Marcelo Fábio Gouveia Nogueira

Superovulation and embryo transfer in Bos indicus cattle

Compared to Bos taurus breeds, Bos indicus breeds of cattle present several differences in reproductive physiology. Follicular diameter at deviation and at the time of ovulatory capability are smaller in B. indicus breeds. Furthermore, B. indicus breeds have a greater sensitivity to gonadotropins, a shorter duration of estrus, and more often express estrus during the night. These differences must be considered when setting up embryo transfer programs for B. indicus cattle. In recent studies, we evaluated follicular dynamics and superovulatory responses in B. indicus donors with the objective of implementing fixed-time AI protocols in superstimulated donors. Protocols using estradiol and progesterone/progestrogen releasing devices to control follicular wave emergence were as efficacious as in B. taurus cattle, allowing the initiation of superstimulatory treatments (with lower dosages of FSH than in B. taurus donors) at a self-appointed time. Furthermore, results presented herein indicate that delaying the removal of progesterone/progestogen-releasing devices, combined with the administration of GnRH or pLH 12 h after the last FSH injection, results in synchronous ovulations, permitting the application of fixed-time AI of donors without the necessity of estrus detection and without compromising the results.

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P<0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P<0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells.

Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle

The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle.

Antioxidant responses and deregulation of epigenetic writers and erasers link oxidative stress and DNA methylation in bovine blastocysts

Early mammalian embryos derived from in vitro fertilization are exposed to conditions distinct from the native oviduct‐uterine environment, including atmospheric oxygen that promotes cellular oxidative stress and alters gene expression. High oxygen partial pressure during embryo development is associated with low pregnancy rates and increased embryonic apoptosis. We investigated how bovine embryos responded to high (20%) or low (5%) oxygen partial pressure during in vitro culture, evaluating levels of reactive oxygen species (ROS) as well as changes in the expression of oxidative stress‐ and epigenetic‐related transcripts and miRNAs in blastocysts. Additionally, we determined the global DNA methylation levels in the resulting embryos. Our data indicated that bovine blastocysts produced in vitro under high oxygen partial pressure possessed elevated ROS abundance and exhibited increased expression of CAT, GLRX2, KEAP1, NFR2, PRDX1, PRDX3, SOD1, TXN, and TXNRD1, versus reduced levels of the oxidative stress‐related bta‐miR‐210. These stressed embryos also presented altered expression of the epigenetic‐associated transcripts DNMT3A, H2AFZ, H3F3B, HDAC2, MORF4L2, REST, and PAF1. In addition, we demonstrated that embryos cultured under high oxygen partial pressure have increased global DNA methylation, suggesting that DNA hypermethylation is mediated by the deregulation of epigenetic‐related enzymes due to oxidative stress.

Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?

Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.

Oxidative Stress Alters the Profile of Transcription Factors Related to Early Development on In Vitro Produced Embryos

High oxygen levels during in vitro culture (IVC) can induce oxidative stress through accumulation of reactive oxygen species (ROS), negatively affecting embryo development. This study evaluated the effect of different O2 tensions during IVC on bovine blastocyst development and transcriptional status, considering transcription factors that play an essential role during early embryo development. For this purpose, embryos were produced in vitro by conventional protocols and cultured in two different oxygen tensions, physiological (5%) and atmospheric (20%). Expanded blastocysts were subjected to transcript quantitation analysis by RT-qPCR with Biomark™ HD System (Fluidigm, US), using 67 TaqMan assays specific for Bos taurus. Differences were observed in genes related to oxidation-reduction processes, DNA-dependent transcription factors, and factors related to important functional pathways for embryo development. Blastocyst rate was higher in the 5% O2 group and the number of cells was assessed, with the 5% O2 group having a higher number of cells. ROS concentration was evaluated, with a higher ROS presence in the 20% O2 group. Taken together, these results allow us to conclude that IVC of embryos at atmospheric O2 tension affects the expression of important transcription factors involved in multiple cell biology pathways that can affect embryo development, quality, and viability.

Number of oocytes retrieved per donor during OPU and its relationship with in vitro embryo production and field fertility following embryo transfer

The association of OPU-IVEP is an important instrument to drive genetic progress. In vitro embryo production (IVEP) has remarkably expanded in the last decade compared to in vivo embryo production. Because of the high repeatability of oocyte retrieval within oocyte-donors, studies exploring the relationship between the number of oocytes recovered per OPU section with IVEP efficiency, as well as with field fertility (pregnancy results following embryo transfer; P/ET) are extremely important to guide cow-donor selection and optimize field reproduction efficiency and the herd’s genetic gain. Based on this rationale, our group conducted a retrospective analysis of a large database comprising IVEP records from several cattle breeds, including Bos indicus and Bos taurus for either beef or dairy purposes. A total of 205,140 oocytes recovered from 7,906 OPU procedures of 6,902 donors (5,227 beef and 1,675 dairy) of Brazilian farms were analyzed. Beef breeds analyzed were Nelore (Bos indicus) and Senepol (Bos taurus) and dairy breeds were Gyr (Bos indicus) and Holstein (Bos taurus). According to our analysis, the IVEP in beef cattle had a great improvement throughout the last years, with a remarkable increase in numbers of pregnancies per OPU compared to late 90’s (averaging only 1 pregnancy per OPU in 1998 vs 2,4 in 2014). As for the distribution of oocytes retrieved, both Bos indicus beef (Nelore = 27.2) and dairy (Gyr = 23.8) breeds seem to yield greater average numbers of oocytes per OPU compared to Bos taurus (Senepol = 21.8; Holstein = 19.3). Despite these differences across genetic groups, outstanding donors can be found in all breeds and the number of oocytes retrieved per donor seems consistent across time. For both beef cattle breeds studied, it appears that number of oocytes retrieved at OPU had a negative but minor effect on both cleavage and blastocyst rates, especially for Senepol breed. Conversely, in dairy breeds the number of oocytes recovered per OPU had essentially no effect on cleavage rates, but we captured a trend for lower blastocyst rates with greater numbers of oocytes per OPU. For both, beef and dairy breeds the number of blastocyst per OPU was greater when higher number of oocytes were recovered per OPU, regardless of genetic group. Pregnancy rate following ET in Nelore breeds was lower in donors with greater amounts of oocytes retrieved per OPU. In contrast, in the Senepol breed and both dairy breeds (Gyr and Holstein) pregnancy rates after ET seems to increase when the number oocytes recovered per OPU increases. In addition, the semen utilized had a major impact of IVEP efficiency: top ranking sires yielded outstanding blastocyst rates, while poor performers produced very low blastocyst rates. The season of the year also had effect on IVEP, with Bos indicus breeds showing less variation in IVEP results throughout the year. In conclusion, despite the evolution of IVEP in the last two decades, the number of oocytes recovered per OPU had a minor effect both on blastocyst rate and pregnancy rates after ET. However as more oocytes are collected, the number of produced blastocysts improves. Thus, it seems important to identify donors with greater oocyte recovery-per-OPU potential, especially in cattle breeds yielding fewer oocytes per OPU, such as Holstein, to assure greater IVEP efficiency. It is also clear that cattle breed, semen used during IVEP and season of the year can potentially influence IVEP and field fertility results. A holistic approach controlling the quality of the performed OPU, consistency in lab routines, as well as selecting donors with high genetic value (through genomics) and greater oocyte population (through AMH assays or ultrasound) are highly advisable.

A method using artificial neural networks to morphologically assess mouse blastocyst quality.

BackgroundMorphologically classifying embryos is important for numerous laboratory techniques, which range from basic methods to methods for assisted reproduction. However, the standard method currently used for classification is subjective and depends on an embryologist’s prior training. Thus, our work was aimed at developing software to classify morphological quality for blastocysts based on digital images.MethodsThe developed methodology is suitable for the assistance of the embryologist on the task of analyzing blastocysts.The software uses artificial neural network techniques as a machine learning technique. These networks analyze both visual variables extracted from an image and biological features for an embryo.ResultsAfter the training process the final accuracy of the system using this method was 95%. To aid the end-users in operating this system, we developed a graphical user interface that can be used to produce a quality assessment based on a previously trained artificial neural network.ConclusionsThis process has a high potential for applicability because it can be adapted to additional species with greater economic appeal (human beings and cattle). Based on an objective assessment (without personal bias from the embryologist) and with high reproducibility between samples or different clinics and laboratories, this method will facilitate such classification in the future as an alternative practice for assessing embryo morphologies.

A Method Based on Artificial Intelligence To Fully Automatize The Evaluation of Bovine Blastocyst Images

Morphological analysis is the standard method of assessing embryo quality; however, its inherent subjectivity tends to generate discrepancies among evaluators. Using genetic algorithms and artificial neural networks (ANNs), we developed a new method for embryo analysis that is more robust and reliable than standard methods. Bovine blastocysts produced in vitro were classified as grade 1 (excellent or good), 2 (fair), or 3 (poor) by three experienced embryologists according to the International Embryo Technology Society (IETS) standard. The images (n = 482) were subjected to automatic feature extraction, and the results were used as input for a supervised learning process. One part of the dataset (15%) was used for a blind test posterior to the fitting, for which the system had an accuracy of 76.4%. Interestingly, when the same embryologists evaluated a sub-sample (10%) of the dataset, there was only 54.0% agreement with the standard (mode for grades). However, when using the ANN to assess this sub-sample, there was 87.5% agreement with the modal values obtained by the evaluators. The presented methodology is covered by National Institute of Industrial Property (INPI) and World Intellectual Property Organization (WIPO) patents and is currently undergoing a commercial evaluation of its feasibility.