In Vitro Embryo Production Research Articles

136 GameteGuard in maturation medium improves bovine embryo quality and quantity

The number of invitro-produced (IVP) bovine embryos generated in the United States increased 11% from 2017 to 2018, demonstrating a demand for new technology to rapidly and efficiently improve herd genetics. Unfortunately, embryo production on a per oocyte basis remains low, limiting the number of high-quality commercial embryos available for transfer or freezing. Reactive oxygen species (ROS) are a major contributor to low embryo production rates of invitro gametes and are especially problematic in maturation media. This is due to high numbers of mitochondria in the bovine oocyte producing ROS exacerbated by the invitro environment. GameteGuard® is an organic plant-based additive that provides protection against reactive oxygen species. The aims of this research were to evaluate the effects of GameteGuard addition to invitro bovine embryo production media to (1) mitigate the impact of ROS on oocyte maturation and competence, and (2) increase the production of high-quality embryos in our IVP system. Four rounds of invitro embryo production (Colorado State University protocol and media) with or without maturation medium supplemented with GameteGuard were performed (n=1175 total oocytes). Oocytes were aspirated from abattoir ovaries; those with homogeneous ooplasm and multiple layers of even cumulus cells were matured using control (n=585) or GameteGuard-supplemented (n=590) maturation medium. Oocytes were fertilized with frozen/thawed semen from proven IVF bulls (n=4) and then cultured in a two-step media system to Day 7. Blastocysts were evaluated on Day 7 according to IETS embryo grading standards. A subset of 80 blastocysts were fixed and stained after grading using an In Situ Cell Death Detection Kit (Fluorescein; Millipore Sigma) and Hoechst 33342 (Invitrogen) as apoptotic and counter-stains, respectively. Stained blastocysts were assessed by immunofluorescence microscopy to obtain the percentage of apoptotic cells and total cell count. Because multiple bulls were used, a linear mixed model was required for data analysis. Treatment was considered the fixed effect and bulls as a random effect to determine the impact of the treatment on stage and quality of blastocysts. Cleavage rates were not different (P>0.5) but GameteGuard supplementation of maturation medium resulted in a 22% increase in grade-1 embryos per total blastocysts produced (P<0.01; n=79/106 control vs. n=120/137 GameteGuard grade 1 blastocysts). Additionally, the percent of grade 2 and 3 embryos per total blastocysts decreased, (P<0.01; 27%, n=27/106 control and 11%, n=14/137 GameteGuard grade 2 and 3 blastocysts), suggesting that supplementation with GameteGuard promoted development of better-quality embryos overall. The percentage of apoptotic cells was also significantly higher in control blastocysts compared with treated blastocysts (P<0.02; 11.4%, n=38 control and 12.8%, n=42 GameteGuard). GameteGuard supplementation of the maturation medium significantly increased the production of high-quality IVP bovine embryos, thereby providing potential marketable gain for embryo producers.

46 Use of time-lapse imaging technology to assess relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos with their developmental competence invitro

The assessment of morphology and digital image opacity may provide valuable information on embryo viability because such traits are linked to embryonic gene expression, metabolism and ultrastructure. Time-lapse imaging has been used in research to monitor the dynamic nature of the developing pre-implantation embryo, which includes capturing alterations in various morphological parameters over time. The present study examined the effectiveness of time-lapse technology in assessing several morphometric and phototextural parameters for predicting the developmental potential of ovine embryos. The development of 37 long wool sheep embryos from IVF to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Image-Pro Plus software was then used to measure zona pellucida thickness, embryo diameter, cellular grey-scale pixel intensity and heterogeneity, and total area of the perivitelline space. A one-way analysis of variance (ANOVA) was done using SigmaPlot® 11.0 for all attributes at various time points during embryo development [i.e. presumptive zygote stage, t(0); first cleavage, t(2) or t(3); second cleavage, t(4) or t(6); and third cleavage, t(7) or t(8)]. Our results indicate that most parameters analysed did not differ among embryos varying in their developmental fate, with the exception of the perivitelline space area, which was greater (P<0.05) for non-dividing embryos than for future blastocysts at the presumptive zygote stage (4040±4137 vs. 857±642µm2, respectively; mean±s.d.). Consequently, the measurement of perivitelline space at t(0) could be used to predict developmental potential of invitro-produced ovine embryos, but further investigation is required.

55 Donor age has the least influence on recovery, quality, and in vitro developmental competence of ovum pickup–based Holstein Friesian oocytes under subtropical conditions

The use of oocytes obtained from younger donors for invitro fertilization followed by embryo transfer represents an opportunity to accelerate genetic gain by reducing generation interval. The present study was designed to evaluate the effect of age of donor on the follicular population, recovery, quality and invitro developmental competence of ovum pickup based Holstein Friesian oocytes under subtropical conditions. A total of eight (n=8) Holstein Friesian (with proper oestrus cyclicity) were selected for the study and divided into 2 groups based on animal age: (1) heifers (n=4), 1.5 to 2 years of age, and (2) adults (n=4), 5 to 6 years of age. The study was conducted near Lahore (31°33′ N, 74°19′ E), Punjab, Pakistan, from November 2019 to February 2020. The animals were wave synchronized using the physiological method of wave synchronization. After 4 days of second dominant follicle puncture, the first ovum pickup was carried out and a total of nine (n=9) OPU sessions were held for each group. The COCs from the follicles were aspirated using a transvaginal ultrasound–guided needle. Following searching and grading, COCs of grade A, B and C were processed for IVM in 100-µL droplets of BO-IVM under mineral oil at 37°C, 5% CO2, and 95% humidity for 24h. The frozen semen of a high-pedigree bull was thawed at 37°C and observed for post-thaw sperm motility. The semen samples of the same bull having motility >50% were processed using the sperm swim-up method throughout the study. The IVF was carried out by placing the COCs and required amount of sperm in 100-µL droplets of BO-IVF at similar conditions for a maximum of 18h. The presumptive zygotes were denuded by gentle pipetting and cultured for a period of 7 days after placing in 100-µL drops of BO-IVC at 37°C, 5% CO2, 5% O2, and maximum humidity. The presumptive zygotes were observed for cleavage rate and blastocyst rate on Days 2 and 7 following COCs-sperm co-incubation. Data on the follicular population, oocytes recovered, and viable oocytes were analysed by the PROC GLIMMIX of SAS (SAS Institute Inc.), and proportional data were analysed by the Chi-squared method using SAS 9.1. COCs of grade AB (35.2 vs. 25.4%) were higher (P>0.05) in the adult group than in the heifer group, respectively. Similarly, COCs with grade CD (57.5 vs. 71.9%) were lower (P<0.05) in the adult group compared with the heifer group, respectively. However, the total follicles (6.55±0.42 vs. 6.39±0.39), number of COCs recovered (3.33±0.32 vs. 3.17±0.41), viable oocytes (3.08±0.29 vs. 3.08±0.39), cleavage rate (60.3 vs. 68.7%), and blastocyst rate (38.7 vs. 48.8%) did not differ (P>0.05) between the groups. To conclude, donor age up to third lactation, under subtropical conditions, does not affect invitro embryo production in Holstein Friesian undergoing repeated OPU.

83 Response to follicle-stimulating hormone superstimulation in heifers with ovarian activity suppressed by active immunization against gonadotrophin-releasing hormone

In the present study, we evaluated the ovarian response to exogenous FSH stimulation in the absence of endogenous LH, using as experimental model heifers immunized against GnRH. Pubertal, cycling Nelore (Bos indicus) heifers were allocated into three experimental groups: (1) non-immunized, FSH stimulated (B−FSH+, n=5), (2) immunized, FSH stimulated (B+FSH+, n=5), and (3) immunized, nonstimulated (B+FSH−, n=5). Active immunization was obtained by 3 subcutaneous injections of 1.0mL anti-gonadotrophin-releasing hormone vaccine (Bopriva, Zoetis), given at 20-day intervals. Effective immunization was characterised by the absence of growing follicles >4mm or corpora lutea (CL) on the ovaries. Follicular wave emergence was synchronized in groups B+FSH+ and B+FSH− by follicle ablation, and in group B−FSH+ by using of a protocol consisting of an injection of 2mg of oestradiol benzoate and 0.5mg of sodium cloprostenol, and insertion of an intravaginal progesterone (P4) device (1g). Four days later (Day 0), groups B−FSH+ and B+FSH+ received 100mg of NIH-FSH-P1 (Folltropin-V, Vetoquinol), injected twice-a-day in 8 decreasing doses, and group B+FSH− received saline. Transvaginal ultrasonography (7.5MHz) was performed daily from Days 0 to 4 and the number and size of follicles were recorded. P4 devices of group B-FSH+ were removed at Day 3. All heifers underwent ovum pickup (OPU) at Day 4, and the cumulus–oocyte complexes (COC) recovered were graded for quality. Viable COC were used for invitro embryo production. The heifers were re-evaluated at Day 11 (7 days after OPU). The GLIMMIX procedure from SAS (SAS Institute Inc.) with repeated-measure statement was used to analyse the effects of group, day, and interactions; and the Chi-squared method was used to analyse binomial data. The results are shown as mean±s.e.m. A progressive increase in average follicle size was observed in groups B−FSH+ and B+FSH+ (P<0.0001), whereas no follicle growth was observed in group B+FSH− (P>0.05). Follicle growth rate was similar between groups B−FSH+ and B+FSH+, and both were greater than group B+FSH− (1.2±0.2 and 1.1±0.1 vs. 0.0±0.1 mm/d; P<0.0001). However, the smaller follicle size in group B+FSH+ at Day 0 resulted in smaller follicle size at Day 4, compared with group B−FSH+ (2.4±0.1 vs. 3.6±0.2 and 6.9±0.7 vs. 8.2±0.6mm, respectively; P<0.05). There was no (P>0.05) difference in the number of COC recovered among groups. The group B+FSH+ yielded fewer (P<0.01) COC of grades I and II and more (P<0.01) degenerated oocytes than groups B−FSH+ and B+FSH− (41.2% vs. 80.0% and 68.0%, and 34.0% vs. 19.8 and 7.0%, respectively). Nevertheless, blastocyst rates were similar (P>0.05) for B−FSH+, B+FSH+, and B+FSH− (57.1%, 45.9% and 44.2%, respectively). Residual follicles or luteal tissue were observed after OPU only in group B−FSH+, resulting in a significant difference in the size of ovaries between Days 0 and 11, compared with that of groups B+FSH+ and B+FSH− (3.7±1.4 vs. 0.2±0.2 and −0.2±0.2cm2, respectively; P<0.05). In summary, exogenous FSH supported follicle growth but did not improve oocyte quality in heifers immunized against GnRH. This research was funded by CAPES.

129 Polar body extrusion following exposure of pig oocytes to different invitro maturation media

The invitro maturation (IVM) rate of pig oocytes is a critical step for subsequent invitro embryo production. The objective of the present study was to compare different IVM media (NCSU 23, NCSU 37, TCM-199, pFF, BO-IVM commercial, and Bovine IVM) on polar body extrusion in pig oocytes following 44h of maturation duration. Ovaries from sows were collected from the local slaughterhouse and transported in warmed (38°C) saline to the laboratory, within 2h following slaughter. The slicing method was used to retrieve oocytes from the ovaries. Collected oocytes (n=353) were selected and washed three times in TCM-199+10% fetal bovine serum. The oocytes were then subjected to IVM as per treatment groups for 44h. Following maturation period, oocytes were removed from each maturation medium and denuded of granulosa cells by vortexing. The oocytes polar body extrusion was evaluated with the aid of OosightTM Imaging Sytem connected to an inverted research microscope. Treatment means were compared using Fisher’s protected least significant difference t-test. Polar body extrusion was 30.0, 18.5, 32.8, 22.1, 32.1, and 27.0% for BO IVM commercial, Bovine IVM, NCSU 23, NCSU 37, TCM-199, and pFF media, respectively (P>0.05). The NCSU 23 and TCM-199 had numerically higher percentages of oocytes with extruded polar body compared with other treatments groups (P>0.05). The lowest (18.5%) polar body extrusion was recorded in Bovine IVM medium. In conclusion, all the IVM media tested can be used during IVM of pig oocytes, with NCSU 23 and TCM-199 having numerically higher polar body extrusion percentages.

137 Comparison of two invitro maturation media on polar body extrusion of cattle oocytes

The invitro embryo production technique is one of the assisted reproduction technologies that has the potential in speeding up genetic improvement in cattle. The developmental competence of invitro-matured oocytes is influenced by several factors during invitro maturation (IVM), such as maturation environment, oocyte quality, type of media, and additives. The objective of the present study was to compare two IVM media (TCM-199 and BO-IVM) on cattle oocyte maturation rate invitro. Cattle ovaries were collected from local slaughterhouse and transported to the laboratory in a thermos flask containing 0.9% saline (Adcock Ingram Critical care) at 37°C. Oocytes were retrieved from the ovaries by the aspiration technique, and then matured invitro in 500µL of TCM-199 (supplemented with 10% fetal bovine serum, FSH, LH, and E2) or in 500µL of commercially available BO- IVM (Bioscience) medium, both covered with mineral oil for 22h at 38.5°C with 5% CO2 and 5% O2. After 22h of IVM, oocyte polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted microscope. The total number of oocytes matured in TCM-199 and BO-IVM media were 401 and 396, respectively. The experiment was replicated 19 times. Data were analysed using the GenStat® program (VSN International). Means of different treatments were separated using Fisher’s protected t-test least significant difference at 5% level of significance. No difference was recorded for polar body extrusion rates in TCM-199 (50.6±13.9) and BO-IVM (47.3±13.7) media. In conclusion, TCM-199 and BO-IVM media did not differ in terms of maturation rate; thus, both can be used for successful cattle oocyte IVM.

145 Comparison of single to multiple injections of follicle-stimulating hormone before ovum pickup in Holstein heifers: Oocyte recovery and embryo production

The demand for invitro-produced embryos from heifers with high genetic merit has increased over time. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and, consequently, embryo production. Multiple injections involve extra labour and stress for both personnel and cattle. The release of FSH can be prolonged by using 0.5% hyaluronan (HA) as a diluent, allowing a decrease in the number of injections. The objective of this study was to compare oocyte recovery and embryo production between single or multiple injections of FSH before OPU of Holstein heifers. During April and May 2020, 20 Holstein heifers (8 to 15 mo old) from Ruann Dairy (Riverdale, CA) were randomly divided and submitted to two different treatments (crossover design). Gonadotrophin-releasing hormone (GnRH; Fertagyl®, Merck, 129µg, IM) was given to synchronize the follicular wave emergence. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 100mg of FSH (Folltropin®, Vetoquinol) 36h after GnRH. The FSH consisted of a 2.5-mL injection of 400mg of FSH diluted in 10mL of 0.5% HA. OPU was performed 48 to 50h after FSH. Treatment 5×FSH consisted of 100mg of FSH divided into 5 equal IM injections (10-14h intervals) 36h after GnRH. The FSH consisted of 5×1-mL injections of 400mg of FSH in 20mL of saline. OPU was performed 18 to 20h after the last FSH injection. All donors received both treatments at a 14-day interval and the recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds. OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated oocytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarized in Table 1. There were no differences between the two treatments on the number of oocytes recovered per OPU (total and grade 1 and 2), percentage of grade 1 and 2 oocytes, cleavage rate, blastocyst rate and number of embryos (total and grade 1). Oocyte recovery and embryo production are highly donor dependent. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH can be efficiently used for IVP in Holstein heifers, without decreasing the number of oocytes recovered and/or embryos produced with the advantage of reducing labour and stress of handling cattle. Table 1. Number and quality of oocytes and cleavage and blastocyst rates Treatment OPU Oocytes per donor Grade 1 and 2 oocytes (%) Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU 1×FSH 20 17.0 45.7 84 39.8 6.2 3.8 5×FSH 20 19.9 46.5 82 35.6 6.3 4.0

Cellular and Molecular Events that Occur in the Oocyte during Prolonged Ovarian Storage in Sheep.

Simple SummaryEstablishing efficient in vitro embryo production (IVP) protocols in sheep usually requires prolonged transportation of post-mortem ovaries since adult animals are often slaughtered in abattoirs far from laboratories. In this study, different analyses were carried out to investigate important cellular and molecular aspects of hypoxic injury on excised ovaries over time in order to understand the factors jeopardizing the development of competent oocytes during prolonged transport times. We observed that, when ovaries were stored for more than 7 h, the quality and developmental potential of oocytes and cumulus cells were greatly reduced. Moreover, the use of medium TCM199 over saline solution also had deleterious effects. Beyond transport time, strategies aimed at reducing these damages may improve oocyte quality and developmental competence.For the past two decades, there has been a growing interest in the application of in vitro embryo production (IVP) in small ruminants such as sheep. To improve efficiency, a large number abattoir-derived ovaries must be used, and long distances from the laboratory are usually inevitable when adult animals are used. In that scenario, prolonged sheep ovary transportation may negatively affect oocyte developmental competence. Here, we evaluated the effect of ovary storage time (3, 5, 7, 9, 11 and 13 h) and the medium in which they were transported (TCM199 and saline solution) on oocyte quality. Thus, live/dead status, early apoptosis, DNA fragmentation, reduced glutathione (GSH) and reactive oxygen species (ROS) content, caspase-3 activity, mitochondrial membrane potential and distribution, and relative abundance of mRNA transcript levels were assessed in oocytes. After in vitro maturation (IVM), cumulus cell viability and quality, meiotic and fertilization competence, embryo rates and blastocyst quality were also evaluated. The results revealed that, after 7 h of storage, oocyte quality and developmental potential were significantly impaired since higher rates of dead oocytes and DNA fragmentation and lower rates of viable, matured and fertilized oocytes were observed. The percentage of cleavage, blastocyst rates and cumulus cell parameters (viability, active mitochondria and GSH/ROS ratio) were also decreased. Moreover, the preservation of ovaries in medium TCM199 had a detrimental effect on cumulus cells and oocyte competence. In conclusion, ovary transport times up to 5 h in saline solution are the most adequate storage conditions to maintain oocyte quality as well as developmental capacity in sheep. A strategy to rescue the poor developmental potential of stored oocytes will be necessary for successful production of high-quality embryos when longer ovarian preservation times are necessary.

Estimate of in vitro embryo production based on sperm subpopulations in Senepol bulls

In cattle, in vitro embryo production (IVEP) is an important reproductive biotechnology responsible for the rapid expansion of the Senepol breed in our country. This breed has shown important results when used in crossbreeding and estimate IVEP in Senepol based on seminal analysis would be valuable for the semen cryopreservation industry, research institutes and breeders. Combining the evaluation of sperm subpopulations with analysis of other sperm attributes may help to improve fertility predictions in cattle. Therefore, the objectives of the present study were to: 1) identify and characterize motile sperm subpopulations in cryopreserved Senepol semen following the washing process carried out before in vitro fertilization, and 2) to determine an model for estimate IVEP based on sperm subpopulations in conjunction with other sperm quality analyzes. Samples of 38 cryopreserved semen from 28 Senepol bulls, chosen based on retrospective data from 386 IVEP routines, underwent the semen washing and were evaluated by the computer-assisted sperm analysis system. Sperm morphology was evaluated by wet preparation technique, and plasma and acrosomal membranes integrity, mitochondrial potential, oxidative status and chromatin resistance were analyzed by flow cytometry. After multivariate analysis of principal components and grouping, three sperm subpopulations were identified: SBP1 (fast and progressive motility), SBP2 (hyperactivated motility) and SBP3 (slow non-progressive motility). After categorization of IVEP in high, medium and low embryo yield, logistic regression analysis was applied to associate the results of subpopulations and other sperm quality variables with IVEP. The SBP1 and SBP2 variables affected embryo production, and an IVEP estimation model was generated for Senepol bulls based on these two subpopulations: embryo yield = 0.1563 + 0.0328 (SBP1) + 0.0173 (SBP2). SBP1 and SBP2 represents the absolute value of the percentage of subpopulations in semen. If the calculated value (by this equation) is close to 1, the embryo yield will be low; if is close to 2, will be medium; if is close to 3, will be high. In conclusion, three subpopulations were found for Senepol semen and, despite all analyzed variables, only SBP1 and SBP2 were included in the model to estimate IVEP in this breed.

Upregulation of interferon-alpha gene in bovine embryos produced in vitro in response to experimental infection with noncytophatic bovine-viral-diarrhea virus.

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20–25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.

Ethanolic Extract of Dried Leaves from the Cerrado Biome Increases the Cryotolerance of Bovine Embryos Produced In Vitro.

In vitro embryo production (IVP) induces excessive production of reactive oxygen species (ROS), which affects blastocyst quality. Therefore, the supplementation of culture media with antioxidants is an alternative to overcome oxidative stress damage. However, there is a growing demand for the use of antioxidant compounds that are more natural and less toxic in cell cultures. The present study is aimed at evaluating the effect of ethanolic extracts from cerrado leaves on IVP. First, the antioxidant capacity and the amount of phenolic compounds of the leaves were evaluated. Then, the best ethanolic extract concentration composed of cagaita (Eugenia dysenterica) and murici (Byrsonima crassifolia) to be used during the in vitro culture of in vitro-produced embryos was determined. Afterward, we evaluated the influence of the extract of both plants on ROS and glutathione (GSH) production, while also evaluating the apoptosis and ROS metabolism gene expression. In a subsequent step, the effect of the ethanolic extracts of dried cagaita and murici leaves during embryonic cultivation on the cryotolerance of expanded blastocysts was studied. The results showed a significant reduction in the proportion of apoptotic cells from embryos cultivated with 0.01 mg/mL of the cagaita ethanolic extract, besides inducing an increase in the GPX4 and PRDX3 transcription levels. The murici ethanolic extract induced an increase in the transcription abundance of these genes but did not reduce the proportion of apoptotic cells. In addition, expanded blastocysts cultivated with extracts at a concentration of 0.01 mg/mL and cryopreserved had higher hatching rates and lower degeneration rates when compared to the frozen group previously supplemented with the extracts. Moreover, the apoptosis rate of embryos cultured for 12 h after cryopreservation was lower in groups previously exposed to extracts during in vitro cultivation. Such extracts may be used as alternatives to increase the cryotolerance of in vitro-produced embryos.

Bovine in vitro embryo production using media prepared with Milli-Q® Water or nanowater

The aim of this study was to compare the efficiency of in vitro embryo production (IVP) following the collection of bovine ovaries and 22-h in vitro maturation (IVM) of oocytes in media prepared with Milli-Q® Water (n = 509 oocytes) or nanowater (NW; n = 304 oocytes). The mean cleavage (63.8 ± 4.6 % vs. 63.6 ± 6.1 %, respectively; mean ± SEM) and blastocyst formation rate (16.3 ± 3.4 % vs. 16.7 ± 6.7 % of presumptive zygotes, respectively) did not vary (P > 0.05; Student t-test) between the two types of media diluents. NW is a safe substitute for Milli-Q® Water for IVM of bovine oocytes.

Production of embryos and a live offspring using post mortem reproductive material from bison (Bison bison bison) originating in Yellowstone National Park, USA

Bison from Yellowstone National Park (YNP) have an important genetic history. As one of the few wild herds of bison with no evidence of cattle DNA introgression and a large enough population to maintain genetic diversity, they are considered a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is to remove bison from the population during the winter migration out of the park. This interagency management cull provides an opportunity to collect a large number of oocytes from a wild bison population for genetic banking and research purposes. During the winters of 2014–2018, which is the nonbreeding season for bison, oocytes were collected post mortem and used to determine the effects of donor reproductive maturity and pregnancy status on oocyte quality and in vitro fertilization (IVF) outcomes, and to demonstrate the feasibility of producing healthy offspring. Cumulus oocyte complexes (COCs) were placed into an in vitro embryo production (IVP) system, and on days 7, 7.5, and 8 of in vitro culture (Day 0 = day of in vitro fertilization) embryos were assessed for developmental stage and quality prior to vitrification. Embryos were then stored in liquid nitrogen until the breeding season when a subset were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. There were no significant differences in the ability of recovered COCs to support blastocyst development based on female reproductive maturity or pregnancy status (juvenile 79/959 (8.2%) vs sexually mature 547/6544 (8.4%); non-pregnant 188/2302 (8.2%) vs pregnant 556/6122 (9.1%)). Following the transfer of 15 embryos to 10 recipients, one healthy female calf was born. This work demonstrates that live offspring can be generated from COCs collected from YNP bison post mortem in the non-breeding season, and that gamete recovery can be a valuable tool for conservation of valuable genetics for this species while mitigating diseases like brucellosis.

Multistep analysis reveals the relationship between blood indices at the time of ovum pick-up and in vitro embryo production in heifers

The inflammatory factors of complete blood count (CBC) are associated with a decrease in the in vitro embryo production (IVP) outcome in women. The relation between the blood indices and in vitro fertilization (IVF) outcomes in bovines remains to be elucidated. Using ovum pick-up (OPU), oocytes were retrieved from heifers (n = 60) and inseminated separately with sperm. The blastocyst formation was recorded on day 7 after insemination for each animal and the blood indices were evaluated at the time of OPU. Then, heifers were classified on the basis of (1) blastocyst formation, cleaved vs. failed, or (2) inflammation, low-grade inflammation (lymphocyte counts > 5.6 × 109/L) vs. no inflammation (lymphocyte counts < 5.6 × 109/L). Oocytes derived from heifers with higher lymphocytes, red blood cells (RBC), platelets, hematocrit, red cell distribution width (RDW-SD) and plateletcrit values and lower monocytes, eosinophils, mean corpuscular hemoglobin (MCH) and MCH concentration (MCHC) successfully developed to the blastocyst stage. Heifers with low-grade inflammation numerically had a higher percentage of blastocyst formation than normal heifers. The principle component analysis (PCA) showed that blastocyst formation had the strongest positive association with RDW-cv and RDW-SD, while having a strong negative association with mean corpuscular volume (MCV), hemoglobin, MCHC and MCH. The PCA determined that the number of grade A COCs and the percentage of COCs reached the cleavage stage had a negative association with white blood cells (WBC), lymphocytes, basophils and monocytes, and a positive correlation with platelet to lymphocyte ratio, platelet distribution width (PDW) and plateletcrit. Network mapping detected close similarities between BFR and RDW-SD, MPV, and lymphocytes. The area under the receiver operating characteristic curve (AUC) identified that, eosinophils (AUC 0.80), RDW-SD (AUC 0.76), monocytes (AUC 0.76) and lymphocytes (AUC 0.76) had a good predictive ability to detect heifers with high OPU-IVP outcome (≥60%). In conclusion, these findings suggest that CBC indices at the time of OPU were associated with the IVF outcome and may be incorporated into protocols for the identification of heifers with high potential for blastocyst formation.

Specific imprinted genes demethylation in association with oocyte donor’s age and culture conditions in bovine embryos assessed at day 7 and 12 post insemination

The production of bovine embryos through in vitro maturation and fertilization is an important tool of the genomic revolution in dairy cattle. Gene expression analysis of these embryos revealed differences according to the culture conditions or oocyte donor’s pubertal status compared to in vivo derived embryos. We hypothesized that some of the methylation patterns in oocytes are acquired in the last step of folliculogenesis and could be influenced by the environment created in the follicles containing these oocytes. These altered patterns may not be erased during the first week of embryonic development in culture or may be sensitive to the conditions during that time. To quantify the changes related to culture conditions, an in vivo control group consisting of embryos (Day 12 post fertilization for all groups) obtained from superovulated and artificially inseminated cows was compared to in vitro produced (IVP) embryos cultured with or without Fetal Bovine Serum (FBS). To measure the effect of the oocytes donor’s age, we also compared a fourth group consisting of IVP embryos produced with oocytes collected following ovarian stimulation of pre-pubertal animals. Embryonic disk and trophoblast cells were processed separately and the methylation status of ten imprinted genes (H19, MEST, KCNQ1, SNRPN, PEG3, NNAT, GNASXL, IGF2R, PEG10, and PLAGL1) was assessed by pyrosequencing. Next, ten Day 7 blastocysts were produced following the same methodology as for the D12 embryos (four groups) to observe the most interesting genes (KCNQ1, SNRPN, IGF2R and PLAGL1) at an earlier developmental stage. For all samples, we observed overall lower methylation levels and greater variability in the three in vitro groups compared to the in vivo group. The individual embryo analysis indicated that some embryos were deviant from the others and some were not affected. We concluded that IGF2R, SNRPN, and PEG10 were particularly sensitive to culture conditions and the presence of FBS, while KCNQ1 and PLAGL1 were more affected in embryos derived from pre-pubertal donors. This work provides markers at the single imprinted control region (ICR) resolution to assess the culture environment required to minimize epigenetic perturbations in bovine embryos generated by assisted reproduction techniques, thus laying the groundwork for a better comprehension of the complex interplay between in vitro conditions and imprinted genes.

Synchronization of stage of follicle development before OPU improves embryo production in cows with large antral follicle counts

In the present study, there was an evaluation of in vitro embryo production (IVEP) in Bos indicus donor cows with small or large antral follicle counts (AFCs) when there was synchronization of follicular dynamics among cows before ovum pick-up (OPU). Donor cows classified as having small or large AFC were submitted to OPU/IVEP program (Experiment-I) or had follicular-stage synchronization imposed before OPU/IVEP (Experiment-II). In Experiment-I, the cows with a large AFC had a greater (P < 0.01) mean of embryos developing to the blastocyst stage compared to those with a small AFC. In Experiment-II, percentage of viable oocytes/OPU were not affected (P = 0.33) by synchronization of follicular dynamics, but the AFC had an effect (P < 0.0001). There was an interaction (P = 0.01) indicating the larger AFC, with or without imposing of a synchronization treatment regimen, resulted in the most desirable outcome. The number of embryos was affected (P < 0.001) by follicular-stage synchronization and AFC, with there being an interaction (P = 0.002) with the most desirable results for the large AFC-synchronized group. Number of pregnancies was greater (P ≤ 0.02) for recipient females with embryos from synchronized donors and with a large AFC. There was an interaction (P = 0.03) with there being a greater pregnancy percentage for cows with synchronized follicular stages and the large AFC. Bos indicus donor with a large AFC when associated with the synchronization of stage of follicular dynamics pre-OPU results in improvement of the efficacy of IVEP.

Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season.

Simple SummaryThe domestic cat is commonly used as a model for the development of assisted reproductive technologies, including in vitro embryo production (IVEP) in felid species. Seasonal reproduction is a feature of domestic cats as well as of several species of wild feline. Likewise, the number and the quality of blastocysts produced in in vitro systems is linked to season. Maintaining stable in vitro embryo production throughout the year is crucial not only for research purposes but also for programs aimed at protecting endangered felines. We assess whether using Brilliant Cresyl Blue (BCB) selection in addition to the classical morphological selection could improve the IVEP outcomes during non-breeding season. Blastocyst yield and quality of the embryos (hatching rate and blastocyst cell numbers) were higher after IVM/IVF in oocytes defined as BCB+ (colored cytoplasm) based on the BCB test than in oocytes only morphologically selected. Furthermore, no adverse effects on bioenergetic/oxidative status were observed in oocytes subjected to BCB staining. In conclusion, BCB test implementation in IVEP programs might ensure a steady output of domestic cat blastocysts throughout the year.In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB− (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB− and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season.

Effect of estrus expression or treatment with GnRH on pregnancies per embryo transfer and pregnancy losses in beef recipients synchronized with estradiol/progesterone-based protocols

Two experiments were designed to determine the effect of expression of estrus or GnRH treatment on pregnancies per embryo transfer (P/ET) and pregnancy losses in beef recipients that were synchronized with estradiol/progesterone based protocols for fixed-time embryo transfer (FTET). Experiment 1 evaluated the effect of expression of estrus and GnRH treatment in the absence of estrus on P/ET. Beef cows (n = 729) were treated with 2 mg estradiol benzoate (EB) and an intravaginal device containing 0.5 g of progesterone. Devices were removed 8 d later and all cows received prostaglandin F2α (PGF2α), 400 IU eCG, and 0.5 mg estradiol cypionate (ECP) at that time. Expression of estrus was determined at 48 and 56 h after device removal using tail-paint and cows that did not show positive signs of estrus by 48 h received GnRH or no treatment at random. The overall estrus rate was 76.0% (554/729); 68.0% had positive signs of estrus by 48 h after progesterone device removal and 28.0% of those not in estrus by 48 h showed estrus by 56 h. The proportion of recipients receiving in vivo-derived (IVD) or in vitro-produced (IVP) embryos and P/ET were greater in recipients that showed estrus by 48 and 56 h (94.0% and 48.4%, respectively) than in those that did not show estrus 41.0% and 29.0%, respectively; P < 0.01). However, GnRH treatment of recipients not showing estrus by 48 h did not improve P/ET. Experiment 2 evaluated the effect of expression of estrus on P/ET and pregnancy loses up to parturition in recipients synchronized with two estradiol-based protocols. Beef cows (n = 403) were divided at random to receive the same synchronization protocol as in Experiment 1 (ECP) or a J-Synch protocol (device removal on day 6 and without using estradiol cypionate to induce ovulation). In this experiment, pregnancy was determined at 30 and 60 d by ultrasonography, and all pregnant recipients were followed until parturition to determine pregnancy losses during gestation. Although the number of recipients receiving IVP embryos was greater in the ECP group (90.5% vs. 83.5%; P = 0.03), P/ET did not differ (ECP: 37.0% and J-Synch: 39.0%; P = 0.43). Overall, 88.0% (357/407) of the recipients synchronized showed estrus and a greater P/ET (P = 0.05) was found in the recipients that showed estrus (39.0%) vs. those that did not show estrus (26.0%), regardless of treatment group. Pregnancy losses were lower (P = 0.004) and the calving rate was higher (P = 0.01) in recipients that showed estrus (25.0% and 29.3%, respectively) than in those that did not (88.8% and 2.9%, respectively). In summary, expression of estrus was associated with a greater P/ET in recipients treated with two different estradiol/P4-based synchronization protocols. The expression of estrus was associated with a greater proportion of recipients receiving embryos, P/ET and calving rate. Treatment with GnRH did not improve P/ET in the recipients that did not show estrus, questioning the its use in recipients synchronized with estradiol/progesterone based FTET protocols.

Reproductive Outcomes and Endocrine Profile in Artificially Inseminated versus Embryo Transferred Cows.

The increasing use of in vitro embryo production (IVP) followed by embryo transfer (ET), alongside with cryopreservation of embryos, has risen concerns regarding the possible altered pregnancy rates, calving or even neonatal mortality. One of the hypotheses for these alterations is the current culture conditions of the IVP. In an attempt to better mimic the physiological milieu, embryos were produced with female reproductive fluids (RF) as supplements to culture medium, and another group of embryos were supplemented with bovine serum albumin (BSA) as in vitro control. Embryos were cryopreserved and transferred while, in parallel, an in vivo control (artificial insemination, AI) with the same bull used for IVP was included. An overview on pregnancy rates, recipients' hormonal levels, parturition, and resulting calves were recorded. Results show much similarity between groups in terms of pregnancy rates, gestation length and calves' weight. Nonetheless, several differences on hormonal levels were noted between recipients carrying AI embryos especially when compared to BSA. Some calving issues and neonatal mortality were observed in both IVP groups. In conclusion, most of the parameters studied were similar between both types of IVP derived embryos and the in vivo-derived embryos, suggesting that the IVP technology used was efficient enough for the safe production of calves.

Seasonality and photoperiod influence in vitro production of buffalo embryos.

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January-March, April-June, July-September and October-December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April-June) and maximum during fourth quarter (October-December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31±0.10 vs. 3.65±0.27) and maturation rate (33.32±2.1 vs. 63.15±7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.