dos touros apresentaram redução da sensibilidade da próstata e no que diz respeito a ausência de sensibilidade dessa glândula, cerca de 7,63%. Conclui-se que o exame ultrassonográfico pode auxiliar em melhor acuidade no exame das glândulas sexuais acessórias em touros de rodeio.

The objective of this study was to determine the occurrence of alterations of the rodeo bull accessory sex glands by transrectal ultrasound. Crossbred bulls (n = 118) were evaluated by palpation and transrectal ultrasound. The accessory sex glands (prostate, ampoules and seminal vesicles) were palpated and delimited for size and higidity. Afterwards, these glands were scanned and measured from end to end with ultrasound (Sonoscape®, China), linear probe, B-mode, with frequency of 7.5 Hz, transrectally. The occurrence of right and left seminal vesicle alterations was 44.91%. Mild vesicularasymmetry was prevalent at 47.17%, moderate at 5.66% and severe at 47.17%. The mean ampoule measurement was 9 mm. Changes in right and left ampoules were 6.78%, in which prostate changes were 17.8%. Besides, about 10.17% of bulls had reduced prostate sensitivity and about 7.63% lack of sensitivity of this gland. It is concluded that the ultrasound examination may help in better accuracy in the examination of the accessory sex glands in rodeo bulls.

Intensified use of TAI and sexed semen on commercial farms

The livestock sector has achieved many technological advances, which have resulted in continued improvements in animal production systems and in the reproductive efficiency of herds. The associated use of reproductive biotechnology and genetic improvements combined with adequate sanitary and nutritional management are essential conditions for sustainable intensified animal production and financial autonomy within farms. Timed artificial insemination (TAI) represents one of the strategies with the greatest impact of expansion in providing genetic improvements and increased reproductive efficiency at a decreased cost. Despite the high proportion of cows receiving TAI, this market still exhibits considerable potential for expansion. After a TAI procedure, approximately 40 to 60% of females become pregnant. This result can vary depending on such factors as the hormonal protocol employed, female category, body condition score, ovarian status, farm management and aspects related to bulls and semen. The fertility and genetic quality of the bull plays an important role in the herd because a single bull can influence the entire production system. Another important strategy is the use of sex-sorted semen associated with TAI, primarily when associated with management practices to improve the pregnancy rate. This paper presents a review of the intensification of TAI, supplying practical information regarding the implementation of TAI commercial programs.

Intensified use of TAI and sexed semen on commercial farms

The livestock sector has achieved many technological advances, which have resulted in continued improvements in animal production systems and in the reproductive efficiency of herds. The associated use of reproductive biotechnology and genetic improvements combined with adequate sanitary and nutritional management are essential conditions for sustainable intensified animal production and financial autonomy within farms. Timed artificial insemination (TAI) represents one of the strategies with the greatest impact of expansion in providing genetic improvements and increased reproductive efficiency at a decreased cost. Despite the high proportion of cows receiving TAI, this market still exhibits considerable potential for expansion. After a TAI procedure, approximately 40 to 60% of females become pregnant. This result can vary depending on such factors as the hormonal protocol employed, female category, body condition score, ovarian status, farm management and aspects related to bulls and semen. The fertility and genetic quality of the bull plays an important role in the herd because a single bull can influence the entire production system. Another important strategy is the use of sex-sorted semen associated with TAI, primarily when associated with management practices to improve the pregnancy rate. This paper presents a review of the intensification of TAI, supplying practical information regarding the implementation of TAI commercial programs.(AU)

Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles / Intervalo na substituição do meio de cultivo in vitro afeta a integridade e o desenvolvimento de folículos pré-antrais equinos

The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher's test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.(AU)

A eficiência de um sistema de cultivo está relacionada à elaboração e substituição do meio de cultivo com condições adequadas ao desenvolvimento folicular. Pesquisas recentes sugerem que o meio de cultivo in vitro deve ser substituído após períodos de tempo específicos para várias espécies. No entanto, o intervalo adequado para a troca de meio de cultivo in vitro ainda não foi estabelecido na espécie equina. O objetivo desta investigação foi avaliar o efeito de intervalos de troca média de 24 horas (T24) ou 48 horas (T48) para cultivo de folículos pré-antrais aos 2 ou 6 dias. No final do período de cultivo, os fragmentos foram processados ​​usando histologia clássica. Os folículos pré-antrais equinos foram classificados de acordo com a integridade morfológica e o estágio de desenvolvimento. A análise dos dados foi realizada utilizando o teste de Fisher com um nível de significância de p<0,05. De um total de 399 folículos avaliados, 174 (43,6%) foram folículos primordiais, 225 (56,4%) estavam em desenvolvimento e 63,76% estavam morfologicamente intactos. No cultivo in vitro realizado ao longo de dois dias, não houve diferença significativa em relação à integridade folicular após a substituição do meio (p>0,05). Comparado com a substituição média aos seis dias de cultivo, houve diferença estatisticamente significativa para T24 (68,9%, p<0,05). Portanto, sugerimos alterar o meio para as espécies equinas às 48 horas após o início da cultura, seguindo as subsequentes substituições diárias.(AU)

Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles / Intervalo na substituição do meio de cultivo in vitro afeta a integridade e o desenvolvimento de folículos pré-antrais equinos

The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher's test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.(AU)

A eficiência de um sistema de cultivo está relacionada à elaboração e substituição do meio de cultivo com condições adequadas ao desenvolvimento folicular. Pesquisas recentes sugerem que o meio de cultivo in vitro deve ser substituído após períodos de tempo específicos para várias espécies. No entanto, o intervalo adequado para a troca de meio de cultivo in vitro ainda não foi estabelecido na espécie equina. O objetivo desta investigação foi avaliar o efeito de intervalos de troca média de 24 horas (T24) ou 48 horas (T48) para cultivo de folículos pré-antrais aos 2 ou 6 dias. No final do período de cultivo, os fragmentos foram processados ​​usando histologia clássica. Os folículos pré-antrais equinos foram classificados de acordo com a integridade morfológica e o estágio de desenvolvimento. A análise dos dados foi realizada utilizando o teste de Fisher com um nível de significância de p<0,05. De um total de 399 folículos avaliados, 174 (43,6%) foram folículos primordiais, 225 (56,4%) estavam em desenvolvimento e 63,76% estavam morfologicamente intactos. No cultivo in vitro realizado ao longo de dois dias, não houve diferença significativa em relação à integridade folicular após a substituição do meio (p>0,05). Comparado com a substituição média aos seis dias de cultivo, houve diferença estatisticamente significativa para T24 (68,9%, p<0,05). Portanto, sugerimos alterar o meio para as espécies equinas às 48 horas após o início da cultura, seguindo as subsequentes substituições diárias.(AU)

Cell proliferation in ovarian follicles fromBos taurus indicus females with different antral follicle count

The aim of this study was to compare the cell proliferation rate of follicles from Bostaurus indicusfemales with different antral follicle counts (AFCs). Thirty pairs of ovaries were classified as having low(≤ 31 follicles), intermediate (≥ 46 and ≤ 76 follicles) or high AFC (≥ 91 follicles). The ovaries were cut into 1 x 1 x 0.3 cm fragments, fixed in 10% buffered formalin solution and subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA). PCNA detection was detected in all stages of follicular development, being 1,388 (82.23%) primordial, 197 (11.67%) primary, 29 (1.72%) secondary and 74 (4.83%) antral follicles. The PCNA detection rate was higher (P ≤ 0.05) for antral and secondary follicles in ovaries with intermediate and low AFCs. This study showed that higher PCNA at early stages of follicular growth is detected in animals with high AFC, whereas a higher PCNA is seen in secondary and antral follicles of animals with low AFC.

Cell proliferation in ovarian follicles fromBos taurus indicus females with different antral follicle count

The aim of this study was to compare the cell proliferation rate of follicles from Bostaurus indicusfemales with different antral follicle counts (AFCs). Thirty pairs of ovaries were classified as having low(≤ 31 follicles), intermediate (≥ 46 and ≤ 76 follicles) or high AFC (≥ 91 follicles). The ovaries were cut into 1 x 1 x 0.3 cm fragments, fixed in 10% buffered formalin solution and subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA). PCNA detection was detected in all stages of follicular development, being 1,388 (82.23%) primordial, 197 (11.67%) primary, 29 (1.72%) secondary and 74 (4.83%) antral follicles. The PCNA detection rate was higher (P ≤ 0.05) for antral and secondary follicles in ovaries with intermediate and low AFCs. This study showed that higher PCNA at early stages of follicular growth is detected in animals with high AFC, whereas a higher PCNA is seen in secondary and antral follicles of animals with low AFC.(AU)

Proliferative activity of oocytes in multi-oocyte follicles of bovine ovary / Atividade proliferativa em folículos multi-oócitos na espécie bovina

We characterized the proliferative activity of multi-oocyte follicles with anti-nuclear antigen of proliferating cells (PCNA). Ovaries (n = 12) from heifers were processed for histology. From 789 multi-oocytes follicles observed, only 11 were considered appropriated for immunostaining, since they presented all nuclei of the oocytes clearly visible. All multi-oocyte follicles were positive for PCNA, but some oocytes showed no proliferative activity. We conclude that oocytes in multi-oocyte follicles seem to be in different stages of the cell cycle.

O objetivo deste trabalho foi caracterizar a atividade proliferativa de folículos multi-oócitos com antígeno marcador de proliferação celular (PCNA). Ovários (n = 12) de novilhas foram processados para histologia. A partir de 789 folículos multi-oócitos identificados, somente 11 foram considerados adequados para imunohistoquímica por apresentarem todos os núcleos claramente visíveis. Todos os folículos multi-oócitos foram positivos para PCNA, mas alguns oócitos não apresentaram marcação. Concluímos que oócitos presentes em folículos multi-oócitos parecem estar em diferentes fases do ciclo celular.

Proliferative activity of oocytes in multi-oocyte follicles of bovine ovary / Atividade proliferativa em folículos multi-oócitos na espécie bovina

We characterized the proliferative activity of multi-oocyte follicles with anti-nuclear antigen of proliferating cells (PCNA). Ovaries (n = 12) from heifers were processed for histology. From 789 multi-oocytes follicles observed, only 11 were considered appropriated for immunostaining, since they presented all nuclei of the oocytes clearly visible. All multi-oocyte follicles were positive for PCNA, but some oocytes showed no proliferative activity. We conclude that oocytes in multi-oocyte follicles seem to be in different stages of the cell cycle.(AU)

O objetivo deste trabalho foi caracterizar a atividade proliferativa de folículos multi-oócitos com antígeno marcador de proliferação celular (PCNA). Ovários (n = 12) de novilhas foram processados para histologia. A partir de 789 folículos multi-oócitos identificados, somente 11 foram considerados adequados para imunohistoquímica por apresentarem todos os núcleos claramente visíveis. Todos os folículos multi-oócitos foram positivos para PCNA, mas alguns oócitos não apresentaram marcação. Concluímos que oócitos presentes em folículos multi-oócitos parecem estar em diferentes fases do ciclo celular.(AU)