Cytoplasmic granules in bovine oocytes do not affect embryonic or fetal development.

Oocyte cytoplasmic evaluation is based on homogeneity and granular appearance. Our study investigated if a granular cytoplasm, highly heterogeneous, would affect oocyte competence in bovine. In two experiments, bovine cumulus-oocyte complexes (COCs) with homogeneous cytoplasm (control, CC) and granulated cytoplasm (granular, GC) were selected from a regular pool of COCs. Experiment 1 was performed with slaughterhouse ovaries, and Experiment 2 was carried out in Girolando COCs obtained from ovum pick-up. Granular oocytes had higher caspase 3 levels (66.17 ± 11.61 vs 172.08 ± 16.95, P < 0.01) and similar GAP junction activity (5.64 ± 0.45 vs 6.29 ± 0.29). ZAR1 relative mRNA amount was lower in granular oocytes (178.27 ± 151.63 vs 0.89 ± 0.89, P = 0.01) and no effect was detected for MATER, PPP2R1A, ENY2, IGF2R, and BMP15 genes. Despite molecular differences, no detrimental effect was detected on oocyte competence in GC oocytes. Cleavage (Experiment 1: 59.52 ± 7.21% vs 59.79 ± 6.10% and Experiment 2: 68.88 ± 4.82 vs 74.41 ± 5.89%) and blastocyst (Experiment 1: 29.28 ± 4.14% vs 23.15 ± 2.96% and Experiment 2: 21.11 ± 3.28% vs 21.02 ± 6.08%) rates were similar between CC and GC (Experiments 1 and 2, respectively). Post-transfer embryo development revealed that pregnancy (CC: 24.27 ± 9.70% vs GC: 26.31 ± 7.23%) and calving (23.68% vs 33.33%) rates and fetal growth were not affected by the presence of cytoplasmic granules. Our results demonstrated that oocytes with granular cytoplasm present equivalent efficiency for IVF and calf production compared with homogenous cytoplasm oocytes. This could be observed through similar cleavage, blastocyst rates, and fetal growth development. In addition to differences in oocyte gene expression related to oocyte quality, it seems not to affect oocyte developmental competence.

Luteinizing hormone secretion, ovulatory capacity, and oocyte quality in peripubertal Gir heifers.

Induction of puberty in cattle breeds that attain puberty in later stages, such as Gir, allows the earlier beginning of reproductive life and it might increase oocyte quality. Here, the ovulatory capacity of prepuberal Gir heifers was studied and its relationship to follicular growth, luteinizing hormone (LH) secretion and oocyte quality was evaluated. Peripubertal Gir heifers were treated with a progesterone-based protocol and according to ovulatory response were separated into groups: not-ovulated (N-OV) and ovulated (OV). Serial blood samples were taken 24 h after estradiol treatment on day 12 to evaluate LH secretion. Cumulus-oocyte complexes (COCs) were collected using ovum pick-up and assessed for brilliant cresyl blue (BCB) staining rate, IVF-grade oocytes rate, and mean oocyte diameter, in comparison with cow oocytes. Gene expression of developmental competence markers (ZAR1, MATER, and IGF2R) was also analyzed. The largest follicle diameters were similar between N-OV and OV groups on the day of estradiol treatment (d12) and the next day and decreased (P = 0.04) in the N-OV group thereafter. LH pulse secretion was different between groups (N-OV = 3.61 ± 0.34 vs OV = 2.83 ± 0.21 ng/ ml; P = 0.04). COC assessment showed that the number of recovered oocytes, BCB+ rate, IVF-grade oocytes and oocyte size was similar (P > 0.05) among groups, resembling adult cow patterns. ZAR1, MATER and IGF2R gene expression in oocytes were also similar (P > 0.05) in N-OV and OV groups. In conclusion, our results demonstrate a lower LH secretion profile in peripubertal Gir heifers prone to ovulate after induction protocol, and that oocyte quality is not affected on a short-term basis by ovulation itself.

Modulation of lipid metabolism through multiple pathways during oocyte maturation and embryo culture in bovine.

Lipid accumulation occurs in cultured embryos and is associated with reduced cryotolerance. Here we report the use of a multiple pathway lipid modulator cocktail (l-carnitine, linoleic acid and forskolin) to improve cryosurvival. First, we stained oocytes and embryos with Oil Red to examine the time course of lipid accumulation during in vitro fertilization (IVF) and embryo culture. Then we evaluated the effects of the lipid modulators cocktail on lipid content, developmental rates and survival after vitrification. In our conditions, lipid accumulation was detected (P < 0.05) at the end of in vitro maturation (IVM) and after 4 days of embryo culture (D4-D5). In experiment 1, we used lipid modulator cocktail during IVM. Reduced (P < 0.05) lipid accumulation was detected in oocytes (Control: 49.9 ± 1.6, Lip. Mod. IVM: 45.0 ± 1.8) but no changes were present at blastocyst stage (Control: 62.4 ± 2.6, Lip. Mod. IVM: 66.8 ± 2.7). Treated oocytes presented decreased (P < 0.05) blastocyst rates and lower (P < 0.05) re-expansion after vitrification. In experiment 2, lipid modulators cocktail was used during embryo culture (from D4-D7 or D6-D7). Treatment had an effect on lipid metabolism, as lipid content was increased (P < 0.05) in D7 blastocysts in treated groups (Control: 52.7 ± 3.1a, D4: 65.9 ± 2.6b, D6: 78.1 ± 2.7b). However, no effect was present for cleavage, blastocyst and cryosurvival rates. No difference was detected in mean cell number comparing the three groups (Control: 78.9 ± 9.6, D4: 82.6 ± 16.5, D6: 68.3 ± 7.8), but apoptosis rate was increased (P < 0.05) in vitrified-warmed blastocysts from treated groups (Control: 14.77*, D4: 22.28, D6: 22.22). We concluded that the combined use of lipid modulators was efficient to promote changes in lipid content of oocytes and embryos in bovine, but those changes did not reflect positively on embryo development or cryosurvival.

Effect of Nellore (Bos indicus) donor age on in-vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of age of Nellore (Bos indicus) donors on the efficiency of in vitro embryo production (IVEP) and pregnancy rate. Thirty-six donors, including 11 female calves (13 ± 0.61 months), 17 prepubertal heifers (25 ± 0.78 months) and 8 cows (83 ± 28 months), were submitted to 3 procedures of ovum pickup (OPU) on random days of the estrous cycle at intervals of 21 days. Caspase-3 and IGFBP2 were quantified in oocytes and blastocysts for the evaluation of oocyte and embryo quality. The produced embryos were vitrified (n = 445) and transferred to synchronized recipients. Cows produced a larger number of follicles (cows: 54.5 ± 6.2; calves: 20.0 ± 0.57; prepubertal heifers: 20.8 ± 0.46), total oocytes (cows: 45.97 ± 7.22; calves: 28.93 ± 6.14; prepubertal heifers: 27.21 ± 4.94) and cleaved oocytes (cows: 21.14 ± 4.22; calves: 13.09 ± 3.72; prepubertal heifers: 12.4 ± 3.19). The cleavage rate was similar between age categories; however, cows tended (p < 0.07) to produce a larger number of blastocysts (9.74 ± 2.26) per OPU than calves (5.57 ± 1.99) and prepubertal heifers tended to have a higher blastocyst yield (35.4%) than calves (27.1%) (p < .07). The expression levels of IGFBP2 and caspase-3 were higher in oocytes derived from calves compared to the other two categories. The pregnancy rate was higher in calves (43.1%) and cows (40.4%) than in prepubertal heifers (33.8%) (p = .03). Despite the larger numbers of follicles and viable oocytes in cows, the blastocyst production results and pregnancy rates obtained indicate that the use of young females as oocyte donors in IVEP is feasible and may contribute to reduce the generation interval.

Citrullination regulates pluripotency and histone H1 binding to chromatin.

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

In-straw warming protocol improves survival of vitrified embryos and allows direct transfer in cattle.

Vitrification is a superior method for cryopreservation of IVF embryos, but due to complicated warming protocols, it is not commonly used for commercial bovine embryos routine. To overcome the need of laboratory embryo preparation during warming, we developed an in-straw warming protocol compatible with most vitrification devices for embryo transfer without sucrose gradient steps and embryo evaluation. Surprisingly, one of the tested protocols improved embryo survival (95.0%* vs 83.1% expansion rate and 74.2%* vs 51.5% hatching rate) compared to conventional in-plate warming. Embryo quality was also increased, taken by the higher total cell numbers (160.7 ± 8.6* vs 99.0 ± 7.9) and lower apoptosis index (4.9 ± 0.6* vs 11.5 ± 2.4) 48 h after warming. Pregnancy rates were similar between vitrified-warmed embryos and fresh embryos (40% vs 43%). Based on our results, we suggest in-straw warming should always be used for vitrified embryos due to beneficial effects. Direct transfer can be safely performed using this protocol.

Variants in the CYP19A1 gene can affect in vitro embryo production traits in cattle.

PURPOSE: This study aimed to associate DNA variants in promoter and exon flanking regions of the CYP19A1 gene with in vitro embryo production traits in cattle. The role of transcription factor binding sites created or lost due to DNA sequence variation and their possible effect on gene expression was also evaluated. METHODS: We collected date from Gyr dairy oocyte donor cows (Bos taurus indicus) at a commercial in vitro embryo production farm and analyzed the genotype-phenotype association with in vitro production traits. Using Sanger sequencing and web-based software, we assessed important CYP19A1 gene regions in oocyte donor cows and analyzed the effects of variants on the transcription factor binding sites. RESULTS: Two SNP mutations significantly associated with oocyte production, oocyte viability, embryo development, and pregnancies were found (T > C in the untranslated exon 1 flanking region ([GenBank: AJ250379.1]: rs718446508 T > C), and a T > C in the 5'-upstream region (1.1 promoter) ([GenBank: AC_000167.1]: rs41651668 T > C). Six new transcription factor binding sites were created. A binding site for transcription factors associated with the development of the placenta and embryo implantation was eliminated due to variations in the DNA sequence identified. CONCLUSIONS: The CYP19A1 gene contributes to genetic variation of in vitro embryo production traits in cattle. The complexity of the physiological phenomena related to estrogen pathways and their influence on reproduction in cattle allow indication of the mutations evaluated here as possible genetic markers for embryo production traits, which should be validated in the next steps of marker-assisted selection.

Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.

As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos.

Morphology of 16-cell embryo in bovine: Inside cells, compaction, fragmentation and effects of X-sorted semen.

In mouse embryos, inside cells are allocated in 16-cell embryos through a well-orchestrated sequence of events involving compaction and polarization. The emergence of inside cells is of great importance as itl later gives rise to the inner cell mass and epiblast. In this study, we report the sequence of critical events in embryology (compaction, inside cells allocation and fragmentation) in bovine 72 h.p.i. 9-16 cell embryos, while also investigating the effects of X-sorted semen on these events. We found a wide distribution of total cell numbers among embryos, attributed to an asynchronous cleavage pattern and blastomere death. Additionally, 13% of embryos displayed irregular shapes. The establishment of the inside cell compartment increased (p < 0.01) in embryos with more cells. However, only 53.8% of 16-cell embryos presented inside cells. Compaction was present in 32.4% embryos and was positively correlated (p = 0.03, OR 3.02) with the establishment of inside cells, occurring independently of cell number. Fragmentation was present in 36% embryos, being more frequent (p = 0.01) in embryos with lower cell numbers. A possible association between irregular shape and fragmentation was considered (p = 0.06). The use of X-sorted semen had no effect on most evaluated parameters. However, it did have a marked effect on cleavage rate (p < 0.01) and the arrest of 2- and 4- cell embryos. In conclusion, bovine embryos exhibit an asynchronous cleavage pattern, high levels of fragmentation, and demonstrate compaction and inside cell allocation later in development compared to mouse embryos. Semen X-sorting has major effects on cleavage and embryo arrest. Further studies are needed to elucidate the association between irregularly shaped embryos and fragmentation, as well as the effects of sex on inside cell allocation.

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts.

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development. We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the same was detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos.

Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA.

Embryo biopsies for genomic selection in tropical dairy cattle.

Genomic selection has transformed the livestock industry, enabling early-life selection of animals. Biopsy sampling of pre-implantation embryos has been described since 1968. However, it was only after 2010, with the advancement of molecular biology techniques such as whole genomic amplification and SNP Chips, that next-generation sequencing became commercially available for bovine embryos. It is now possible to make decisions about which embryos to transfer not only based on recipients' availability or embryo morphology but also on genomic estimates. This technology can be implemented for a wide spectrum of applications in livestock. In this review, we discuss the use of embryo biopsy for genomic selection and share our experience with Gir and Girolando Brazilian breeding programs, as well as future goals for implementing it in Brazilian bovine in vitro embryo production practices.

Outstanding Gir oocyte donors: How does individual factor affect in vitro embryo production efficiency?

The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n = 40), average (0.17-0.48, n = 168), and low (<0.17, n = 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p < 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p < 0.05) in high efficiency donors and a decrease (p < 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p = 0.017) by donor's efficiency (low: 0.60 ± 0.09 $$ 0.60\pm 0.09 $$ , average: 1.17 ± 0.07 $$ 1.17\pm 0.07 $$ , high: 2.57 ± 0.26 $$ 2.57\pm 0.26 $$ ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs.

Imputation accuracy for genomic selection using embryo biopsy samples in Gir.

The aim of this study was to establish a platform for genomic selection of in vitro-fertilized (IVF) Gir embryos. Multiple displacement amplification (MDA)-based embryo biopsy samples were genotyped, and genomic estimated breeding values (GEBV) for milk yield (305MY) were calculated. The concordance of GEBV and accuracy between embryo biopsies and the respective liveborn were assessed. Imputation was performed using two panels (Z-Chip and Bovine HD, Illumina) based on a database of 73,110 lactating cow's database and pedigree files from 147,131 animals. Biopsied embryos had similar pregnancy rates (39% vs 40%), pregnancy loss rates (18% vs 20%), and pregnancy length compared to Control embryos. After genotyping, low call rate means were detected for biopsy samples compared to the respective calf samples (0.80 vs 0.98). Imputation presented 0.83 (Z-Chip) and 0.96 (HD) accuracy (CORRanim). Embryo GEBV accuracy levels were higher in BovineHD imputation (0.82) than Z-Chip imputation (0.55) or no imputation (0.62), and the correlation between embryo/calf pairs' accuracy was 0.85 for BovineHD imputation, 0.11 for Z-Chip imputation, and 0.02 for no imputation. GEVB estimates correlation between embryo/calf pairs was 0.87 for BovineHD imputation, 0.80 for Z-Chip imputation, and 0.41 before imputation. The call rate of embryo samples did not affect the correlation between embryo/calf pairs for accuracy and GEBV before and after BovineHD imputation. Embryos obtained on the same farm presented GEBV 305MY differences of up to 800 kg, emphasizing the expected impact of embryo genomic selection for the Gir breed.

Reproductive development of dairy heifers in an integrated livestock-forest system during the summer.

This study aimed to assess the cortisol, body and reproductive development of prepubertal Holstein and Holstein-Gir ¾ heifers at 27 months of age maintained in an integrated livestock-forest (ILF) system for 60 summer days compared to the monoculture system in full sun (FS). The ILF system promoted changes (P=0.02) in the cortisol levels of Holstein-Gir ¾ heifers and did not affect weight gain in any of the breed groups studied. Animals in ILF system presented a lower (P=0.006) vulvar development for the rima height parameter and similar for the vulva width parameter. The ovarian follicular population of Holstein-Gir ¾ heifers in the ILF system was lower (P=0.004); however, for the Holstein heifers, no statistical difference was found, and numbers were higher (P=0.08) in the ILF system. None of the other ovarian parameters studied had any changes, and we also found important racial differences. Weight gain (P=0.003), vulvar development (P<0.001), and mean follicular size (P=0.008) were higher in the Holstein-Gir ¾ animals. Based on such results, the effect of the ILF system at 27 months of age on stress and reproductive parameters in the Holstein breed is considered positive, although negative effects have been detected on reproductive parameters in the Holstein-Gir ¾ breed.

Perspectives of gene editing for cattle farming in tropical and subtropical regions.

Cattle productivity in tropical and subtropical regions can be severely affected by the environment. Reproductive performance, milk and meat production are compromised by the heat stress imposed by the elevated temperature and humidity. The resulting low productivity contributes to reduce the farmer's income and to increase the methane emissions per unit of animal protein produced and the pressure on land usage. The introduction of highly productive European cattle breeds as well as crossbreeding with local breeds have been adopted as strategies to increase productivity but the positive effects have been limited by the low adaptation of European animals to hot climates and by the reduction of the heterosis effect in the following generations. Gene editing tools allow precise modifications in the animal genome and can be an ally to the cattle industry in tropical and subtropical regions. Alleles associated with production or heat tolerance can be shifted between breeds without the need of crossbreeding. Alongside assisted reproductive biotechnologies and genome selection, gene editing can accelerate the genetic gain of indigenous breeds such as zebu cattle. This review focuses on some of the potential applications of gene editing for cattle farming in tropical and subtropical regions, bringing aspects related to heat stress, milk yield, bull reproduction and methane emissions.

Administration of PGF2α at the moment of timed-AI using sex-sorted or conventional semen in suckled nelore cows with different intensity of estrus behavior.

The aim of this work was to evaluate pregnancy rates (PR) and ovulatory characteristics of Nelore cows receiving PGF2α at the time of AI (artificial insemination) in a progesterone(P4)/estradiol-based timed-AI protocol. We also compared the effects of PGF2α treatment at AI in cows inseminated with conventional or sex-sorted semen, with the absence or expression of estrus. In experiment 1, a total of 701 suckled, multiparous Nelore cows from two commercial beef farms were submitted to the same protocol. All cows received a 12.5 mg (IM) injection of dinoprost tromethamine (Dinoprost; Lutalyse®; PGF treatment) at days 7 and 9 of a timed-AI protocol. Following P4 device removal (day 11; D11), AI was performed 48 h later with conventional or sex-sorted semen from two different sires. At AI, cows received an additional dose of 12.5 mg (IM) of Dinoprost (PGF treatment) or 2.5 mL (IM) of sterile saline (Control). Estrus behavior was determined at D11 by activation of an estrus detection device (Estrotect®). The overall PR was 32.8% (n = 348) at Farm 1 and 42.3% (n = 353) at Farm 2 (P = 0.01). Despite PR differences between farms, the same factors affected PR at Farms 1 and 2. Body condition score (P = 0.02), estrus behavior (P = 0.01), and type of semen (P < 0.001) were factors affecting PR. Conventional semen had a 2.73x greater chance of successful pregnancy than sex-sorted semen. Cows displaying estrus had a 2.5x greater chance of successful pregnancy than cows that did not display estrus. No treatment effect (P = 0.67) was detected in cows receiving conventional or sex-sorted semen. However, there was a tendency (P = 0.08) for an interaction between treatment (PGF or control) and estrus behavior (estrus or no estrus). PGF2α at the time of AI tended to increase PR of cows that did not display estrus (P < 0.10). In experiment 2, 29 suckled, multiparous Nelore cows were compared using B-mode and Doppler ultrasongraphy to assess the ovulatory characteristics of cows receiving the 12.5 mg (IM) injection of Dinoprost (PGF treatment) or saline solution (control) at D11. No significant effects of PGF2α treatment at D11 were observed in follicular characteristics and/or ovulation performance. It was concluded that fertility of sex-sorted semen was lower than conventional semen, regardless of the PGF2α treatment. The 12.5 mg treatment of Dinoprost at AI did not accelerate the occurrence of ovulation; however, it was interesting to note that PGF2α treatment at timed-AI appeared to increase the fertility of cows that did not display estrus, independent of semen type.

The SPOM-adapted IVM system improves in vitro production of bovine embryos.

This study aimed to test the effects of an IVM SPOM adaptation (SPOM-adapted IVM) on the production, total number of cells (TNC), apoptosis, and cryotolerance (post-warming survival and cytoskeleton actin integrity) of bovine IVP embryos. Two experiments were conducted with two experimental groups based on IVM treatment: A control group (TCM 199 without FCS) and an SPOM-adapted group (TCM 199 with forskolin and IBMX in pre-IVM and IVM with cilostamide). The first experiment evaluated embryo in vitro production, TNC, and apoptosis rate on D9 of development. In the second experiment, embryos were vitrified/warmed at D7 (control fresh and vitrified; SPOM-adapted fresh and vitrified) and assessed regarding post-warming survival rates and cytoskeleton actin integrity. Statistical analysis was performed using GraphPad INSTAT software at a significance level of 5%. An increase (p < 0.05) in blastocyst production was observed in the SPOM-adapted group comparing to the control group. There was no difference (p > 0.05) in the TNC or apoptosis rate between the groups. Regarding cryopreservation, no differences were found (p > 0.05) in actin integrity or post-warming survival rates between the vitrified groups. In both vitrified groups, we observed a significantly lower uninjured pattern of actin integrity compared to the fresh groups (p < 0.05). We conclude that the SPOM-adapted IVM system is beneficial for blastocyst production and does not affect the quality and cryotolerance of the produced embryos.

Individual assessment of bovine embryo development using a homemade chamber reveals kinetic patterns of success and failure to reach blastocyst stage.

Most early developmental data are lost in bovine embryo culture systems. We developed and validated a method for culture of bovine embryos in groups that allow individual assessment. An autoclavable low-cost multiembryo chamber (MEC) was prepared using a polyester mesh fixed to a glass coverslip. Embryonic development was not affected by MEC. Compared to conventional bovine culture system (oil-covered drops, control), cleavage (C, 71.2 ± 7.8%; MEC, 74.3 ± 6.0%), blastocyst rate (C, 29.9 ± 4.4%; MEC, 28.3 ± 5.0%) and blastocyst cell number (C, 94.1 ± 9.7; MEC, 92.9 ± 5.3) were similar. Caspase 3 positive cell index in blastocysts was increased in MEC group, but apoptosis rate was below 5% (C, 2.9 ± 0.5; MEC, 4.6 ± 0.6). Using MEC, we performed a retrospective analysis for 'failure' and 'success' embryos, based on their ability to reach the blastocyst stage. We detected the majority of 'success' embryos displayed 8 cells at 48 h post-insemination (hpi) (48.7%), but blastocysts derived from this pattern presented lower cell numbers (91.3 ± 4.2 vs. 107.9 ± 4.9) and higher apoptosis index (6.2 ± 0.6 vs. 4.4 ± 0.5) than blastocysts from 4-cell embryos at 48 hpi. Most (72.0%) embryos that were at morula stage 120 hpi reached blastocyst stage at 168 hpi. Those blastocysts presented more number of cells than blastocysts derived from embryos exhibiting 16 cells at 120 hpi (108.6 ± 4.1 vs. 83.9 ± 4.8). Combination of embryo kinetics data at 48 and 120 hpi revealed high chances of blastocyst formation for patterns: 8 cells/morula, 4 cells/morula, 8 cells/16 cells and 4 cells/16 cells. Blastocysts formed from 4-cell/morula and 8-cell/morula patterns represented 69% of all 168 hpi blastocysts. Blastocysts derived from 4 cells/16 cells displayed decreased apoptosis (3.1 ± 0.6). Our results suggest that MEC can be used for bovine embryo culture without detrimental effects on development and can help to predict blastocyst formation and quality of in vitro fertilization (IVF) embryos. Abbreviations: BSA: bovine serum albumine; COC: cumulus-oocyte complex; FERT-TALP: Tyrode's albumin lactate pyruvate fertilization; FBS: fetal bovine serum; IVF: in vitro fertilization; MEC: multiembryo chamber; PBS: phosphate buffered saline; SOF-AA: synthetic oviductal fluid with amino acids medium; TCM: Tissue Culture Medium.

Follicular dynamics and in vivo embryo production in Santa Inês ewes treated with smaller doses of pFSH.

To evaluate follicular dynamics, there was assessment of superovulatory response and in vivo embryo production in ewes treated with relatively smaller doses of exogenous pFSH than typically used in combination with a dose of eCG at the beginning of the gonadotropin treatment period. Santa Inês ewes (n = 24) were randomly divided into three groups, based on mg dose of pFSH administered: G200 (n = 8), G133 (n = 8) and G100 (n = 8) in eight decreasing doses at 12 -h intervals. All ewes were treated with 300 IU of eCG concomitantly starting with first pFSH administration. Ovulatory follicular dynamics and follicular wall vascularization (FWV) were evaluated using a B-mode and color Doppler ultrasonic machine, respectively. Superovulatory response and embryo production were evaluated 6 days after estrous detection. In the G200 group, the preovulatory follicle size (PFS) were less (P <  0.05), ovulation time later (P <  0.05), and PFS rate greater (P <  0.05); while in the G100 group ovulation rate, and number and percentage of unfertilized eggs were greater (P <  0.05) than in the G133 group (P <  0.05). Number and percentage of viable embryos were greater in the G200 and G100 compared to G133 group (P <  0.05). The dose of 100 mg of FSH was as efficacious as the traditional dose of 200 mg, in combination with a dose of eCG, for superovulatory response and viable embryo production but there was a greater percentage of unfertilized eggs with this treatment.