Assisted Reproductive Technologies (ART) and genome editing to support a sustainable livestock

This article provides an overview of assisted reproductive technologies (ART) and genome engineering to improve livestock production systems for the contribution of global sustainability. Most ruminant production systems are conducted on grassland conditions, as is the case of South American countries that are leaders in meat and milk production worldwide with a well-established grass-feed livestock. These systems have many strengths from an environmental perspective and consumer preferences but requires certain improvements to enhance resource efficiency. Reproductive performance is one of the main challenges particularly in cow-calf operations that usually are conducted under adverse conditions and thus ART can make a great contribution. Fixed-time artificial insemination is applied in South America in large scale programs as 20 to 30% of cows receive this technology every year in each country, with greater calving rate and significant herd genetic gain occurred in this region. Sexed semen has also been increasingly implemented, enhancing resource efficiency by a) obtaining desired female replacement and improving animal welfare by avoiding newborn male sacrifice in dairy industry, or b) alternatively producing male calves for beef industry. In vitro embryo production has been massively applied, with this region showing the greatest number of embryos produced worldwide leading to significant improvement in herd genetics and productivity. Although the contribution of these technologies is considerable, further improvements will be required for a significant livestock transformation and novel biotechnologies such as genome editing are already available. Through the CRISPR/Cas-based system it is possible to enhance food yield and quality, avoid animal welfare concerns, overcome animal health threats, and control pests and invasive species harming food production. In summary, a significant enhancement in livestock productivity and resource efficiency can be made through reproductive technologies and genome editing, improving at the same time profitability for farmers, and global food security and sustainability.(AU)

Prohibition of hormones in animal reproduction: what to expect and what to do?

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. The development of Fixed-Time Artificial Insemination (FTAI) protocols at the end of the 20th century has increased exponentially the number of animals inseminated over the last 20 years. The main reasons for this growth were the possibility of obtaining acceptable pregnancy rates without heat detection and, above all, the induction of cyclicity in suckled cows in postpartum anestrus and prepubertal heifers at the beginning of the breeding season. Most FTAI treatments in South America have been based on the use of progesterone (P4) releasing devices and estradiol to synchronize both follicular wave emergence and ovulation, with pregnancy rates ranging from 40 to 60%. These protocols are implemented on a regular basis, allowing producers access to high-quality genetics, and increasing the overall pregnancy rates during the breeding season. In addition, it provided the professionals involved in these programs with a new source of income and the diversification of their practices into activities other than their usual clinical work. Many of these practices are now apparently at risk from restrictions on the use of estradiol by the European Union (EU) and other countries. However, the development of alternative protocols based on GnRH, with P4 devices and eCG and other new products that are not in the market yet will allow us to adapt to the new times that are coming. Logically, the challenge has already been raised and we must learn to use alternative protocols to try to continue increasing the use of this technology in beef and dairy herds. The objective of the present review is to describe the main aspects of banning estradiol in livestock production, the negative impacts on reproductive efficiency, and to present some alternative FTAI protocols for dairy and beef cattle.(AU)

Recent advances in treatments for resynchronization of ovulation in small ruminants: a review

Hormonal methodologies to control small ruminants' estrous cycle are worldwide used and evolved, adjusting the application to the precise female physiological moments to enhance reproductive performance. The estrous cycle can be induced and/or synchronized, aiming for fixed-time artificial insemination, or based on estrus behavior signs for insemination, natural or guided mating. Successive protocols can be performed to resynchronize ovulation and increase reproductive outcomes in females that failed to conceive. These recently developed treatments aim to resynchronize the ovulation as earlier as non-pregnancy is detected. The present review aimed to summarize the recent advances and main findings regarding resynchronization protocols used in small ruminants. Lastly, we present future perspectives and new paths to be studied in the subject. The resynchronization treatment is still a growing field in small ruminant reproduction, nevertheless, some enhancements are found in the reproductive outcome, showing that such protocols can be successfully used in sheep and goat production.(AU)

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P 0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year /1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year /5. No cases of maternal cannibalism were found on Year /1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year /5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P 0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year /5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P 0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year /1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year /5. No cases of maternal cannibalism were found on Year /1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year /5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P 0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year /5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.(AU)

Evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in South American cattle

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. Luteal function in cattle has been studied in detail, and prostaglandin F2α has been used for several years for the elective induction of luteal regression. More recently, follicle wave dynamics has been studied and protocols designed to induce follicular wave emergence and ovulation have reduced, and even eliminated, the need for estrus detection. The addition of progestin-releasing devices, estradiol, GnRH and equine chorionic gonadotropin (eCG) have provided opportunities for fixed-time AI (FTAI) and possibilities for increased pregnancy rates. In embryo transfer programs, these same treatments have eliminated the need for estrus detection, permitting fixed-time embryo transfer and the initiation of superstimulatory treatments without regard to day of the estrous cycle. Collectively, new treatment protocols have facilitated the application of assisted reproductive technologies, and this is especially true in South America. Over the last 20 years, the use of AI in South America has increased, due largely to the use of FTAI. There has been more than a 10-fold increase in the use of FTAI in Brazil with more than 11 million treatments in 2016, representing 85% of all AI. Similar trends are occurring in Argentina and Uruguay. Production of in vivo-derived (IVD) embryos has remained relatively stable over the years, but in vitro embryo production (IVP) has increased dramatically over the past 10 to 15 years, especially in Brazil where more than 300,000 IVP embryos were produced in 2010. World-wide, more than 666,000 bovine IVP embryos were produced in 2016, of which more than 57% were produced in South America. The use of assisted reproductive technologies has facilitated the dissemination of improved genetics and increased reproductive performance; other South American countries are now following suit.(AU)

From reproductive technologies to genome editing in small ruminants: an embryo’s journey

The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.

Evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in South American cattle

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. Luteal function in cattle has been studied in detail, and prostaglandin F2α has been used for several years for the elective induction of luteal regression. More recently, follicle wave dynamics has been studied and protocols designed to induce follicular wave emergence and ovulation have reduced, and even eliminated, the need for estrus detection. The addition of progestin-releasing devices, estradiol, GnRH and equine chorionic gonadotropin (eCG) have provided opportunities for fixed-time AI (FTAI) and possibilities for increased pregnancy rates. In embryo transfer programs, these same treatments have eliminated the need for estrus detection, permitting fixed-time embryo transfer and the initiation of superstimulatory treatments without regard to day of the estrous cycle. Collectively, new treatment protocols have facilitated the application of assisted reproductive technologies, and this is especially true in South America. Over the last 20 years, the use of AI in South America has increased, due largely to the use of FTAI. There has been more than a 10-fold increase in the use of FTAI in Brazil with more than 11 million treatments in 2016, representing 85% of all AI. Similar trends are occurring in Argentina and Uruguay. Production of in vivo-derived (IVD) embryos has remained relatively stable over the years, but in vitro embryo production (IVP) has increased dramatically over the past 10 to 15 years, especially in Brazil where more than 300,000 IVP embryos were produced in 2010. World-wide, more than 666,000 bovine IVP embryos were produced in 2016, of which more than 57% were produced in South America. The use of assisted reproductive technologies has facilitated the dissemination of improved genetics and increased reproductive performance; other South American countries are now following suit.

From reproductive technologies to genome editing in small ruminants: an embryos journey

The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.(AU)

Programs for fixed-time artificial insemination in South American beef cattle

Fixed-time artificial insemination (FTAI) has been widely applied in South America within the last 20 years for the genetic improvement of commercial beef herds. Most FTAI treatments for beef cattle used in South America are based on the use of progesterone (P4) releasing devices and estradiol to synchronize follicle wave emergence, with pregnancies per AI (P/AI) ranging from 40 to 60%. More recent protocols focusing on extending the interval from device removal to FTAI (i.e. increasing the growing period of the ovulatory follicle) have been reported to improve P/AI in beef cattle. These new protocols and the more traditional FTAI protocols have also been adapted for use with sexed-sorted semen with acceptable P/AI in beef cattle. Finally, color-flow Doppler ultrasonography has been incorporated recently to determine the vascularity of the CL and thereby detect pregnancy as early as Day 22 after the first AI for resynchronization of ovulation for a second FTAI in nonpregnant animals. In summary, FTAI protocols have facilitated the widespread application of AI in South American beef cattle by allowing for the insemination and re-insemination of herds during a defined breeding season, without the necessity of clean up bulls to achieve high pregnancy rates.(AU)

Programs for fixed-time artificial insemination in South American beef cattle

Fixed-time artificial insemination (FTAI) has been widely applied in South America within the last 20 years for the genetic improvement of commercial beef herds. Most FTAI treatments for beef cattle used in South America are based on the use of progesterone (P4) releasing devices and estradiol to synchronize follicle wave emergence, with pregnancies per AI (P/AI) ranging from 40 to 60%. More recent protocols focusing on extending the interval from device removal to FTAI (i.e. increasing the growing period of the ovulatory follicle) have been reported to improve P/AI in beef cattle. These new protocols and the more traditional FTAI protocols have also been adapted for use with sexed-sorted semen with acceptable P/AI in beef cattle. Finally, color-flow Doppler ultrasonography has been incorporated recently to determine the vascularity of the CL and thereby detect pregnancy as early as Day 22 after the first AI for resynchronization of ovulation for a second FTAI in nonpregnant animals. In summary, FTAI protocols have facilitated the widespread application of AI in South American beef cattle by allowing for the insemination and re-insemination of herds during a defined breeding season, without the necessity of clean up bulls to achieve high pregnancy rates.

Estrous synchronization treatments in sheep: Brief update / Atualização nos protocolos de sincronização do cio em ovinos

Fixed-time artificial insemination (FTAI) is the most effective method to increase the number offemales inseminated in a single day, eliminating the necessity of estrus detection. Usually the treatments forFTAI in sheep are based on the use of progesterone-releasing devices and equine chorionic gonadotropin (eCG)administration at device removal, with an acceptable pregnancy rate. The current information about the ovarianphysiology supports the idea of shortening the traditional progesterone exposure with intravaginal devices from14 days to 5-7 days (Short-term protocols). These protocols ensure appropriate progesterone concentrations toinduce follicular turnover and ovulation of a non-persistent follicle. One im dose of PGF2alpha is required attime of device removal with eCG administration, ovulation occurs around 60 h later and FTAI is performed at 48or 54 h by cervical or intrauterine route, respectively. Several experiments have been conducted during last yearsto adjust this new protocol. In general, reported pregnancy rate is greater -or at least similar- than traditional 14days protocols, and remaining progesterone in the silicone intravaginal devices may be enough for theirreutilization with interesting results. Several improvements on follicular dynamics, time of ovulation,pharmacological associations, insemination time, sperm dose, among others, are summarized in this review inFTAI protocols facilitate a further adoption of insemination and genetic improvement in this species.(AU)

Estrous synchronization treatments in sheep: Brief update / Atualização nos protocolos de sincronização do cio em ovinos

Fixed-time artificial insemination (FTAI) is the most effective method to increase the number offemales inseminated in a single day, eliminating the necessity of estrus detection. Usually the treatments forFTAI in sheep are based on the use of progesterone-releasing devices and equine chorionic gonadotropin (eCG)administration at device removal, with an acceptable pregnancy rate. The current information about the ovarianphysiology supports the idea of shortening the traditional progesterone exposure with intravaginal devices from14 days to 5-7 days (Short-term protocols). These protocols ensure appropriate progesterone concentrations toinduce follicular turnover and ovulation of a non-persistent follicle. One im dose of PGF2alpha is required attime of device removal with eCG administration, ovulation occurs around 60 h later and FTAI is performed at 48or 54 h by cervical or intrauterine route, respectively. Several experiments have been conducted during last yearsto adjust this new protocol. In general, reported pregnancy rate is greater -or at least similar- than traditional 14days protocols, and remaining progesterone in the silicone intravaginal devices may be enough for theirreutilization with interesting results. Several improvements on follicular dynamics, time of ovulation,pharmacological associations, insemination time, sperm dose, among others, are summarized in this review inFTAI protocols facilitate a further adoption of insemination and genetic improvement in this species.

Transgenesis and gene edition in small ruminants / Transgênese e edição gênica em pequenos ruminantes

This review summarizes the main achievements with the use of transgenesis and genome editingtechnologies in sheep and goats. Transgenesis, also referred to as recombinant DNA (rDNA) technology, madepossible by the first time 30 years ago the addition of novel traits from a given species into a different one. Onthe other hand, more recently genome editing appears a much more precise method of making changes to thegenome of a plant, animal, or other living organism, allowing for the addition, substitution, or deletion ofspecific nucleotides in an organisms genome. With transgenesis, the introduction of new DNA into anorganisms genome was generally without control of the site of the genome in which the insertion of that rDNAconstruct would occur. With genome editing in contrast, researchers and developers of products can makespecific changes in precise locations of the genome. This concept was absolutely improved with the novelCRISPR/Cas system, making genome edition cheaper, more efficient, easier and affordable for every Laboratoryaround the world. This revolution that originally emerged from molecular biology and passed to biomedicine,has recently been applied to livestock and agriculture. In addition, the application of this technology in sheep,goats, pigs and cattle, also has been possible by the advance of assisted reproductive technologies for embryoproduction, micromanipulation, cryopreservation and transfer. In general, multidisciplinary approaches includingbasic research and technical improvements, participation of private actors and adequate regulation should bemerged to take advantage of this potent biotechnology in different countries.(AU)

Transgenesis and gene edition in small ruminants / Transgênese e edição gênica em pequenos ruminantes

This review summarizes the main achievements with the use of transgenesis and genome editingtechnologies in sheep and goats. Transgenesis, also referred to as recombinant DNA (rDNA) technology, madepossible by the first time 30 years ago the addition of novel traits from a given species into a different one. Onthe other hand, more recently genome editing appears a much more precise method of making changes to thegenome of a plant, animal, or other living organism, allowing for the addition, substitution, or deletion ofspecific nucleotides in an organism’s genome. With transgenesis, the introduction of new DNA into anorganism’s genome was generally without control of the site of the genome in which the insertion of that rDNAconstruct would occur. With genome editing in contrast, researchers and developers of products can makespecific changes in precise locations of the genome. This concept was absolutely improved with the novelCRISPR/Cas system, making genome edition cheaper, more efficient, easier and affordable for every Laboratoryaround the world. This revolution that originally emerged from molecular biology and passed to biomedicine,has recently been applied to livestock and agriculture. In addition, the application of this technology in sheep,goats, pigs and cattle, also has been possible by the advance of assisted reproductive technologies for embryoproduction, micromanipulation, cryopreservation and transfer. In general, multidisciplinary approaches includingbasic research and technical improvements, participation of private actors and adequate regulation should bemerged to take advantage of this potent biotechnology in different countries.