Introduction

The text of the FAO Convention on Biological Diversity (CBD)¹, in force since 1993, states that the term "biotechnology" refers to any technological application that uses biological systems, living organisms (or derivatives thereof) to make or modify products or processes for a specific use. In this sense, and for the purposes of the topics to be covered in this review, reproductive biotechnology will be understood as the technological applications that affect the physiological processes of animal reproduction, their gametes and embryos, for the purpose of achieving productive improvements.

This document will cover only the bovine species and will place particular emphasis on the research carried out by institutions and universities in Mexico, particularly the National Institute for Research in Forestry, Agriculture and Livestock (INIFAP), in its 35 years of existence, and its predecessor: the National Institute of Livestock Research (INIP).

While the authors do not claim this to be an exhaustive review of all the reproductive biotechnologies that have been applied to cattle, it is address those that have had the greatest impact on productivity and briefly mention those that have had minimal or no application in Mexico, such as transgenesis and cloning.

The topics of estrus synchronization and ovulation induction are discussed first, as they are the topics on which research has been conducted for the longest time (50 yr) and for which INIP-INIFAP has generated the largest number of technologies. Biotechnologies developed for semen collection, dilution, and cryopreservation have underpinned the massive use of bulls of high genetic merit through artificial insemination (AI). This topic will be discussed, with emphasis on recent developments such as semen sexing and computer-assisted sperm analysis. Issues related to embryo manipulation, including multi-ovulation and embryo transfer (MOET), in vitro embryo production (IVP), and transvaginal oocyte aspiration (TVA) have seen important developments in recent years, which will be addressed in this paper. In addition to including research results on these topics, in some cases, mention will be made of government or producer organization programs that marked a milestone in the dissemination and adoption of these reproductive biotechnologies.

Estrus synchronization and ovulation induction

In cattle, a common problem is prolonged postpartum anestrus, a condition characterized by a delay in the return to estrous cyclicity after parturition due to various factors^2-6. Many efforts have been made to resolve this condition: hormonal treatments have been studied to induce estrus and ovulation^7,8 by controlling breastfeeding^9,10, the frequency and quality of its stimuli¹¹, feeding¹² and the different mating seasons¹³. This section reviews results of ovulation induction in anestrus females and synchronization of estrus and ovulation in cycling females, carried out by INIP (1971-1985) and the current INIFAP (1986-2021), as well as by other institutions.

Use of the progesterone-releasing intravaginal device (PIDR) in estrus synchronization

The introduction of the PIDR into the market revolutionized estrus synchronization³⁷, and, therefore, researchers from INIFAP^38,39 started using this device in cows in anestrus and cycling cows with good body condition (BC), obtaining a CR of 44.8, 77.1 and 100.0 % at 3, 30 and 60 d after PIDR withdrawal, respectively³⁸. In Bos taurus x Bos indicus³⁹ heifers, the CR with PIDR and FTAI at 84 h after the removal of the device was higher than with conventional AI at 12 h after the detection of estrus (36.4 vs 18.2 %, respectively). Similarly, the CR with CRESTAR and FTAI at 84 h after device removal was higher than with conventional AI (27.3 vs 18.2 %, respectively). In this work, the advantage of FTAI at 84 h after the removal of the device was demonstrated, since pregnancy was achieved in some heifers that did not exhibit estrus. A frequent practice in estrus and ovulation synchronization protocols with FTAI in which PIDR and PGF2α are used in association with estradiol benzoate (EB) (which is applied when inserting the PIDR on d 0 and the day after its removal) is the substitution of GnRH for EB. This is because, like EB, GnRH synchronizes the emergence of a new wave of follicular development, which culminates in the ovulation of the mature follicle, giving good results. However, if a significant proportion of the cows are suspected to be in anestrus, it is recommended to additionally apply 400 IU of equine chorionic gonadotropin eCG on d 7 of the protocol. Furthermore, EB can be replaced with GnRH (100 µg i.m.), on the first application (d 1 of PIDR), but the second application must be carried out at the time of FTAI. An example for cycling cows is shown in Figure 1 ⁴⁰.

Figure 1 General outline of estrous synchronization in cows and heifers with FTAI⁴⁰  

Semen processing and artificial insemination

AI was the first reproductive biotechnology applied to improve production through the more efficient use of bulls of high genetic merit. The widespread use of this technique and the achievement of its full potential required the cryopreservation of semen for long periods of time.

The first report of semen cryopreservation was made in 1776⁵⁶, where it was observed that when spermatozoa from human, guinea pig and frog were cooled in snow for up to 30 min, they became inactive, but could be reactivated. In 1940⁵⁷, egg yolk began to be used to protect bull sperm cells from thermal shock upon cooling. In 1941⁵⁸, the sperm extender was improved by using egg yolk with sodium citrate, which allowed semen preservation at 5 °C for up to 3 d. In 1949, bull spermatozoa were frozen for the first time by using glycerol in the diluent. The great discovery of the cryoprotective action of glycerol opened a successful era in the cryopreservation not only of gametes of various species, but also of other cells and tissues⁵⁹. The so-called Cornell diluent, created by Foote and Bratton in 1950⁶⁰, contained a mixture of the antibiotics penicillin, streptomycin, and polymyxin B, and was used for many years as standard. In 1952, in Cambridge, the first calf was born from frozen-thawed bovine sperm⁶¹.

Reports of fertility with frozen spermatozoa from bulls led to an intense development of cryopreservation methods to try to improve the results obtained, so other disciplines (such as molecular biology) associated with the cryopreservation process began to be used. Such is the case of proteomics⁶² and genetic engineering⁶³. On these issues, a study was carried out in Mexico with INIFAP researchers, where the addition of recombinant FAA (fertility-associated antigen) and recombinant TIMP-2 (tissue inhibitor of metalloproteinases 2) to frozen bovine semen was evaluated and proved to significantly increase its fertility⁶⁴.

Computer-assisted sperm evaluation

Fertility is a multiparametric event related to semen quality and quantity, the exact timing and method of AI, the intrinsic fertility of the bull and proper herd management⁸⁷. Morphology, motility, viability, acrosome, and DNA concentration and integrity have been used as parameters to evaluate the sperm quality of bovine semen^87-90. Some of these parameters (such as morphology and motility) can be assessed manually by conventional methods using a visible light microscope. However, these evaluations are subject to subjective criteria and technical errors that diminish their accuracy and repeatability.

In the mid-1980s, computer-assisted sperm analysis (CASA) systems were introduced commercially to maximize the accuracy and repeatability of semen evaluations⁹¹. The main function of these CASA systems is the objective evaluation of semen quality. The basic components of this technology consist of a microscope to visualize the sample, a digital camera to capture images and a computer with specialized software to analyze the images.

Motility is one of the most important sperm characteristics associated with the ability to fertilize⁹². With the use of the CASA system, various motility parameters describing specific sperm movements are obtained. Total and progressive motility percentages are the most important parameters in the evaluation of sperm kinetics⁸⁷. Total motility refers to the fraction of spermatozoa that show any movement, whereas spermatozoa with progressive motility have a forward movement, essentially in a straight line.

Other specific kinetic parameters determined by the CASA system are useful to evaluate several sperm characteristics simultaneously and objectively. These kinetic parameters consist mainly of three values of the speed of movement, three speed indices, and three parameters that reflect the oscillation characteristics of the spermatozoa⁹³. The three values of the velocity of motion are the curvilinear velocity (VCL), the rectilinear velocity (VSL) and the mean trajectory velocity (VAP). From these three values, three indices are calculated, linearity (LIN=VSL/VCL), straightness (STR=VSL/VAP) and trajectory oscillation (VAP/VCL), thus characterizing the quality of sperm movement. The parameters that show the oscillation characteristics of spermatozoa are the lateral displacement of the head, the frequency of tail beating, and the mean angular displacement.

CASA system movement parameters have been used for the identification of sperm subpopulations and their subsequent correlation with freezing resistance^94,95. In addition, the effects of different media during in vitro processing on sperm function have evaluated⁹⁶.

On the other hand, with the use of the CASA system, several groups have reported a significant correlation between the total (r=0.26-0.61) and progressive (r=0.26-0.33) motility of bull semen and its field-associated fertility^97-101. The CASA system collects a wide range of sperm kinetics parameters. Some research groups showed a positive correlation between VSL (r=0.17-0.67), LIN (r=0.28-0.46) and STR (r=0.33), and field fertility^98,102,103. Correlations between motility parameters and fertility tend to be low to medium, and their use in isolation to predict the fertility of a semen sample is not recommended^101-103. However, the combination of several motility parameters provides a better tool for predicting fertility, as the multi-parameter regression of the CASA system explained up to 0.98 (r² value) of the variation in fertility, compared to 0.34 explained by total motility alone¹⁰³. On the other hand, recent analyses of sperm movement in the third dimension and the study of flagellar movement are new functional parameters that could be related to fertility¹⁰⁴.

Sperm morphology is one of the most important tests in semen quality control, as it reflects the physiological or pathological state of the functionality of the testicles, epididymis and accessory glands of the reproductive tract¹⁰⁵; is also considered a better test to evaluate sperm DNA and genetic characteristics, compared to sperm motility^106,107. Several studies have shown a significant correlation between sperm morphology (r=0.22-0.76) and field fertility^94,98,108. However, most of the morphological analyses are performed through conventional methods, which remains a problem due to the subjectivity in the evaluation, as well as the inconsistency observed within and between technicians^109,110. For this reason, morphological analysis has not been considered reliable in predicting field fertility^{(94, 98)}. The development of specific modules for the morphological analysis of spermatozoa within the CASA system has allowed the individual evaluation of morphometric characteristics of the sperm head in terms of size (area, perimeter, length and width) and shape (ellipticity, elongation, regularity and roughness)¹¹¹. Some systems even provide information about the mid piece (area and width) and data concerning the insertion of the mid piece into the head, such as distance and angle of insertion¹¹². Within these parameters, the width of the sperm head showed a significant correlation (r=0.53) with field fertility¹¹³. On the other hand, sperm subpopulations have been reported based on their morphometric structure¹¹⁴. The evaluation of new parameters, as well as the identification of sperm subpopulations, could provide information on an optimal fertility-enhancing population⁹⁰.

There are other parameters in the study of sperm function, such as vitality, acrosome integrity, and DNA fragmentation. Viability classifies spermatozoa as alive or dead and shows the existence of damage to the sperm plasma membrane¹¹⁵. Acrosome integrity is one of the most important sperm function tests, since only a spermatozoon with an intact acrosome can penetrate the oocyte¹¹⁶. In the case of sperm DNA integrity, its importance in fertilization and in the early stages of embryonic development has been demonstrated, having been recognized as a parameter indicative of sperm fertilization potential^117,118. Several studies have shown a significant correlation between vitality (r=0.19-0.40), acrosome integrity (r=0.52) and DNA fragmentation (r=-0.49), and the field fertility of bulls^94,95,97,99. In addition, when several parameters (sperm kinetics, vitality, DNA fragmentation and morphology) were combined in regression models, the correlation with fertility increased to more than 90%¹². Although most of these tests are performed through the use of flow cytometry, new generations of CASA system modules have been developed for the automatic assessment of vitality, acrosome integrity, and DNA fragmentation⁹². However, few studies have been conducted in cattle using these new modules. Therefore, motility and morphology are shown to be the most important modules of the CASA system in terms of use.

These studies show the great potential of CASA systems for estimating semen quality, studying sperm function, and predicting fertility. However, the parameters provided by the CASA system have also been shown to have limitations and cannot be used in isolation as reliable predictors of the fertilization ability of the sperm. In addition, spermatozoa are complex cells that require a large number of criteria in order to be considered to achieve fertilization¹¹⁹. Therefore, the use of various parameters provided by the CASA system within a regression model is presented as the best option to attempt to predict the fertility of a semen sample. However, there is no consensus as to which parameters of sperm functionality to use in the spermatozoa⁹⁹, perhaps due to the differences found between working groups with respect to the parameters correlated with fertility.

Currently, sperm analysis through a CASA system is widely used within the quality control protocols of semen processing centers, mainly the motility module. These protocols establish thresholds for certain variables such as total and progressive motility; ejaculates below these thresholds are usually discarded before or after freezing⁹⁹.

In Mexico, several associations, institutions and companies related to the livestock sector utilize CASA systems on a regular basis mainly in the quality control of commercial doses of bovine, ovine, and porcine semen. However, studies of post-cryopreservation sperm kinetics of Pelibuey and Blackbelly rams¹²⁰, Mexican Pelon pigs¹²¹ and Merino sheep from Socorro Island¹²², and evaluations of cryopreserved semen samples from Chiapas sheep¹²³ and Tamaulipas Creole cattle¹²⁴ stored at the National Center for Genetic Resources of INIFAP (CNRG-INIFAP) have also been carried out. On the other hand, CASA systems have become more attractive as part of the practical evaluation of the sperm quality in semen samples of many domestic species. However, there are multiple reasons why a semen evaluation using a CASA system may vary, including system and equipment maintenance, sample handling, and technician experience⁸⁹. For this reason, it is vitally important to corroborate the effectiveness of the analysis. To ensure that the CASA system works properly, validation of technicians, protocols and equipment is crucial. In this sense, the CNRG-INIFAP has a test validated before the Mexican accreditation entity called “Evaluation of semen from domestic animals”, which is carried out with a CASA system. This allows semen evaluations to be performed with high quality standards¹²⁵.

CASA systems have evolved in recent years to become powerful tools for rapid and objective evaluation of sperm quality and function of bovine semen samples. CASA systems will continue to be developed to perform new, repeatable and increasingly accurate tests in order to contribute to fertility improvement in the field.

Multi-ovulation and embryo transfer (MOET)

This concept groups together a series of reproductive biotechnologies whose purpose is to increase the reproductive capacity on the maternal side and which are used as tools for genetic improvement. These biotechnologies include: selection of donors (both female and male) of high genetic merit, superovulation of donors, embryo collection and evaluation, embryo transfer to recipient cows, or embryo cryopreservation. This latter technique has had its greatest development in cattle; however, it is also widely applied in such species as equines, sheep, goats, and deer, among others¹²⁶. In addition to the aforementioned purpose (genetic improvement), the MOET has also been used for the conservation of animal genetic resources, as, according to FAO¹²⁷, it is an excellent option to conserve genetic diversity and is the fastest way to restore a population at risk. For this reason, INIFAP has the National Center for Genetic Resources, where work has been done to generate germplasm banks of bovine breeds with different risk statuses¹²⁸.

The first birth from embryo transfer in mammals was achieved in 1890 with rabbit embryos¹²⁹, and the first successful surgical transfer in cattle was achieved in 1951¹³⁰. The set of biotechnologies involving MOET developed between 1940 and 1960, laying the foundation for the embryo transfer industry, which began in 1970 as a tool for the mass introduction of continental European breeds to North America¹³¹. The modern activity of the MOET is the result of the efforts of two groups: a) scientists, who initially developed the procedures and techniques of embryo transfer, and b) field veterinarians, who applied this technology commercially, making it practical and available for the cattle industry, and later for other productive species as well¹³¹.

In the early 1970s, the first embryo transfer centers were established in North America, including Alberta Livestock Transfer (Alberta, Canada), Modern Ova Trends (Ontario, Canada), Colorado State University (Colorado, United States), Carnation Genetics (California, United States), and Codding Embryological Science, Inc. (Oklahoma, United States)¹³². In our country, the first embryo transfers (ET) in cattle were performed in 1978 by North American technicians, but the results are unknown. In February 1979, the Embryo Transfer Clinic was inaugurated in Ajuchitlán, Querétaro, as part of the National Center for Animal Reproduction, under the National Institute for Artificial Insemination and Animal Reproduction (INIARA), of the then Ministry of Agriculture and Hydraulic Resources (Secretaría de Agricultura y Recursos Hidráulicos, SARH). In that same year, MOET's first Holstein calf was born there¹³². The first TE zebu calf was born in 1981 at Carnation in Mexico¹³³.

The Center for Genetic Improvement and Embryo Transplantation (CEMEGEN), which is attached to the state-owned dairy company Leche Industrializada Conasupo (LICONSA), began its activities in November 1986, producing embryos of the Holstein Friesian, Brown Swiss, Jersey, Simmental, Simbrah, and F1 Holstein x Gyr dairy (Gyrholando) and Holstein x Guzerat breeds. It was estimated that, by 1993, 20,000 embryos per year would be produced from 2,200 donors. The center ceased operations in 1994; between 1987 and 1993 it produced almost 42,000 embryos. In addition, 18 training courses in the technique were given to approximately 300 professionals, and work was carried out for 23 Bachelor's and 4 Master's degree theses. Most of the embryos produced were frozen and subsequently transferred to different states of Mexico¹³⁴. In 1990, the National Center for Animal Reproduction became part of the National Commission for Genetic Improvement and Animal Reproduction (CONAMEGRA), established through an agreement between the Ministry and the National Livestock Confederation (CNG). Several TE works were carried out for farmers in different parts of the country, and an agreement was established with LICONSA for the commercialization, between 1993 and 1994, of 1,343 embryos produced at CEMEGEN, at a price of $100.00 per embryo ―an amount well below the cost of production. CONAMEGRA sold this genetic material to 33 producers from 11 Mexican states, its own technicians thawed 447 embryos, transferred them, and obtained 51 % of pregnancies¹³⁵. In 1993, INIFAP received a donation of 1,558 embryos from CEMEGEN; these were F1 and – Holstein x Zebu embryos, destined for the genetic programs of its experimental stations in the tropics. That same year, 800 embryos and 300 head of cattle were donated to the College of Posgraduates (Colegio de Posgraduados).

In 1994, LICONSA terminated the loan agreement with UNAM, auctioned the livestock, dismissed and paid off its staff, and returned its facilities to UNAM, thus putting an end to the operation of CEMEGEN¹³⁵.

The collection and transfer of embryos was initially a very complex process, since both the collection of embryos from the donor and their subsequent transfer to recipient females was done by surgical methods and using general anesthesia; this involved an enormous logistical effort, as the donors and the recipients had to be prepared for surgery at the same time¹³². In the first instance, both donor and recipient surgeries were performed under local anesthesia; subsequently, procedures for the non-surgical collection of embryos and their transfer to recipients were developed, which facilitated the more widespread use of the technique¹³⁶. The results obtained using non-surgical methods gradually approached those obtained with surgical methods, which is why the latter fell into disuse¹³¹.

Regarding ovarian stimulation to induce multiovulation, there has always been a high variability in the superovulatory response, despite efforts to control the sources of variation, which is one of the main problems affecting the efficiency and profitability of MOET at the commercial level¹³⁷. A study carried out with successive superovulations in zebu cows in the "El Macho" experimental station, INIFAP, where the variables of time, FSH dose and age were considered, concluded that the initial response of a zebu cow to superovulation cannot be said to be a good predictor of subsequent responses by the same cow¹³⁸.

To date, there have been considerable advances in the study of ovarian physiology, as well as factors inherent to the donor; however, there remain some aspects to be understood in order to generate more efficient ovarian stimulation protocols¹³⁹.

In its beginnings, superovulation was induced with eCG; however, a few years later it was discovered that crude pituitary extract (FSH with 20% LH) generated a better superovulatory response than eCG¹⁴⁰. Pituitary extracts are now widely used; there is a high variability in the amount of LH present in crude extracts, while purified extracts have less variation in the amount of LH. Multiple studies have been conducted to evaluate in vivo embryo production using crude and purified extracts¹³¹. In a 1995 study conducted at CEMEGEN in Mexico, the crude extract was found to produce more embryos than the purified extract in dairy cattle¹⁴¹. Conversely, in a study conducted in 2014 at INIFAP with beef cattle, the purified extract generated more embryos than the crude extract¹⁴²; this may be due to the fact that the hepatic metabolism of dairy cattle is more accelerated compared to that of beef cattle¹⁴³.

Despite many efforts to increase embryo production per cow per year by increasing ovarian response to superovulatory treatment, little progress has been made¹³¹. The use of PIDR in combination with BE and prostaglandins has allowed the development of a protocol called Rapid Donor Recycling. This protocol reduces the interval between superovulations by almost half (33 to 35 d) with no decline in response, either in the number of embryos produced or in their quality, in successive superovulations for up to one and a half years^131,142. Thus, the production of transferable embryos per donor per year is nearly doubled.

The first successful cryopreservation of embryos was reported in murines in 1972¹⁴⁴. One year later, the first calf was born from a previously cryopreserved blastocyst using a 2M dimethyl sulfoxide solution with a freezing and thawing rate of 0.2 °C/min and 36 °C/min, respectively¹⁴⁵. The first calves born from cryopreserved embryos in Mexico came from embryos frozen in Colorado, USA, and transferred to Nayarit, Mexico, into an experimental station of INIFAP¹⁴⁶.

For more than 10 yr, glycerol was the cryoprotectant of choice for bovine embryo cryopreservation. However, in 1992, a direct transfer cryopreservation system using ethylene glycol as cryoprotectant was announced. This had a rapid and positive effect worldwide on the embryo transfer industry to the present day¹³¹.

According to data from the American Embryo Transfer Association, in 2009, approximately 99 % of the beef bovine embryos and 94 % of the dairy bovine embryos were frozen in ethylene glycol for direct transfer¹³¹.

In vitro production of bovine embryos (IVP)

IVP biotechnology has a variety of applications in basic and applied science; in the first instance, it has supported the production of embryos used for a wide variety of research, including the treatment of infertility in humans. It is certainly a tool to increase the productivity of farm animals by increasing the reproductive potential of animals of higher genetic merit; in addition, it plays a relevant role in the conservation of the genetic resources of animals, especially endangered ones¹⁴⁷.

Generically known as IVF (in vitro fertilization) or IVP (in vitro production), it is a reproductive biotechnology, which, like MOET, is composed of several biotechnologies such as: in vitro maturation of eggs, sperm capacitation, in vitro fertilization, and the culture of zygotes and embryos up to pre-eclosion stages (7 to 8 d post-fertilization)¹⁴⁸. IVP consists of retrieving eggs or oocytes from ovarian follicles to be matured and fertilized under laboratory conditions; the resulting zygotes are cultured until post-compaction stages (morula or blastocyst), at which time they are transferred to a recipient cow or cryopreserved for subsequent transfer. Oocytes can be drawn from trace ovaries or live animals, by means of ultrasound-guided transvaginal aspiration (TVA)¹⁴⁸.

This biotechnology dates back to the 1970s, thanks to which research and achievements in the areas of culture media development, oocyte maturation, sperm capacitation and fertilization (which occurred in that decade and the following one¹²⁶) led to the birth, in 1987, of the first calf produced entirely in vitro¹⁴⁹. Although this biotechnology was initially oriented primarily to research and was based on tests carried out on ovaries obtained at the slaughterhouse, with the incorporation of TVA (making it possible to obtain immature oocytes from living donors), the commercial application was seen as a more promising tool than MOET for the mass production of offspring from progenitors of high genetic merit. The above is confirmed by observing that, worldwide, while the number of embryos collected in vivo and transferred has remained stable in recent years, the number of transferred IVF embryos has had an average annual growth rate of 12 % and, for the first time, in 2017, the number of viable embryos produced in vitro, exceeded the number of transferable embryos collected in vivo¹⁵⁰; that trend has continued as of the 2019 International Embryo Technology Society (IETS) report, with data from 2018¹⁵¹. In this same report, it is noted that the vast majority of viable IVF embryos were obtained from oocytes collected through TVA (98.9 %), in contrast to the few embryos obtained from trace ovaries¹⁵¹. The reason for this is the uncertainty of obtaining germplasm suitable for commercial production from the genetic and sanitary point of view. It is known that there are a significant number of research laboratories where viable IVP embryos are produced and discarded once they have fulfilled their function; however, this datum is not recorded by the IETS statistics committee¹⁵².

Globally, there are two events that have been driving forces in IVP: sexed semen biotechnology and genomic evaluations. The first is that thanks to IVP it is possible to maximize the use of sexed semen by fertilizing a large number of oocytes (100+) with a single dose of semen, and the second is that it makes it possible to intensify the power of selection by shortening generation intervals (TVA in calves, enabling the use of their germplasm before the age of service) and increasing the reproductive capacity on the maternal side. Thus, these three biotechnologies (IVP, sexed semen, genomic evaluations) play a relevant role in genetic improvement and the cattle industry in general¹⁵².

IVP had a period of great growth at the beginning of this century, especially in Brazil, where, in 2009, 85 % of the available embryos came from in vitro production; this amount was equivalent to 50 % of the worldwide production. The success of the Brazilian companies encouraged their expansion to other Latin American countries, including Mexico, where they settled, working independently or in partnership with Mexican companies or producer organizations. It has not been possible to replicate what has been achieved in Brazil in Mexico because this country does not have the competitive advantages that Brazil has (breeds, demand, size of production units, availability of receivers)¹⁴⁸. Nevertheless, there continues to be moderate activity by these and other domestic companies; thus, in 2018, the transfer of almost 28,000 embryos was reported in Mexico, almost all from IVP, and, in contrast, only over 4,000 embryos obtained in vivo were transferred¹⁵¹.

Although a considerable amount of research has been generated regarding the main components of this biotechnology (development of sequential culture media, control of potentially toxic agents, exclusion of serum components, inclusion of amino acids, vitamins, chelating agents and hormones, among others)^153-156, and so has research in oocyte maturation and in vitro fertilization processes^152-157, the fact is that it has not been possible to exceed the limit of 40 to 50 % of blastocysts obtained from fertilized oocytes, a value not very different from the 30 to 40 % that existed 20 yr ago. This has drawn attention to the lack of homogeneity in the oocyte source as the most likely cause of limited IVP success rates^155,157,158. It is therefore imperative to continue to promote IVP-IVT (in combination with other biotechnologies such as the use of sexed semen and genomic evaluations) as a valuable productive tool, and also to continue to conduct research for IVP in larger quantities with respect to the eggs left to mature, which achieve a better post-transfer development and have greater tolerance to cryopreservation.

Due to the complexity and high requirements of infrastructure and equipment, as well as personnel trained in this biotechnology, IVP research in Mexico has been incipient. Only a handful of universities ―namely, Universidad Nacional Autónoma de México, Universidad Autónoma Metropolitana, Universidad Veracruzana, Universidad Autónoma de Nuevo León, Universidad Autónoma de Tamaulipas, Colegio de Posgraduados, and Universidad Autónoma de Chihuahua―, some producer organizations and livestock companies, and INIFAP have worked on certain IVP research topics. The following are some of the results obtained in research conducted in Mexico on topics related to bovine IVP: in relation to agents and culture conditions to reduce the production of reactive oxygen species (ROS) in the culture medium, in a study conducted at INIFAP, it was found that by reducing the O₂ tension to 2 %, the number of cells in the blastocysts increased and the production of ROS was reduced¹⁵⁹. In another study also carried out at INIFAP, pterostilbene, a phytoalexin, was used as an antioxidant agent at a concentration of 0.33 μM, and it was found to reduce the production of ROS and the occurrence of intracytoplasmic lipids in 7-d-old embryos¹⁶⁰. The latter is presented as an alternative for improving IVP embryo cryopreservation. In a collaborative work between INIFAP and UANL, the effect of the addition of a heat shock protein (HSP70) on the development of bovine embryos produced in vitro was evaluated, and the addition of HSP70 to the culture media was found to have favorable effects on the percentage of blastocysts and cell number¹⁶¹. A system for individual embryo culture called "WOW" was also evaluated at INIFAP, and, although it produces a similar number of embryos compared to group culture in microdroplets, the WOW system was found to produce a higher percentage of embryos with a better morphological quality¹⁶². In another study carried out at UNAM, the effectiveness of two vitrification devices for cryopreserving bovine embryos was compared. Cryotop® was found to be a more effective vitrification support than Open Pulled Straw®, resulting in higher post-vitrification viability¹⁶³. Another study carried out between the Colegio de Posgraduados and the Universidad Veracruzana tested the alternative of utilizing the culture media used in the human IVF system to cultivate bovine embryos and found that it is possible to produce blastocysts with similar results¹⁶⁴. It should be noted that the culture media used in humans are often more readily available on the domestic market. Yet another study, carried out at the Autonomous University of Chihuahua, proved that the addition of IGF-I at different times during the IVP process did not produce a beneficial effect on the percentage of blastocysts¹⁶⁵.

The future of IVP in cattle faces important challenges that must be resolved in order to ensure its usefulness as a productive tool, as well as support for research in multiple areas of knowledge. The evolution of culture media to provide near-physiological conditions for gametes and embryos has been one of the areas with the greatest progress¹⁵², despite which, difficulties are still encountered today in producing similar embryos to those obtained in vivo, resulting in low pregnancy rates at transfer, low tolerance to cryopreservation and alterations in fetal and placental development¹⁵⁷. Research efforts have focused on the development of chemically defined culture media¹⁶⁶, use of oxidation level regulating compounds¹⁶⁷, use of delipidating compounds or lipid metabolism regulators^160,168, and mechanisms such as the use of sequential media to remove the presence of molecules that are toxic to the embryo¹⁶⁹, as well as on the development of cryopreservation methods that are friendlier to the embryo produced in vitro, such as vitrification¹⁷⁰. On the other hand, with the development of biotechnologies such as embryonic nuclear transplantation (16-cell stage blastomers) and somatic transplantation (cumulus cells, fibroblasts, etc.)¹⁷¹, an important future was foreseen for the genetic modification of embryos for productive, medical and research purposes (gene function studies, xenotransplantation, recombinant protein production, genetic improvement, and food production)¹⁷² and their eventual cloning. However, despite some encouraging results, the random insertion of the transgenes generated highly variable and unpredictable results, rendering the use of this technology unviable¹⁷³. Today, with the advent of gene editing technologies (CRISPR - Cas - 9), it is already possible to perform precise gene editing, including epigenetic reprogramming, which augurs a golden age in the genetic modification of farm animals for the aforementioned purposes, with the strong support of IVP biotechnologies¹⁷⁴. There will still be ethical impediments and legal pitfalls to be resolved.

Conclusions

As a final reflection, it can be said that, although research institutions in Mexico, and especially INIP-INIFAP, have accompanied the development of reproductive biotechnologies in the world in the last five decades. With the passage of the years, these have evolved meteorically, and it has been increasingly difficult to maintain a solid research base that allows us to be aligned with the technological developments that are taking place. Although the information generated by Mexican institutions on current topics such as in vitro embryo production is modest, the national livestock industry is demanding immersion in these technologies. This should encourage research institutions to generate technological components that will allow the efficient use of this and other technologies under local conditions.