In Vitro Embryo Production Research Articles

Nobiletin as a novel agent to enhance porcine in vitro embryo development and quality

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3′,4′ hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 μM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.

Oviduct and endometrial epithelium improve in vitro produced bovine embryo developmental kinetics.

Standard in vitro produced (IVP) bovine embryo culture media limit embryonic development. Culturing IVP bovine embryos in standard IVP bovine embryo culture media conditioned with oviduct and/or endometrial cells improves blastocyst formation and reduces the time to formation. In vitro embryo production in cattle greatly impacts blastomere biochemistry, embryo rate of development and pre- and post-transfer survival. In vivo, the bovine embryo migrates through the oviduct isthmus before entering the uterus on approximately day 4 of development where it remains unattached within the uterine lumen until day 20 of gestation. During this time, the embryo is sequentially exposed to oviduct followed by endometrial secretions that support embryonic development. Considering this, we tested the effect of culturing in vitro produced (IVP) bovine embryos sequentially in oviduct epithelial- (OEp; days 1-3) followed by endometrial epithelial- (EEp) or EEp and fibroblast cell (EEp/F; days 4-8)-conditioned media on embryonic development using a time-lapse monitoring system. Compared to control, culturing IVP embryos in EEp- or EEp/F-conditioned media without prior culture in OEp-conditioned media increased blastocyst formation (P < 0.05) and reduced the time to blastocyst formation (P < 0.05). Culturing IVP bovine embryos in OEp-conditioned media followed by EEp- or EEp/F-conditioned media, however, had the greatest impact on embryo developmental kinetics and increased morula and blastocyst formation (P < 0.05) and reduced time to formation (P < 0.05). Day 8 blastocyst cell numbers, diameter and quality were not significantly different, although, blastocyst quality scores were less (indicative of better quality) for all cell-conditioned media compared to control. In conclusion, IVP bovine embryo development may be improved using a sequential embryo culture system involving bovine oviduct followed by endometrial cell-conditioned media.

Genetic parameters for bull effects on in vitro embryo production (IVP) and relationship between semen quality traits and IVP performance

In vitro production of embryos (IVP) is increasingly applied in dairy cattle breeding and promises widespread use of females of superior genetic merits. One of the current challenges with implementation of IVP is the variability in blastocyst rates. Several factors contribute to these variabilities, among which is known to be the bull used for oocytes fertilization. The extent of genetic control of bulls’ effect on IVP performances is yet to be investigated. This study estimates genetic parameters for bull effects on IVP performance traits including blastocyst rate, hatching rate and an index trait combining Blastocyst rate, Kinetic Score, and Morphology score (BL_M_K). The IVP experiments were performed using oocytes aspirated from slaughterhouse ovaries from Holstein cows, fertilized with semen from 123 Holstein bulls. A total of 77 in vitro fertilization (IVF) experiments with 163 records (different IVF groups) were available for the analysis. The results indicate low to moderate heritability and moderate to high repeatability estimates for bull effects on IVP performance traits. Our study also showed that some semen quality traits had significant effects on IVP performance. This included strong genetic correlations between pre-cryopreservation sperm viability and blastocyst rate as well as BL_M_K score at days 7 and 8. Despite the generally weak bull effect correlations and the high standard errors of the estimates, our results provide initial evidence of a measurable genetic component in the bull's impact on IVP performance traits. However, the high standard errors underscore the need for further studies with a larger sample size.

Factors influencing ovum pick-up technique results in cattle

The Ovum Pick-Up (OPU) technique, which is used in in vitro embryo production (IVP) to retrieve immature oocytes from live donor animals, is one of the most important biotechnological procedures used in cattle breeding. The most important advantage of this technology is that it allows for the reproducible retrieval of immature oocytes from living donor animals. It is particularly useful in dairy cattle breeding to address infertility issues and boost the production of superior animals with high genetic value. The OPU technique offers several advantages, including its applicability to cows ranging from six-month-old calves to the first three months of pregnancy, its effectiveness in animals with genital tract infection or acyclic cattle, and its ability to yield a higher number of embryos within the same period compared to the Multiple Ovulation and Embryo Transfer (MOET) technique. Understanding and improving the technical and biological factors influencing the OPU procedure is necessary to increase and optimize donor animal use in IVP and the number of quality oocytes obtained. This review aims to examine the specifics of the OPU approach and the factors influencing its performance in light of contemporary literature, as well as to propose fresh ideas to researchers.

Unveiling maturation biomarker dynamics: A comprehensive review on In Vitro embryo production in cattle

In vitro embryo production (IVEP) is a revolutionary reproductive biotechnology in bovine species which enables rapid genetic gain and conservation of genetic resources. IVEP has numerous applications in field of animal breeding, conservation and research, making it a powerful tool for enhancing reproductive efficiency and genetic progress in livestock industry. The process involves manipulating gametes, including oocytes and sperm, in a laboratory setting. Success of IVEP largely relies on optimization of each step, including gamete quality, sperm preparation techniques, fertilization conditions and culture medium. Advancements in culture media formulations by additions of growth factors, antioxidants along with co-culture systems and advanced imaging techniques have improved efficiency and success rates. In this article, the authors have reviewed the importance as well as biomarkers associated with in vitro maturation for a more successful outcome of IVEP in bovines.

An Overview of Reactive Oxygen Species Damage Occurring during In Vitro Bovine Oocyte and Embryo Development and the Efficacy of Antioxidant Use to Limit These Adverse Effects.

The in vitro production (IVP) of bovine embryos has gained popularity worldwide and in recent years and its use for producing embryos from genetically elite heifers and cows has surpassed the use of conventional superovulation-based embryo production schemes. There are, however, several issues with the IVP of embryos that remain unresolved. One limitation of special concern is the low efficiency of the IVP of embryos. Exposure to reactive oxygen species (ROS) is one reason why the production of embryos with IVP is diminished. These highly reactive molecules are generated in small amounts through normal cellular metabolism, but their abundances increase in embryo culture because of oocyte and embryo exposure to temperature fluctuations, light exposure, pH changes, atmospheric oxygen tension, suboptimal culture media formulations, and cryopreservation. When uncontrolled, ROS produce detrimental effects on the structure and function of genomic and mitochondrial DNA, alter DNA methylation, increase lipid membrane damage, and modify protein activity. Several intrinsic enzymatic pathways control ROS abundance and damage, and antioxidants react with and reduce the reactive potential of ROS. This review will focus on exploring the efficiency of supplementing several of these antioxidant molecules on oocyte maturation, sperm viability, fertilization, and embryo culture.

Impact of negative energy balance and postpartum diseases during the transition period on oocyte quality and embryonic development in dairy cows.

Transition period is a critical time for dairy cows because a large proportion of clinical and subclinical diseases are observed in the first month after parturition. Occurrence of negative energy balance is associated with depressed immunity and these conditions can affect oocyte quality and further embryonic development. The aim of this study was to assess the effects of negative energy balance-associated disorders on in vitro embryo production (IVP) in dairy cattle. We hypothesized that subclinical metabolic and/or inflammatory disorders have a negative effect on oocyte developmental competence and morphokinetic parameters of the resulting embryos. The study was conducted on 30 lactating Holstein-Friesian cows which were assigned into four groups: healthy (HEAL, n = 6), metabolic disease (META, n = 8), inflammatory disease (INFL, n = 8), or combined metabolic and inflammatory disease (COMB, n = 8). Ovum pick-up (OPU) was performed twice weekly on all cows over a period of four weeks (n = 8 OPU sessions/cow) starting on the fifth week postpartum, and the collected oocytes were subjected to routine IVP. Donor's health status did not affect the number of oocytes/OPU or the recovery rate (p > 0.05). The number of quality 1 oocytes collected from INFL and COMB cows was lower compared to HEAL cows (p < 0.05). Also, the percentage of quality 1 embryos was reduced in META and COMB compared to HEAL cows (p < 0.05). Cleavage, blastocyst and hatching rates were similar among groups (p > 0.05). Presence of disease did not affect the time required by zygotes to reach specific developmental stages, as recorded by means of time-lapse monitoring. Nevertheless, there was a higher probability of direct cleavage after IVF in oocytes of COMB cows compared to those of HEAL cows (p < 0.05). In conclusion, oocytes and embryos derived from dairy cows diagnosed with subclinical metabolic and/or inflammatory diseases during the transition period showed reduced quality but similar developmental potential and morphokinetics when compared to healthy cows. These results shed light on the consequences of subclinical disease on embryonic development in dairy cows which might be important for embryo transfer programs.

Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Parâmetros genéticos da produção in vitro de embriões das raças Nelore e Senepol

Abstract This study aimed to estimate genetic parameters for traits of in vitro embryo production (IVEP) of Nellore and Senepol cattle. Data from 1,247 rounds of in vitro fertilization (1,029 Nellore, 218 Senepol) were used, totaling 148,311 oocytes (116,972 Nellore, 31,339 Senepol), 47,301 embryos (38,722 Nellore, 8,579 Senepol), and 6,323 pregnancies (5,534 Nellore, 789 Senepol). The variables percentage of viable oocytes (Pooc), percentage of cleaved embryos (Pcleav), percentage of produced embryos (Pemb), percentage of pregnancy (Ppreg) per round per bull, mean number of viable oocytes per donor (MOD), mean number of embryos produced per donor (MED), and mean number of pregnancies per donor (MPD) were analyzed from data provided by a partner company between the years 2019 and 2022. The SAS program was used to analyze fixed effects and Pearson linear correlation. The components of variance for heritabilities were calculated using the MTDFREML program. MOD, MED, and MPD values of 29.94, 10.01, and 2.53 were obtained for Nellore and 30.12, 8.17, and 2.34 for Senepol, respectively. In general, Nellore provided better embryo production compared to Senepol. Heritability estimates showed low to medium magnitude, with values Nellore and Senepol of 0.16 and 0.04 (Pcleav), 0.14 and 0.08 (Pemb), 0.02 and 0.15 (Ppreg), 0.07 and 0.02 (MED), and 0.05 and 0.00 (MPD), respectively. However, it indicates the presence of genetic variability and the possibility of selection. Therefore, there is genetic variability for IVEP traits in both breeds, indicating that they can be used as selection criteria because they are heritable and that Nellore presents better performance for IVEP traits compared to Senepol.

3D culture applied to reproduction in females: possibilities and perspectives.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

Recent advancements to increase success in assisted reproductive technologies in cattle.

Assisted reproductive technologies (ART) are fundamental for cattle breeding and sustainable food production. Together with genomic selection, these technologies contribute to reducing the generation interval and accelerating genetic progress. In this paper, we discuss advancements in technologies used in the fertility evaluation of breeding animals, and the collection, processing, and preservation of the gametes. It is of utmost importance for the breeding industry to select dams and sires of the next generation as young as possible, as is the efficient and timely collection of gametes. There is a need for reliable and easily applicable methods to evaluate sexual maturity and fertility. Although gametes processing and preservation have been improved in recent decades, challenges are still encountered. The targeted use of sexed semen and beef semen has obliterated the production of surplus replacement heifers and bull calves from dairy breeds, markedly improving animal welfare and ethical considerations in production practices. Parallel with new technologies, many well-established technologies remain relevant, although with evolving applications. In vitro production (IVP) has become the predominant method of embryo production. Although fundamental improvements in IVP procedures have been established, the quality of IVP embryos remains inferior to their in vivo counterparts. Improvements to facilitate oocyte maturation and development of new culture systems, e.g. microfluidics, are presented in this paper. New non-invasive and objective tools are needed to select embryos for transfer. Cryopreservation of semen and embryos plays a pivotal role in the distribution of genetics, and we discuss the challenges and opportunities in this field. Finally, machine learning (ML) is gaining ground in agriculture and ART. This paper delves into the utilization of emerging technologies in ART, along with the current status, key challenges, and future prospects of ML in both research and practical applications within ART.

Evolution over the last 40 years of the assisted reproductive technologies in cattle - the Brazilian perspective for embryo transfer and resynchronization programs (part II).

The productivity of the beef and dairy industries depends directly on the reproductive efficiency and genetic gain of the herd, which are directly associated with the appropriate use of Assisted Reproductive Technologies (ARTs). The objective of this review is to show from a Brazilian perspective the evolution over the last 40 years of ARTs related to ovulation resynchronization programs and embryo transfer in cattle. Despite significant improvements and high fertility obtained in timed artificial insemination (TAI) protocols (Sales et al., 2024 - Part I), the improvement of the use of in vitro-produced embryos, development of resynchronization programs, and the advance in Doppler ultrasonography (Doppler-US) for reproductive assessments of bovine females were the ARTs that presented the greatest relevance on reproductive effectiveness in cattle. In the last seven years, the embryo transfer (ET) technology using in vitro-produced (IVP) embryos took over the conventional ET of in vivo produced embryos after donor's superovulation. Also, procedures and pregnancy rates after ET of IVP embryos were improved in dairy and beef operations. The Doppler-US allows the identification of non-pregnant females at an early stage based on the evaluation of blood perfusion of the corpus luteum. Recent studies in beef and dairy cows indicate satisfactory accuracy when Doppler-US is used at 20-22 days after TAI. Consequently, super-early resynchronization programs have been developed and are being implemented in commercial programs, thereby facilitating earlier conception through the use of semen from superior bulls, providing genetic and economic improvements in herds. Likewise, the assessment of luteal function by Doppler-US allows the selection of embryo recipients with greater receptivity, and consequently may increase the effectiveness of timed ET programs.

Effects of mycobacterium cell wall fraction on embryo development following in vitro embryo production and pregnancy rates following embryo transfer in virgin dairy heifers

An experiment was conducted to determine whether administration of mycobacterium cell wall fraction (MCWF; Amplimune, NovaVive) could enhance embryo developmental competence following in vitro embryo production (IVP) and pregnancy establishment after embryo transfer (ET). Nulliparous, Holstein heifers (n = 40; age 8–15 months) were submitted to two rounds of ovum pick-up (OPU) and IVP in a crossover design. Thirty-six h after follicle wave synchronization, treatments (saline or MCWF, 5 mL, im) were administered in conjunction with a single dose of follicle stimulating hormone (175 IU) and OPU was performed 48–52 h later. Recovered cumulus-oocyte complexes were used for IVP to assess embryo development. For ET, nulliparous, Holstein heifers (n = 225; age 12–18 months) were used as recipients. At 12–24 h after detection of spontaneous estrus, recipients were randomly treated with either saline or MCWF (5 mL, im). The effect of MCWF on pregnancy per ET (P/ET) was assessed in a 2 × 2 factorial design with recipients treated with or without MCWF receiving a fresh IVP embryo from a donor treated with or without MCWF at day 7 or 8 after detected estrus. Blood samples were collected from a subset of donors (n = 8) and recipients (n = 26 to 33 per treatment) prior to treatment and at 6 and 24 h post-treatment to determine serum concentration of interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and interferon-γ. Blood samples were also collected from a group of recipients (n = 31 to 39 per treatment) to assess serum concentration of progesterone at days 4, 7, and 16 post-treatment. Pregnancy status was determined at days 40 and 100 of gestation. Donor treatment with MCWF tended (P < 0.07) to increase the proportion of oocytes that developed into transferable embryos, but there was no effect of MCWF on other parameters of embryo development. The P/ET at days 40 and 100 of gestation and pregnancy loss were not affected by donor treatment or recipient treatment with MCWF and there was no interaction. Serum concentration of proinflammatory cytokines among donors and recipients and serum concentration of progesterone among recipients were not increased by treatment with MCWF. Results of the present study indicate that treatment of donors with MCWF has minimal impact on subsequent embryo development following IVP. Moreover, regardless of whether donors or recipients were treated with MCWF, there was no effect on P/ET following transfer of IVP embryos.

Effect of follicular wave synchronization using a progesterone-releasing intravaginal device-based protocol on in vitro embryo production in Bos taurus cows subjected to 14-day intervals ovum pick-up

Follicular wave synchronization (FWS) before ovum pick-up (OPU) is one of the strategies used to improve the efficiency of in vitro embryo production (IVP). This study aimed to evaluate the effect of FWS on the total follicular number, cumulus-oocyte complex (COC) recovery, and in vitro embryo development in Angus cows (n = 33) subjected to OPU with 14-day intersession intervals. Additionally, it was also evaluated the presence of carryover effects given the short intersession interval used. The experiment was run as a 2-treatment (FWS vs. Control) x 2-period (1 vs. 2) crossover design. Animals in the FWS group received an intravaginal progesterone implant (1gr), estradiol benzoate (2 mg), and D-cloprostenol (150 μg) on day 0 and the OPU was performed on day 5. Control group animals did not receive any hormone treatment. The FWS increased the number of 6–10 mm follicles (P = 0.05), but it decreased the COC recovery rate (P < 0.01). The FWS did not affect the total or frozen embryo numbers (P = 0.49 and P = 0.17; respectively), but it increased the total blastocyst cell number (P < 0.01). A carryover effect was found on the total and < 6 mm follicles number (P = 0.10 and P < 0.01; respectively), and on the regular, atretic, viable, and total number of COC (P = 0.01, P = 0.08, P = 0.02 and P < 0.01; respectively). We concluded that the FWS increased the quality of embryos after OPU with 14-day intersession intervals in Angus cows and that this kind of OPU/IVP scheme enabled the existence of a carryover effect, especially on the follicle number and COC morphology.

Effects of long-acting injectable progesterone supplementation at early dioestrus on pregnancy maintenance in beef and dairy recipient cattle.

We tested in the present study the hypothesis that supplementation with long-acting P4 (iP4) at different times of the initial dioestrus improves pregnancy rates in dairy and beef recipients submitted to fixed-time embryo transfer (FTET). Recipients from commercial farms had their oestrous cycle synchronized with an E2/P4-based protocol in three experiments (Exp. 1 to 3). In Exp. 1, dairy heifers (n = 76) and cows (n = 104) were randomly assigned to two experimental groups: the control group (n = 89) and the iP4D4 group (n = 91). For Exps. 2 and 3, suckled beef recipients were used. In Exp. 2, recipients were assigned to two experimental groups: Control group (n = 147) and iP4D7 group (n = 144); whereas in Exp. 3, recipients were randomly assigned to three experimental groups: Control group (n = 85), iP4-D4 group (n = 86) and iP4D7 group (n = 81). Recipients in the iP4D4 and iP4-D7 groups received an i.m. administration of 150 mg iP4, on D4 or D7 (D0 was the day of expected oestrus). On D7, all recipients were evaluated by transrectal ultrasonography and those that had a CL received a fresh or vitrified invitro-produced embryo. In Exp. 2 and 3, the CL area was also determined by ultrasonography at the time of FTET. The pregnancy diagnosis was performed at 30 days in Exp. 1, 57 days in Exp. 2, and between 40 and 72 days of pregnancy in Exp. 3. In Exp. 1, the pregnancy rate did not differ (p > .1) between the Control group (38.2% [34/89]) and iP4D4 group (49.5% [45/91]); yet, a parity effect indicated a greater (p < .05) pregnancy rate in heifers (57.9% [44/76]) than cows (30.8% [32/104]). In Exp. 2, the pregnancy rate was greater (p < .05) in the iP4D7 group (45.0% [65/144]) than in the Control group (34.0% [50/147]). Also, a greater (p = .08) pregnancy rate was observed for recipients with a small CL (≤2.75 cm2 ) that were treated with iP4 on the day of FTET than the control recipients (46.4% [32/69] vs. 32.6% [28/86]). In Exp. 3, no significant effects (p > .1) of the treatment group or CL size were detected on pregnancy rates at days 30 and 60. In conclusion, the beneficial effects of iP4 supplementation at early dioestrus on pregnancy maintenance may vary according to the experimental conditions, but its use at the time of FTET can be used as an alternative to enhance the fertility of beef recipients in challenging conditions in commercial herds.

Effect of Intra-ovarian Injection of Mesenchymal Stem Cells or its Conditioned Media on Repeated OPU-IVEP Outcomes in Jersey Heifers and Its Relationship with Follicular Fluid Inflammatory Markers.

Repeated Ovum Pick Up (OPU) could have a detrimental effect on ovarian function, reducing In Vitro Embryo Production (IVEP). The present study examined the therapeutic effect of adipose-derived Mesenchymal Stem Cells (MSCs) or its Conditioned Medium (ConM) on ovarian trauma following repeated OPU. Resolvin E1 (RvE1) and Interleukin-12 (IL-12) were investigated as biomarkers. Jersey heifers (n=8) experienced 11 OPU sessions including 5 pre-treatment and 6 treatment sessions. Heifers received intra-ovarian administration of MSCs or ConM (right ovary) and Dulbecco's Modified Phosphate Buffer Saline (DMPBS; left ovary) after OPU in sessions 5 and 8 and 2 weeks after session 11. The concentrations of RvE1 and IL-12 in follicular fluid was evaluated on sessions 1, 5, 6, 9, and 4 weeks after session 11. Following each OPU session, the IVEP parameters were recorded. Intra-ovarian administration of MSCs, ConM, and DMPBS did not affect IVEP parameters (p>0.05). The concentration of IL-12 in follicular fluid increased at the last session of pre-treatment (Session 5; p<0.05) and remained elevated throughout the treatment period. There was no correlation between IL-12 and IVEP parameters (p>0.05). However, RvE1 remained relatively high during the pre-treatment and decreased toward the end of treatment period (p<0.05). This in turn was associated with decline in some IVEP parameters (p<0.05). Intra-ovarian administration of MSCs or ConM during repeated OPU did not enhance IVEP outcomes in Bos taurus heifers. The positive association between RvE1 and some of IVEP parameters could nominate RvE1 as a promising biomarker to predict IVEP parameters following repeated OPU.

Field outcomes of laparoscopic ovum pick-up combined with in vitro embryo production in sheep: Effects of long-acting recombinant ovine FSH pre-stimulation, collection frequency, and donor breed

Laparoscopic ovum pick-up (LOPU) combined with in vitro embryo production (IVEP) is a technology platform that improves the utilization rate of the elite ewe's ovarian oocytes and increases the number of obtained offspring. This study aimed to evaluate the effects of FSH pre-stimulation, serial oocyte collection, and breed on LOPU-IVEP under field conditions. Donors were randomly assigned to five groups (group A: decreasing doses of pituitary FSH (p-FSH); group B: constant doses of p-FSH; group C: two doses of long-acting recombinant ovine FSH (ro-FSH); group D: single administration of a long-acting ro-FSH in; group E: no FSH stimulation). Oocyte yield following LOPU (average recovered oocytes: 20.9 ± 0.5; average viable oocytes: 17.2 ± 0.4) and oocyte developmental competence (average blastocysts: 7.0 ± 0.2) in group C were significantly better than these of group D and group E, and similar to these of groups A and B. Meanwhile, there were no differences in oocyte yield and developmental capacity using repeated LOPU session at 1-, 2-, and 3-month intervals (p > 0.05). Finally, we compared LOPU-IVEP outcomes among five sheep breeds. The results indicated that East Friesian × Chinese Mongolian crossbred sheep and purebred East Friesian sheep had the more recovered oocytes and viable oocytes compared with the Suffolk, Dorper, and Texel breeds, and average number of blastocysts in East Friesian × Chinese Mongolian sheep group was also highest among the groups (8.1 ±0.3, p < 0.05). In summary, the results of this study indicate long-acting ro-FSH pre-stimulation combined with 12 times LOPU sessions over one year maximizes embryo production of elite donor ewes under field conditions.

298 Interleukin-6 Family Members Improve Pre-Implantation Bovine Blastocyst Composition

Abstract Inner cell mass (ICM) size is a predictor of bovine blastocyst quality and post-transfer pregnancy success. In vitro produced (IVP) embryos have reduced ICM cell numbers compared with in vivo produced embryos, and we propose this is one reason pregnancy success is compromised in IVP embryos. Our recent work found that human recombinant interleukin-6 (IL6) supplementation during in vitro embryo culture increased ICM cell numbers. We hypothesized that other human recombinant members of the IL6 cytokine family, specifically interleukin-11 (IL11) and leukemia inhibitory factor (LIF), will have a similar response. In vitro maturation, fertilization and culture procedures were used to produce embryos that were treated with 100 ng/mL recombinant human recombinant IL6, IL11, LIF, or carrier [control; 1% (wt/vol) SOF-BSA] beginning at day 6 post-fertilization (25 embryos/50 mL drop; 3 drops/treatment; 5 replicates). A subset of day 8 blastocysts (n = 28 to 29) was fixed and processed for cell counting. Analyses were performed by least-squares ANOVA using the general linear model. No treatment differences in overall blastocyst rates, trophectoderm (TE) cell numbers, and total cell numbers were observed at day 8. Treatment with IL6 (P = 0.09) and LIF (P = 0.06) tended to increase ICM cell numbers when compared with the control, whereas IL11 was not different from the control. Treatment with IL6 increased the ICM:TE ratio (P &amp;lt; 0.05) but the other cytokines did not influence this ratio. Several treatment by stage interactions were observed. In regular day 8 blastocysts (n = 10 to 14), none of the treatments affected ICM numbers or the ICM:TE ratio, but LIF increased TE number (P &amp;lt; 0.05) and IL6 increased total cell number (P &amp;lt; 0.05). When grouping expanded and hatched blastocysts together (n = 14 to 18), each treatment increased ICM cell numbers and IL6 increased the ICM:TE ratio (P &amp;lt; 0.05). Furthermore, both IL11 (P = 0.08) and LIF (P = 0.08) tended to increase the ICM:TE ratio. Only LIF increased total cell numbers (P &amp;lt; 0.05). None of the other treatments affected total cell numbers. These observations suggest that IL6, IL11, and LIF are capable of influencing ICM cell numbers in IVP bovine embryos, but they may possess different abilities in affecting TE and overall cell numbers. These findings differ from previous findings from our lab. We suspect that transitioning from bovine recombinant preparations with undefined purity and bioactivity to using purified human recombinant proteins with high bioactivity allowed us to detect these responses for each cytokine. Collectively, this work implicates several members of the IL6 cytokine family as embryokines that may be used to improve bovine IVP embryo quality. Funding for this work was provided by USDA-NIFA (2017-67015-2646 and 2021-67015-34485) and by NIH (R21-OD026516-01).

Phenotypic and genetic relationships among anogenital distance, anti-Müllerian hormone, and in vitro embryo production in Gyr dairy cattle

Anti-Müllerian hormone (AMH) concentration and number of recovered oocytes (ROOC) are phenotypic parameters associated with in vitro embryo production (IVEP). More recently, anogenital distance (AGD) has been proposed as a proxy for fertility in dairy cattle that is easy to collect at a low cost. The aim of this study was to characterize the AGD and its phenotypic and genetic associations with AMH and IVEP in Bos indicus Gyr dairy cattle. The hypothesis was that the number of ROOC, in vitro-produced embryos, and AMH concentration would increase as the AGD decreases. From July to December 2021, a single morphometrical measurement of AGD was collected in 552 donors from 6 herds in Brazil. A subset of donors had AMH assayed on the same day. Only ovum pick-up events that occurred up to 12 mo preceding and 7 mo succeeding the AGD measurement were used to assess the association between AGD, AMH, and IVEP. Thus, 472 donors (1,551 ovum pick-up events and 140 donors with AMH) were considered in the analysis. A raw average was calculated for each individual donor's ROOC, viable oocytes, total produced embryos, viability rate, and embryo rate (defined as total produced embryos/viable oocytes). Comparisons were conducted within the age categories of 3 to <6 yr or 6 to <10 yr. Phenotypic associations were performed in SAS software (SAS Institute Inc., Cary, NC). Genetic correlations were estimated using the BLUPF90 family of programs. The AGD (128.7 mm ± 14; mean ± standard deviation) had a normal distribution and was highly variable (83 to 172 mm) among the Gyr population. Our experimental hypothesis was partially supported by a phenotypic association of a greater number of total produced embryos (R2 = 0.023) as AGD decreased. Our results failed to support an increase in AMH concentration along with a decrease in AGD. In addition, positive and low genetic correlations were observed between AGD and viable oocytes (r = 0.08), and embryo rate (r = 0.20). A greater number of viable oocytes and embryos were observed in donors in the high compared with intermediate and low ROOC categories within both age categories. The age interval of 3 to <6 yr showed a greater number of recovered and viable oocytes for the high AMH compared with the low category, but no differences were observed among the AGD categories. In summary, for the Gyr breed, AGD was phenotypically inversely associated with a quantity-related parameter, such as the total number of produced embryos. In contrast, AGD showed a low genetic correlation with qualitative-related outcomes such as viable oocytes and embryo rate. Further studies should be performed to validate these retrospective analyses and to better understand the association between AGD and IVEP.

Mitochondrial bioenergetic profiles of warmed bovine blastocysts are typically altered after cryopreservation by slow freezing and vitrification

The widespread use of cryopreserved in vitro produced (IVP) bovine embryos is limited due to their low post-warming viability compared to their ex vivo derived counterparts. Therefore, the present study aimed to analyse in detail the consequences of cryopreservation (vitrification and slow freezing) on the bioenergetic profile of the embryo and its mitochondria. To accomplish that, day 7 IVP embryos were separated in a non-cryopreserved control group (fresh, n = 120, 12 replicates) or were either slow frozen (slow frozen, n = 60, 6 replicates) or vitrified (vitrified, n = 60, 6 replicates). An in-depth analysis of the bioenergetic profiles was then performed on these 3 groups, analysing pools of 10 embryos revealing that embryo cryopreservation both via vitrification and slow freezing causes profound changes in the bioenergetic profile of bovine embryos. Noteworthy, fresh embryos demonstrate a significantly (P < 0.05) higher oxygen consumption rate (OCR) compared to vitrified and slow frozen counterparts (0.858 ± 0.039 vs. 0.635 ± 0.048 vs. 0.775 ± 0.046 pmol/min/embryo). This was found to be largely due to significantly reduced mitochondrial oxygen consumption in both vitrified and deep-frozen embryos compared to fresh counterparts (0.541 ± 0.057 vs. 0.689 ± 0.044 vs. 0.808 ± 0.025 pmol/min/embryo). Conversely, slow-frozen thawed blastocysts showed 1.8-fold (P < 0.05) higher non-mitochondrial OCR rates compared to fresh embryos. Maximum mitochondrial respiration of vitrified and slow-frozen embryos was significantly reduced by almost 1.6-fold compared to fresh embryos and the proportion of ATP-linked respiration showed significantly lower values in vitrified thawed embryos compared to fresh embryos (1.1-fold, P < 0.05). Likewise, vitrification-warming and freeze-thawing reduced reactive glycolytic capacity (1.4 fold, 1.2-fold)as well as compensatory glycolytic capacity to provide energy in response to mitochondrial deficiency (1.3-fold and 1.2-fold, P < 0.05). In conclusion, the present study has, to the best of our knowledge, identified for the first time a comprehensive overview of typical altered metabolic features of the bioenergetic profile of bovine embryos after cryopreservation, which have great potential to explain the detrimental effects of cryopreservation on embryo viability. Avoidance of these detrimental effects through technical improvements is therefore suggested to be mandatory to improve the viability of bovine embryos after cryopreservation-warming.