Human recombinant interleukin-6 improves the morphological quality of cryopreserved in vitro produced bovine blastocysts.

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis.

Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†.

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

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.

Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART).

CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A RNPs targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1,000 nucleotides long. Electroporation of Cas13a RNPs prevents the production of the corresponding proteins. We electroporated Cas9D10A RNPs targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a RNPs targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P < 0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (False Discovery Rate, FDR < 0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is a key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.

Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle.

Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, early embryonic development, compaction and blastulation, embryonic cell lineage specification, elongation, gastrulation, and placentation. Additionally, discussed are potential remediation strategies, but unfortunately, corrective actions are not available for several of the problems being discussed. Further research is needed to produce bovine embryos that have a greater likelihood of surviving to term.

Conjugated linoleic acid supplementation changes prostaglandin concentration ratio and alters the expression of genes involved in maternal-fetal recognition from bovine trophoblast cells in vitro.

Early embryonic mortality caused by maternal-fetal recognition failure in the three weeks after fertilization represents a major cause of reproductive inefficiency in the cattle industry. Modifying the amounts and ratios of prostaglandin (PG) F2α and PGE2 can benefit the establishment of pregnancy in cattle. Adding conjugated linoleic acid (CLA) to endometrial and fetal cells culture affects PG synthesis, but its effect on bovine trophoblast cells (CT-1) is unknown. The aim of this study was to determine the effects of CLA (a mixture of cis- and trans-9, 11- and -10,12-octadecadienoic acids) on PGE2 and PGF2α synthesis and the expression of transcripts involved with maternal-fetal recognition of bovine trophectoderm. Cultures of CT-1 were exposed to CLA for 24, 48 and 72 h. Transcript abundance was determined by qRT-PCR and hormone profiles were quantified by ELISA. The PGE2 and PGF2α concentrations were reduced in the culture medium of CLA-exposed CT-1 compared to that of unexposed cells. Furthermore, CLA supplementation increased the PGE2:PGF2α ratio in CT-1 and had a quadratic effect (P < 0.05) on the relative expression of MMP9, PTGES2, and PTGER4. The relative expression levels of PTGER4 were reduced (P < 0.05) in CT-1 cultured with 100 µM CLA than in the unsupplemented and 10 µM-CLA groups. Treatment of CT-1 with CLA decreased PGE2 and PGF2α synthesis but a biphasic effect of CLA was observed on the PGE2:PGF2α ratio and relative abundance of transcripts with 10 µM CLA providing maximal improvements in each endpoint. Our data suggest that CLA may influence eicosanoid metabolic process and extracellular matrix remodeling.

An updated protocol for in vitro bovine embryo production.

Cattle embryos represent a useful model for understanding parts of human embryogenesis due to various biological similarities. We describe a protocol to mature and fertilize bovine oocytes followed by culture of resulting presumptive zygotes up until the blastocyst stage. Our protocol features a unique procedure for washing and moving oocytes and zygotes between their respective dishes using a cell strainer. A thorough troubleshooting section will help users optimize embryo development with cleavage and blastocyst rates exceeding 70% and 20%, respectively. For complete details on the use and execution of this protocol, please refer to Wooldridge and Ealy (2019).1.

Cleavage kinetics is a better indicator of embryonic developmental competency than brilliant cresyl blue staining of oocytes.

In vitro production of embryos (IVP) is a valuable technology to produce embryos of high genetic value. Despite advances in IVP, the efficiency of culture systems remains low. One method to increase IVP success is the early selection of oocytes or embryos that may have greater developmental potential. Here, we investigated two methods of selection, namely BCB staining and cleavage kinetics, both individually and in conjunction, for improved developmental outcomes in vitro. We hypothesized that a synergistic use of both BCB staining and cleavage kinetics would result in identification of embryos of greater developmental potential. The selection of oocytes by BCB staining does select for those oocytes with higher developmental potential, as noted by a greater blastocyst development between BCB positive (32.6%) and BCB negative (22.0%) on day 8 post-fertilization. However, the utilization of BCB staining and cleavage kinetics in tandem resulted in a complete masking of the effect observed when using BCB alone. We obtained the highest proportion of blastocyst development per selection group using cleavage kinetics alone, in which 53.1% of embryos grouped as Fast produced a blastocyst, which was significantly different from the three other groups (Fast+, Slow, not cleaved). We observed, however, that the separation of embryos by cleavage kinetics did not predict their survival to cryopreservation. In conclusion, in standard culture systems, cleavage kinetics is an effective method for the selection of embryos with increased developmental potential to develop blastocysts, however, it may not be effective to select healthy embryos for transfer following cryopreservation.

Influences of Supplementing Selective Members of the Interleukin-6 Cytokine Family on Bovine Oocyte Competency.

This work explored whether supplementing selective members of the interleukin-6 (IL6) cytokine family during in vitro bovine oocyte maturation affects maturation success, cumulus-oocyte complex (COC) gene expression, fertilization success, and embryo development potential. Human recombinant proteins for IL6, IL11, and leukemia inhibitory factor (LIF) were supplemented to COCs during the maturation period, then fertilization and embryo culture commenced without further cytokine supplementation. The first study determined that none of these cytokines influenced the rate that oocytes achieved arrest at meiosis II. The second study identified that LIF and IL11 supplementation increases AREG transcript abundance. Supplementation with IL6 supplementation did not affect AREG abundance but reduced HAS2 transcript abundance. Several other transcriptional markers of oocyte competency were not affected by any of the cytokines. The third study determined that supplementing these cytokines during maturation did not influence fertilization success, but either LIF or IL11 supplementation increased blastocyst development. No effect of IL6 supplementation on subsequent blastocyst development was detected. The fourth experiment explored whether each cytokine treatment affects the post-thaw survivability of cryopreserved IVP blastocysts. None of the cytokines supplemented during oocyte maturation produced any positive effects on post-thaw blastocyst re-expansion and hatching. In conclusion, these outcomes implicate IL11 and LIF as potentially useful supplements for improving bovine oocyte competency.

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

BACKGROUND: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence. METHODS: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes. RESULTS: The BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex. CONCLUSIONS: If oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.

Short Communication: Maternal obesity alters ovine endometrial gene expression during peri-implantation development.

Exposure to maternal obesity in utero is associated with marked developmental effects in offspring that may not be evident until adulthood. Mechanisms regulating the programming effects of maternal obesity on fetal development have been reported, but little is known about how maternal obesity affects the earliest periods of embryonic development. This work explored how obesity influences endometrial gene expression during the peri-implantation period using a sheep model. Ewes were assigned randomly to diets that produced an obese state or maintained a lean state. After 4 mo, obese and lean ewes were bred and then euthanized at day 14 post-breeding. The uterus was excised, conceptuses were flushed, and endometrial tissue was collected. Isolated RNA from endometrial tissues (n = 6 ewes/treatment) were sequenced using an Illumina-based platform. Reads were mapped to the Ovis aries genome (Oar_4.0). Differential gene expression was determined, and results were filtered (false discovery rate ≤ 0.05 and ≥2-fold change, ≥0.2 reads/kilobase/million reads). Differentially expressed genes (DEGs) were identified (n = 699), with 171 downregulated and 498 upregulated in obese vs. lean endometrium, respectively. The most pronounced gene ontology categories identified were cellular process, metabolic process, and biological regulation. Enrichments were detected within the DEGs for genes involved with immune system processes, negative regulation of apoptosis, cell growth, and cell adhesion. A literature search revealed that 125 DEGs were associated with either the trophoblast lineage or the placenta. Genes within this grouping were involved with wingless/integrated signaling, angiogenesis, and integrin signaling. In summary, these data indicate that the peri-implantation endometrium is responsive to maternal obesity. Transcript profile analyses suggest that the endometrial immune response, adhesion, and angiogenesis may be especially susceptible to obesity. Thus, alterations in uterine transcript profiles during early embryogenesis may be a mechanism responsible for developmental programming following maternal obesity exposure in utero.

Mammals derived from obese mothers can exhibit a host of health issues after birth and into adulthood. It remained unclear how early these adverse effects of maternal obesity could influence pregnancies. This work describes how obesity changes uterine gene expression early in pregnancy in ewes. Uterine tissue was harvested, and RNA was isolated and sequenced. A total of 699 differentially expressed genes were identified. These genes were associated with various cellular and reproductive processes, including placental development and function, cellular metabolic processes, immune system processes, cell death, cell growth, and cell adhesion. These data are supportive of the idea that the peri-implantation endometrium is susceptible to maternal obesity. Changes in the local immune system, uterine function, and early placental development seem to be especially prone to modifications based on the body condition of the mother. Thus, changes in uterine gene expression and uterine biology occurring early in gestation could be one mechanism for developmental programming.

Ruminant conceptus-maternal interactions: interferon-tau and beyond.

Embryonic or fetal loss in cattle is associated with problems that occur during oocyte maturation, early embryonic development, conceptus elongation, maternal recognition of pregnancy (MRP), and/or placental attachment and implantation. Many of these problems manifest as inadequate or asynchronous communication between the developing conceptus and endometrium, resulting in pregnancy failure. This review will provide an overview of how various conceptus-endometrial paracrine signaling systems control the fate of early pregnancy in cattle and other ruminants. We begin by summarizing the actions of interferon-tau, the classic MRP signal in ruminates, and then explore how other secretory factors derived from either the conceptus or endometrium influence establishment and maintenance of pregnancy. Insight into how the endometrium responds to male vs. female conceptuses or conceptuses produced by in vitro methods will also be described. Specific focus will be placed on describing how "omic" technologies and other cutting-edge techniques have assisted with identifying novel conceptus and/or endometrial factors and their functions. Recent findings indicate that the endometrial transcriptome and histotroph are altered by conceptus sex, quality, and origin, suggesting that the endometrium is a sensor of conceptus biochemistry. Although the endometrium has a certain level of flexibility in terms of conceptus-maternal interactions, this interplay is not sufficient to retain some pregnancies. However, new information inspires us to learn more and will help develop technologies that mitigate early embryonic loss and reproductive failure in ruminants and other animals.

Early pregnancy losses are common in cattle. This review describes how critical the interplay between the developing conceptus (embryo and extraembryonic membranes) and endometrium is to maintaining pregnancies in cattle and other ruminants. The discovery of interferon-tau more than 40 yr ago initiated a new field of reproductive biology focused on describing how the conceptus and endometrium communicate with one another through the secretion of paracrine factors, extracellular vesicles, and other molecules. The use of "omic" and gene editing technologies has assisted with identifying novel functions for many conceptus and endometrial secreted factors. This review provides examples of how conceptus sex, quality, and in vitro vs. in vivo development influences endometrial function. The endometrium appears to have some flexibility in its response to conceptuses, and this insight could be used to our advantage as we work towards developing schemes to rescue conceptuses that are in danger of experiencing pregnancy loss.

Bioactive supplements influencing bovine in vitro embryo development.

Ovum pickup and in vitro production (IVP) of bovine embryos are replacing traditional multiple ovulation embryo transfer (MOET) as the primary means for generating transferable embryos from genetically elite sires and dams. However, inefficiencies in the IVP process limit the opportunities to produce large numbers of transferable embryos. Also, the post-transfer competency of IVP embryos is inferior to embryos produced by artificial insemination or MOET. Numerous maternal, paternal, embryonic, and culture-related factors can have adverse effects on IVP success. This review will explore the various efforts made on describing how IVP embryo development and post-transfer competency may be improved by supplementing hormones, growth factors, cytokines, steroids and other bioactive factors found in the oviduct and uterus during early pregnancy. More than 40 of these factors, collectively termed as embryokines, are reviewed here. Several embryokines contain abilities to promote embryo development, including improving embryo survivability, improving blastomere cell numbers, and altering the distribution of blastomere cell types in blastocysts. A select few embryokines also can benefit pregnancy retention after IVP embryo transfer and improve neonatal calf health and performance, although very few embryokine-supplemented embryo transfer studies have been completed. Also, supplementing several embryokines at the same time holds promise for improving IVP embryo development and competency. However, more work is needed to explore the post-transfer consequences of adding these putative embryokines for any adverse outcomes, such as large offspring syndrome and poor postnatal health, and to specify the specific embryokine combinations that will best represent the ideal conditions found in the oviduct and uterus.

Ovum pickup and in-vitro production (IVP) of bovine embryos have quickly become commercial options for generating large quantities of transferable bovine embryos from genetically elite sires and dams. However, 2 limitations in this process still exist. First, the percentage of eggs/oocytes that become fertilized and produce transferable embryos remains low. Second, IVP embryos that are transferred to recipients are less able to maintain a viable pregnancy than embryo produced by other means. Various maternal, paternal, embryonic, and culture-related factors will influence IVP success. This review describes how both IVP embryo development and post-transfer embryo competency may be improved by supplementing hormones, growth factors, cytokines, steroids, and other bioactive factors found in the oviduct and uterus during early pregnancy. These factors are collectively termed as embryokines. Several embryokines will promote IVP embryo development, but only a few of these embryokines have been tested for their ability to improve post-embryo transfer pregnancy retention. More work is needed to explore the post-transfer consequences of adding embryokines. However, with that being said, all indications are that we are on the right track with identifying one and likely several embryokines that will improve IVP embryo development and post-transfer pregnancy retention in cattle.

A Synopsis of the NE1727 Multistate Project Collection in the Journal of Animal Science.

This article provides a synopsis of the collection of papers provided by participants of the NE1727 Multistate project. Five reviews and thirteen primary research articles are included that provide contributions the group has made to understanding the role of the corpus luteum in reproduction, describing how the ovary influences fertility, delineating mechanisms controlling oocyte quality and early embryo development, and exploring new reproductive management schemes.

Microbiomes of Various Maternal Body Systems Are Predictive of Calf Digestive Bacterial Ecology.

Body systems once thought sterile at birth instead have complex and sometimes abundant microbial ecosystems. However, relationships between dam and calf microbial ecosystems are still unclear. The objectives of this study were to (1) characterize the various maternal and calf microbiomes during peri-partum and post-partum periods and (2) examine the influence of the maternal microbiome on calf fecal microbiome composition during the pre-weaning phase. Multiparous Holstein cows were placed in individual, freshly bedded box stalls 14 d before expected calving. Caudal vaginal fluid samples were collected approximately 24 h before calving and dam fecal, oral, colostrum, and placenta samples were collected immediately after calving. Calf fecal samples were collected at birth (meconium) and 24 h, 7 d, 42 d, and 60 d of age. Amplicons covering V4 16S rDNA regions were generated using DNA extracted from all samples and were sequenced using 300 bp paired end Illumina MiSeq sequencing. Spearman rank correlations were performed between genera in maternal and calf fecal microbiomes. Negative binomial regression models were created for genera in calf fecal samples at each time point using genera in maternal microbiomes. We determined that Bacteroidetes dominated the calf fecal microbiome at all time points (relative abundance ≥42.55%) except for 24 h post-calving, whereas Proteobacteria were the dominant phylum (relative abundance = 85.10%). Maternal fecal, oral, placental, vaginal, and colostrum microbiomes were significant predictors of calf fecal microbiome throughout pre-weaning. Results indicate that calf fecal microbiome inoculation and development may be derived from various maternal sources. Maternal microbiomes could be used to predict calf microbiome development, but further research on the environmental and genetic influences is needed.

Cytokines That Serve as Embryokines in Cattle.

The term "embryokine" has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos. Embryokines are being explored for their ability to overcome the poor development rates of IVP embryos and to limit post-transfer pregnancy retention efficiencies that exist in IVP embryos. This review will focus on describing two of the best-characterized cytokines, colony-stimulating factor 2 and interleukin 6, for their ability to modify bovine embryo quality and confirmation, promote normal fetal development, and generate healthy calves. Additional cytokines will also be discussed for their potential to serve as embryokines.

Effects of mid-gestational l-citrulline supplementation to twin-bearing ewes on umbilical blood flow, placental development, and lamb production traits.

The objective of the study was to examine how l-citrulline supplementation to ewes during mid-gestation influences placental activity, placental blood flow, lamb body weight, and carcass characteristics. Two studies were completed. A pharmacokinetic study to compare circulating plasma amino acid concentrations after a single intravenous injection of 155 µmol/kg BW l-citrulline or after an isonitrogenous amount of l-alanine (control; 465 µmol/kg BW). Increases (P < 0.05) in circulating citrulline concentrations were detected for 8 h after l-citrulline injection versus the control. Similarly, increases (P < 0.05) in circulating arginine concentrations were detected for 24 h after l-citrulline treatment. The second study used 12 ewes with twin pregnancies. Daily intravenous injections of either l-citrulline or l-alanine were administered for 39 d from d 42-45 to 81-84 of gestation. Ewes were limit-fed at 85% daily energy requirements during the injection period. A decrease (P < 0.0001) in body weight was observed in both treatment groups during this period. No treatment differences were observed in circulating pregnancy-specific protein B concentrations or placental blood flow during the treatment and post-treatment gestational period. No treatment differences were observed in lamb survival nor in lamb birth, weaning and slaughter weights. Treatment did not influence lamb carcass composition or organ weights. However, there was a tendency (P = 0.10) for an increase in antral follicle numbers in ovaries from ewe lambs derived from ewes treated with l-citrulline. In summary, a daily l-citrulline injection increased both circulating citrulline and arginine concentrations in ewes, but daily l-citrulline injections during mid-gestation did not produce any detectable changes in placental activity and blood flow, neonatal and postnatal lamb development, and lamb carcass composition at slaughter. In conclusion, no benefits in placental function and lamb development were observed after providing l-citrulline during mid-gestation in ewes exposed to a mild energy restriction, but there was an indication that follicle numbers in ewe lambs were positively influenced by l-citrulline treatment during fetal development.

Interleukin-6 supplementation improves post-transfer embryonic and fetal development of in vitro-produced bovine embryos.

The use of in vitro produced embryos in dairy and beef cattle has increased in recent years, but compromised post-transfer pregnancy success prevents producers from capturing all the benefits this technology can provide. This study explored whether supplementing interleukin-6 (IL6) during in vitro embryo development influences post-transfer development of the embryo-proper, fetus and placenta during early gestation in cattle. Slaughterhouse-derived cumulus oocyte complexes underwent IVM (day -1) and IVF (day 0). On day 5 post-fertilization, embryos were treated with either 0 (CONT) or 100 ng/mL recombinant bovine IL6. No difference in blastocyst formation was detected on day 7.5 post-fertilization, but an increase (P < 0.05) in inner cell mass cell numbers and tendency for increased (P = 0.08) trophectoderm cell numbers were detected in IL6-treated blastocysts. A subset of the blastocysts was loaded individually into transfer straws, and embryo transfer (ET) was completed using estrous cycle stage-matched, nonlactating commercial beef and dairy cows. A subset of cows from each group underwent timed artificial insemination (TAI). Pregnancy rates were similar among all three treatment groups at day 28 and 70. No differences in crown-rump length (CRL), crown nose length (CNL), abdominal diameter (AD), or placental fluid volume (PFV) were detected between TAI and ET-IL6 groups. Reductions (P < 0.05) in CRL and AD were detected at day 56 and a tendency for a reduction (P = 0.08) in PFV was detected on day 35 when comparing the ET-CONT group with the TAI group. Reductions (P < 0.05) in CRL and PFV on day 28 and CNL and AD on day 56 as well as a tendency for a reduction (P = 0.08) in PFV on day 35 were detected when contrasting ET-CONT with ET-IL6. Circulating plasma pregnancy-associated glycoprotein concentrations were similar among all treatment groups. In summary, IL6 treatment to IVP embryos before ET produced pregnancies that more closely resembled TAI-generated pregnancies than pregnancies generated using conventionally cultured embryos. These findings failed to find any adverse effects of IL6 supplementation on early development of the embryo-proper and fetus or on placental activity. Rather, these observations suggest that IL6 treatment may normalize the developmental trajectory of the embryo-proper and fetus for in vitro produced embryos.

Physical parameters of bovine activated oocytes and zygotes as predictors of development success.

The worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

Interleukin-6 promotes primitive endoderm development in bovine blastocysts.

BACKGROUND: Interleukin-6 (IL6) was recently identified as an embryotrophic factor in bovine embryos, where it acts primarily to mediate inner cell mass (ICM) size. This work explored whether IL6 affects epiblast (EPI) and primitive endoderm (PE) development, the two embryonic lineages generated from the ICM after its formation. Nuclear markers for EPI (NANOG) and PE (GATA6) were used to differentiate the two cell types. RESULTS: Increases (P < 0.05) in total ICM cell numbers and PE cell numbers were detected in bovine blastocysts at day 8 and 9 post-fertilization after exposure to 100 ng/ml recombinant bovine IL6. Also, IL6 increased (P < 0.05) the number of undifferentiated ICM cells (cells containing both PE and EPI markers). The effects of IL6 on EPI cell numbers were inconsistent. Studies were also completed to explore the importance of Janus kinase 2 (JAK2)-dependent signaling in bovine PE cells. Definitive activation of STAT3, a downstream target for JAK2, was observed in PE cells. Also, pharmacological inhibition of JAK2 decreased (P < 0.05) PE cell numbers. CONCLUSIONS: To conclude, IL6 manipulates ICM development after EPI/PE cell fates are established. The PE cells are the target for IL6, where a JAK-dependent signal is used to regulate PE numbers.