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Multi-parous Holstein cows exposed to short day photoperiod (SDPP) of 8 h of light per day during their dry period produced up to 3.2 kg more milk per day compared with cows exposed to long day photoperiod (LDPP) of 16 h of light per day; it is unknown if a similar response would be observed for Jersey cow milk production. The objective of this study was to determine the effect of photoperiod during the dry period on subsequent colostrum and milk production in Holstein and Jersey cattle. Holstein and Jersey cows (n = 33) were dried off 60 d before their due date and randomly assigned to SDPP (Holstein (n = 9), Jersey (n = 8)) or LDPP (Holstein (n = 8), Jersey (n = 8)) until calving. Cows were weighed at the time of enrollment (d 0) and were housed in an enclosed barn at 20°C and exposed to 250 to 450 lx during periods of light and < 10 lx during periods of darkness. At calving, colostrum volume was weighed and tested for relative protein concentration with a Brix refractometer and a sample was collected for component analysis (fat, protein, lactose, solids-not-fat (SNF)) via infrared spectroscopy, as well as immunoglobulin (IgA, IgG, IgG1, IgM), lactoferrin and somatic cell score (SCS) analysis. Post-calving, cows were returned to the free-stall barn and exposed to ambient photoperiod and temperature. Milk production data were collected for 15 weeks post-calving. Data were analyzed using PROC MIXED in SAS (SAS 9.4; SAS Institute, INC., Cary, NC) with treatment, breed, and d 0 weight as fixed effects. PROC MIXED with repeated measures was used to evaluate the relationship of day length and breed with mature milk volume, fat, and protein production. Random effects included replicate, lactation number, genetic inbreeding percentage, previous lactation mature equivalent (ME) 305-d protein production and calf sex. For colostrum, Brix score, colostral protein, fat, IgA, and IgM were increased in Jersey cows compared with Holstein cows. Total colostrum weight, SNF, lactose, lactoferrin, IgG, IgG1 and SCS did not differ by breed or treatment. Post-calving, energy corrected milk (ECM) production was increased in Holstein cows compared with Jersey cows but unaffected by photoperiod treatment. Conversely, milk protein percentage was increased for Jersey cows relative to Holstein cows but was unaffected by photoperiod treatment. Milk fat increased in LDPP Holstein cows compared with SDPP Jersey cows during the first week of lactation, which is likely due to the transition from colostrum to mature milk production. Overall, photoperiod did not affect colostrum production, but differences by breed were detected. Photoperiod during the dry period did not impact mature milk production or protein, but milk fat percentage was affected by photoperiod × breed. Therefore, altered lighting during the dry period does not unfavorably impact colostrum or milk production in Jersey or Holstein cows., (© 2025, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

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) F 2α and PGE 2 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 PGE 2 and PGF 2α 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 PGE 2 and PGF 2α concentrations were reduced in the culture medium of CLA-exposed CT-1 compared to that of unexposed cells. Furthermore, CLA supplementation increased the PGE 2 :PGF 2α 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 PGE 2 and PGF 2α synthesis but a biphasic effect of CLA was observed on the PGE 2 :PGF 2α 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., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ovarian antral follicle count (AFC) is a marker of ovarian stimulatory response to superovulation protocols in cattle. This article reviews novel research from the past 10 years, focusing on the relationship between AFC and embryo production and cow fertility. Substantial evidence indicates a positive relationship between AFC with embryo production; however, conflicting findings exist regarding the relationship of AFC with conception and pregnancy rates. This lack of consistent association with pregnancy outcomes is perplexing given the differences detected in oocytes, embryos, and endometria from high- versus low-AFC animals. Those differences include markers of embryonic viability such as protein level, blastocyst development rates, cleavage rates, and blastocyst cell numbers that differ between high- and low-AFC groups, as well as differential gene expression at the cow and embryo level with genes associated with fertility. In addition, Bos indicus and Bos taurus cattle appear to have different fertility responses based on their AFC category. In summary, clearly more studies are needed to elucidate the true associations between AFC and cow fertility, but the data that have been accumulated thus far indicate that AFC has the potential to be a useful marker of lifetime cow fertility., (© 2023.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hyperinsulinemia concurrent with hypoglycemia is one of a myriad of physiological changes typically experienced by lactating dairy cows exposed to heat stress, the consequences of which are not yet well defined or understood. Therefore, the objective of this experiment was to separate the production-related effects of hyperinsulinemia with hypoglycemia from those of a hyperthermic environment. Multiparous lactating Holstein cows (n = 23; 58 ± 4 d in milk, 3.1 ± 0.3 lactations) were housed in temperature-controlled rooms and all were subjected to 4 experimental periods as follows: (1) thermoneutral (TN; temperature-humidity index of 65.1 ± 0.2; d 1-5), (2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; insulin infused at 0.3 µg/kg of BW per h, glucose infused to maintain 90 ± 10% of baseline blood glucose for 96 h; d 6-10), (3) heat stress (HS; temperature-humidity index of 72.5 ± 0.2; d 16-20), and (4) HS + euglycemic clamp (EC; glucose infused to reach 100 ± 10% of TN baseline blood glucose for 96 h; d 21-25). Cows were fed and milked twice daily. Feed refusals were collected once daily for calculation of daily dry matter intake, and milk samples were collected at the beginning and end of each period for component analyses. Circulating insulin concentrations were measured in daily blood samples, whereas glucose concentrations were measured more frequently and variably in association with clamp procedures. Rectal temperatures and respiration rates were greater during HS than TN, as expected, and states of hyperinsulinemia and hypoglycemia were successfully induced by the HHC and high ambient temperatures (HS and EC). Feed intake differed based upon thermal environment as it was similar during TN and HHC periods, and declined for HS and EC. Milk production was not entirely reflective of feed intake as it was greatest during TN, intermediate during HHC, and lowest during HS and EC. All milk components differed with the experimental period, primarily in response to the thermal environment. Interestingly, TN baseline glucose concentrations were highly correlated with the change in glucose from TN to HS, and were related to glycemic status during HS. Furthermore, although few in number, those cows that failed to become hypoglycemic during HS tended to have a greater reduction in milk yield. The work presented here addresses a critical knowledge gap by broadening our understanding of the physiological response to heat stress and the related changes in glycemic state. This broadened understanding is fundamental for the development of novel, innovative management strategies as the dairy industry is compelled to become increasingly efficient in spite of global warming., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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&#95;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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives were to evaluate the effects of follicular wave and progesterone concentration on growth of the ovulatory follicle, conceptus elongation, uterine IFN-τ concentration, and transcriptome of conceptus and endometrium of pregnant cows on d 17 of gestation. Nonlactating nonpregnant Holstein cows were assigned randomly to one of 3 treatments: ovulation of a first-wave follicle (FW, n = 15); ovulation of a first-wave follicle and progesterone supplementation (FWP4, n = 12); and ovulation of a second-wave follicle (SW, n = 19). Ovulation of a first- or second-wave follicle was achieved by initiating the Ovsynch protocol (d -9 GnRH, d -2 and -1 PGF 2α , d 0 GnRH and artificial insemination, d 0.7 artificial insemination) on d 0 or 6 of a presynchronized estrous cycle, respectively. Cows in FWP4 received 3 intravaginal inserts containing progesterone at 12, 24, and 48 h after the first GnRH injection that were removed on d -2. Cows were killed on d 17 for collection of the reproductive tract. Transcriptome was evaluated by microarray using the Affymetrix Bovine Array. Orthogonal contrasts were built to assess the effects of progesterone concentration during follicle growth (FW vs. FWP4 + SW) and follicular wave (FWP4 vs. SW). Progesterone concentrations (LSM ± SEM) from d -9 to -2 were greater for SW, followed by FWP4 and FW (5.38 ± 0.24, 4.26 ± 0.28, and 1.17 ± 0.27 ng/mL). Diameter of the ovulatory follicle (FW = 19.6 ± 0.6; FWP4 = 15.6 ± 0.6; SW = 15.2 ± 0.5 mm) and concentrations of estradiol from d -2 to 1 (FW = 4.05 ± 0.33; FWP4 = 2.73 ± 0.35; SW = 2.48 ± 0.30 pg/mL) were greater for FW compared with FWP4 and SW. Progesterone concentrations from d 3 to 16 were greater for FW compared with FWP4 and SW. A total of 28 singleton conceptuses were collected (FW, n = 8; FWP4, n = 8; SW, n = 12) and only intact conceptuses were included in the analyses of length (FW, n = 8; FWP4, n = 6; SW, n = 12). Although conceptuses were longer for FW compared with FWP4 and SW (FW = 16.6 ± 2.3; FWP4 = 9.8 ± 2.2; SW = 9.6 ± 2.0 cm), treatment did not affect the amount of IFN-τ in uterine flushing. Transcriptome of conceptuses and endometrium of pregnant cows was not extensively affected by follicular wave (8 and 1 differentially expressed transcripts) or concentration of progesterone during follicle growth (0 and 3 differentially expressed transcripts), showing that these factors did not affect conceptuses and endometrium transcriptome in pregnancies that are maintained to d 17., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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.

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.

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., Competing Interests: Declaration of competing interest None of the authors have any conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Recent research from our group demonstrated that Bos indicus-influenced suckled beef cows had greater resilience to withstand nutrient restriction and establish pregnancy compared with B. taurus cows exposed to the same conditions. To further understand these findings, differences in metabolic profile between these same B. indicus-influenced and B. taurus females were explored. Suckled beef cows (n = 134) were enrolled in a completely randomized design with a 2 × 2 factorial arrangement of treatments. On day -21, Angus (AN; Bos taurus) and Brangus (BN; B. indicus-influenced) cows were randomly assigned to 1) a diet that met daily energy maintenance requirements (MAINT), or 2) a diet that restricted intake to 70% of the daily energy maintenance requirements (RESTR). Cows were exposed to an estrus synchronization protocol and received an embryo 7 d after ovulation was pharmacologically induced on day 0. Blood samples were collected on days -21 and 19 to determine circulating concentrations of non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), insulin, glucose, and IGF-1. Pregnancy status after embryo transfer was determined on day 28. As a consequence of the proposed diets, cows in the RESTR diet had less body condition score (BCS) on day 19 (P = 0.008) across breed types. Moreover, BCS change from day -21 to 19 was included as independent covariate into subsequent analyses, allowing for the comparison of breed types under an equivalent level of body reserve mobilization. A breed × diet interaction was observed for plasma insulin (P = 0.03) and IGF-1 (P = 0.04) on day 19, where AN-RESTR cows had less plasma concentrations on day 19 compared with AN-MAINT cows. Diets did not impact (P > 0.10) plasma insulin and IGF-1 concentrations in BN cows. No diet or breed effects were observed in circulating concentrations of NEFA, BHB, and glucose (P > 0.10). Across breed types and nutritional treatment, there was positive linear effect (P ≤ 0.04) of plasma concentrations of insulin and IGF-1 on the probability of pregnancy to fixed-time embryo transfer. In summary, the negative impacts of nutrient restriction on the somatotropic axis, independently of body tissue mobilization, were heightened in Bos taurus females compared with B. indicus-influenced cohorts, which corroborate with the differences observed in fertility between these subspecies., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Although the presence of bacteria has been characterized throughout the reproductive tracts of multiple species, how these bacteria may interact with the host has yet to be described. Previous reviews have described how pathogenic bacteria interact with the reproductive tract to cause infections such as metritis. This review aimed to summarize the knowledge related to pathogenic and nonpathogenic bacteria in various locations of the bovine reproductive tract and the possible mechanisms underlying host-microbe interactions during gametogenesis and early pregnancy. Lactic acid bacteria such as Lactobacillus seem to be beneficial in multiple areas of the reproductive tract: they have been associated with increased oocyte quality when in follicular fluid and secrete reactive oxygen species that are beneficial during placental angiogenesis. However, other bacteria, including Enterococcus, Staphylococcus, and Streptococcus, may modulate T helper cells that inhibit maternal recognition of pregnancy. Available data on the reproductive microbiome focus on variations in microbial communities and their associations with reproductive performance. However, research on these host-microbiome interactions may provide more insight on how bacteria affect fertility., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P < 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P < 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Several tools exist to diagnose pregnancy in dairy cattle. However, substantial pregnancy loss occurs within the first 60 d of gestation in cattle, and these losses have a profound adverse economic impact on the dairy and beef cattle industries. Detecting these impending pregnancy losses could offer producers an opportunity to reduce costs associated with this source of reproductive inefficiency. Several of the pregnancy diagnostic tools currently available and new technologies are being examined for their ability to predict pregnancies at risk for failing in early pregnancy. This review provides a synopsis of work undertaken recently to predict pregnancy losses in cattle. Currently, opportunities to predict pregnancy loss include (1) using transrectal ultrasonography to detect loss of the fetal heartbeat, floating debris within the placental fluids, and reductions in fetal size; (2) observing reductions in circulating progesterone concentrations; (3) detecting reductions in concentrations of circulating placental products; namely, pregnancy-associated glycoproteins and microRNAs; and (4) detecting reductions in the early pregnancy-dependent increase in interferon-stimulatory gene expression in peripheral blood leukocytes. An achievable goal may be to identify markers of embryo mortality so that researchers and clinicians can focus their efforts on developing intervention strategies for cows identified to be at risk for pregnancy failure., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.

In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To evaluate how the inclusion of Bos indicus genotype influences early fetal development in cattle, a reciprocal embryo transfer approach was used in a completely randomized design with a 2 × 2 × 2 factorial arrangement of treatments to generate 55 pregnancies over 2 consecutive years (n = 55). Recipient cows were randomly assigned to (i) a diet that met daily energy maintenance requirements (MAINT) or (ii) a diet that restricted intake to 70% of the energy maintenance requirements (RESTR). Angus (AN) and Brangus (BN) embryo donors were superovulated and artificially inseminated with female sexed-sorted semen from the same breed. Embryos were then randomly transferred to either AN or BN recipients fed their respective diets for 28 d. Recipients remained on the dietary scheme until day 91 of gestation and were then comingled and fed a common diet that met their energy requirements until calving. Measurements included pregnancy establishment at day 28 of gestation, interferon-stimulated genes (ISG) expression in peripheral blood leukocytes, pregnancy-associated glycoproteins (PAG; using two commercial [A1 and A2] and one in-house assay), and fetal crown-to-rump length (CRL). Recipients in the RESTR diet had lower BWs and BCS (diet × day; P < 0.01) than MAINT recipients. Energy-restricted AN recipients experienced greater (recipient breed × diet, P < 0.01) pregnancy failure by day 28 than the other recipient breed × diet combinations. Restricted recipients that received AN embryos experienced greater pregnancy failure than RESTR recipients receiving BN embryos (embryo breed × diet; P = 0.03). No relevant differences were observed in ISG expression (P > 0.10). Recipients that received BN embryos had greater plasma concentrations of PAG in both A1 (embryo breed × day, P < 0.01) and A2 (embryo breed; P < 0.01). Alternatively, recipients that received AN embryos had greater plasma concentrations of PAG for the in-house assay (embryo breed × day; P < 0.01). In addition, fetuses from AN recipients had greater CRL on day 91 (breed × day, P < 0.01). In summary, Bos taurus cows experienced greater pregnancy failure when nutrient restricted. Furthermore, fetal size and the profile of PAG production during early gestation differed between B. indicus-influenced and B. taurus cattle., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To provide insight into maternal recognition of pregnancy control in equids, the mitogenic and developmental effects of endometrium-expressed growth factors (epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1)) were examined in equine iTr cells, an equine trophectoderm cell line. Initial western blots revealed that HGF and IGF-1 stimulate phosphorylation of AKT serine/threonine kinase 1 (AKT1) and EGF, FGF2, or HGF resulted in phosphorylation of both extracellular signal-regulated kinase 1 (ERK1) and ERK2. Mitotic activity was stimulated (P < 0.05) by EGF, FGF2, and HGF. Chemical disruption of mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) phosphorylation suppresses (P < 0.05) proliferation in control and growth factor treated cells demonstrating a dependence on ERK1/2 for mitotic activity. Treatment of iTr cells with EGF or HGF in the presence of an AKT1 inhibitor prohibits (P < 0.05) growth factor stimulated proliferation. The effect of EGF, FGF2, HGF, and IGF-1 on steroid biosynthetic enzyme gene expression, including prostaglandin-endoperoxide synthase 2 (PTGS2), was determined by real-time PCR. Neither EGF, FGF2, nor IGF-1 affected PTGS2 expression while HGF caused a two-fold increase (P < 0.05) in expression. Co-supplementation with HGF and an AKT1 inhibitor did not block PTGS2 expression, whereas providing an MEK1/2 inhibitor prevented (P < 0.05) the HGF-mediated increase in PTGS2. These results provide novel evidence of a role for HGF in equine trophectoderm proliferation and prostaglandin biosynthesis.

Background: Work in other species suggests that interleukin-6 (IL6) promotes early embryo development. It was unclear whether IL6 serves as an embryokine in cultured bovine embryos. This work was undertaken to elucidate the role of IL6 during in vitro bovine embryo production., Results: Transcripts for IL6 and its two cognate receptor subunits (IL6R, IL6ST) were confirmed in bovine embryos from the 1-cell to blastocyst stages. Supplementing 100 ng/ml recombinant bovine IL6 to in vitro-produced bovine embryos at day 1, 3 or 5 increased (P < 0.05) inner cell mass (ICM) cell number and the ICM:trophectoderm (TE) ratio but not TE cell number. No increase in ICM or TE cell number was observed after supplementation of 1 or 10 ng/ml IL6 beginning at either day 1 or 5. Sequential supplementation with 100 ng/ml IL6 at both day 1 and 5 (for a total of 200 ng/ml IL6) increased (P < 0.05) ICM cell number to a greater extent than supplementing IL6 at a single time period in one study but not a second study. Additionally, providing 200 ng/ml IL6 beginning at day 1 or 5 yielded no further increase on ICM cell numbers when compared to supplementing with 100 ng/ml IL6. IL6 treatment had no effect on cleavage or blastocyst formation in group culture. However, IL6 supplementation increased cleavage and day 8 blastocyst formation when bovine embryos were cultured individually., Conclusions: These results implicate IL6 as an embryokine that specifically increases ICM cell numbers in bovine embryos and facilitates bovine blastocyst development in embryos cultured individually.

This work examined the downstream fetal and placental outcomes of introducing a cocktail of uterine-derived growth factors during bovine embryo culture. Abattoir-derived bovine oocytes were matured and fertilized in vitro. On day 4 post-fertilization, ≥ 8-cell embryos were harvested, pooled and exposed to an embryokine mix, termed EFI, which contained recombinant human epidermal growth factor (10 ng/ml), bovine fibroblast growth factor-2 (10 ng/ml) and human insulin-like growth factor 1 (50 ng/ml) or to a carrier-only control treatment (CON). On day 7, individual, transfer-quality embryos were transferred to recipients. Timed ovulation was completed in mature, non-suckled commercial beef cows. Cows either were artificial inseminated (AI) or received an embryo (ET) on day 7 post-estrus (n = 23-31 cows/treatment over 4 replicate studies). The percentage of grade 1 and 2 morulae and blastocysts was greater (P < 0.05) for EFI-treated embryos than CON. The percentage of pregnant cows diagnosed by transrectal ultrasonography did not differ among the AI and ET groups on days 28, 42 and 56 post-estrus. There also were no differences in the ratio of male to female fetuses determined on day 60 post-estrus by transrectal ultrasonography. On day 21 post-estrus, the relative abundance of three interferon-stimulated gene (ISG) transcripts in peripheral leukocytes were not different based on AI/ET group or the sex of the conceptus. Circulating pregnancy-associated glycoprotein (PAG) concentrations differed (P < 0.05) among days. Also, a difference in PAG concentrations (P < 0.05) were detected between male and female pregnancies in the CON-ET group but not in the AI or EFI-ET groups. Crown-rump length was not affected by AI/ET group on day 42 but were less (P < 0.05) in the CON and EFI-ET groups than the AI group on day 56. These findings implicate EFI supplementation as a means for improving transferable embryo production in a bovine IVP system, but it is not clear if this treatment improves embryo competency after ET., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Emerging data highlighting gut microbiome influences on health support evaluation of how microbial fermentation end-products influence postabsorptive systems. This study aimed to investigate the effect of increased propionate status on progesterone profiles and insulin sensitivity in dairy heifers. Eleven Holstein heifers, synchronized in estrus, were assigned to one of two continuous, 5-day IV treatments: sodium propionate (PRO; n = 5) or saline (CON; n = 6). These infusions culminated in a hyperglycemic clamp with daily blood samples for an additional 7 days. Plasma propionate concentrations increased over the first 9 h in PRO heifers, then decreased until day 3 when they matched CON heifers. Maximum plasma progesterone concentrations tended to be greater in PRO heifers than CON heifers (4.19 vs 3.73 ng/mL; P = 0.087). Plateau insulin concentrations in CON animals were significantly greater than those in PRO animals (249.4 ± 25.1 vs 123.9 ± 35.8; P = 0.008) with a trend for an increased insulin sensitivity index in PRO heifers compared to CON heifers (P = 0.06). These changes in plasma propionate clearance leading to increased progesterone response and changes in insulin sensitivity suggest a role for SCFA metabolism in reproductive hormone regulation.

Background: Embryonic and fetal exposure to maternal obesity causes several maladaptive morphological and epigenetic changes in exposed offspring. The timing of these events is unclear, but changes can be observed even after a short exposure to maternal obesity around the time of conception. The hypothesis of this work is that maternal obesity influences the ovine preimplantation conceptus early in pregnancy, and this exposure will affect gene expression in embryonic and extraembryonic tissues., Results: Obese and lean ewe groups were established by overfeeding or normal feeding, respectively. Ewes were then bred to genetically similar rams. Conceptuses were collected at day 14 of gestation. Morphological assessments were made, conceptuses were sexed by genomic PCR analysis, and samples underwent RNA-sequencing analysis. While no obvious morphological differences existed between conceptuses, differentially expressed genes (≥ 2-fold; ≥ 0.2 RPKM; ≤ 0.05 FDR) were detected based on maternal obesity exposure (n = 21). Also, differential effects of maternal obesity were noted on each conceptus sex (n = 347). A large portion of differentially expressed genes were associated with embryogenesis and placental development., Conclusions: Findings reveal that the preimplantation ovine conceptus genome responds to maternal obesity in a sex-dependent manner. The sexual dimorphism in response to the maternal environment coupled with changes in placental gene expression may explain aberrations in phenotype observed in offspring derived from obese females.

The influence of exposure to overfeeding-induced maternal obesity around the time of conception on early embryogenesis was examined in the day 14 ovine conceptus. The relative abundance of FGFR2 and DNMT1 was influenced by maternal obesity status and conceptus sex, and the abundance of PPARG and PTGS2 transcripts was greater in male conceptuses regardless of the obesity status of the ewe. These observations demonstrated that short-term exposure to maternal obesity impacts early conceptus transcript patterning., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Thirty years ago, a novel type I interferon (IFN) was identified by molecular cloning of cDNA libraries constructed from RNA extracted from ovine and bovine pre-implantation embryos. This protein was eventually designated as IFN-tau (IFNT) to highlight its trophoblast-dependent expression. IFNT function is not immune related. Instead, it interacts with the maternal system to initiate the establishment and maintenance of pregnancy. This activity is indispensable for the continuation of pregnancy. Our review will describe how IFNT evolved from other type I IFNs to function in this new capacity. IFNT genes have only been identified in pecoran ruminants within the Artiodactyla order (e.g. cattle, sheep, goats, deer, antelope, giraffe). The ancestral IFNT gene emerged approximately 36 million years ago most likely from rearrangement and/or insertion events that combined an ancestral IFN-omega ( IFNW ) gene with a trophoblast-specifying promoter/enhancer. Since then, IFNT genes have duplicated, likely through conversion events, and mutations have allowed them to adapt to their new function in concert with the emergence of different species. Multiple IFNT polymorphisms have been identified in cattle, sheep and goats. These genes and gene alleles encode proteins that do not display identical antiviral, antiproliferative and antiluteolytic activities. The need for multiple IFNT genes, numerous alleles and distinct activities remains debatable, but the consensus is that this complexity in IFNT expression and biological activity must be needed to provide the best opportunity for pregnancy to be recognized by the maternal system so that gestation may continue., (© 2017 Society for Reproduction and Fertility.)

Rapid morphological and gene expression changes occur during the early formation of a mammalian blastocyst. Critical to successful retention of the blastocyst and pregnancy is a functional trophectoderm (TE) that supplies the developing embryo with paracrine factors and hormones. The contribution of TE conformational changes to gene expression was examined in equine induced trophoblast (iTr) cells. Equine iTr cells were cultured as monolayers or in suspension to form spheres. The spheres are hollow and structurally reminiscent of native equine blastocysts. Total RNA was isolated from iTr monolayers and spheres and analyzed by RNA sequencing. An average of 32.2 and 31million aligned reads were analyzed for the spheres and monolayers, respectively. Forty-four genes were unique to monolayers and 45 genes were expressed only in spheres. Conformation did not affect expression of CDX2, POU5F1, TEAD4, ETS2, ELF3, GATA2 or TFAP2A, the core gene network of native TE. Bioinformatic analysis was used to identify classes of genes differentially expressed in response to changes in tissue shape. In both iTr spheres and monolayers, the majority of the differentially expressed genes were associated with binding activity in cellular, developmental and metabolic processes. Inherent to protein:protein interactions, several receptor-ligand families were identified in iTr cells with enrichment of genes coding for PI3-kinase and MAPK signaling intermediates. Our results provide evidence for ligand initiated kinase signaling pathways that underlie early trophectoderm structural changes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies., (© 2017 Wiley Periodicals, Inc.)

Plane of nutrition effects on body, tissue and cellular growth in the neonatal calf are poorly understood. The hypothesis that a low plane of nutrition (LPN) would limit skeletal muscle size by reducing fibre growth and muscle progenitor cell activity was tested. At birth, calves were randomly assigned to either a LPN (20% CP, 20% fat; GE=1.9 Mcal/days) or a high plane of nutrition (HPN; 27% CP, 10% fat, GE = 3.8 Mcal/days) in a 2 × 3 factorial design to test the impact of diet on neonatal calf growth, organ weight and skeletal muscle morphometry with time. Groups of calves (n = 4 or 5) were euthanised at 2, 4 and 8 week of age and organ and empty carcass weights were recorded. Body composition was measured by DXA. Longissimus muscle (LM) fibre cross-sectional area (CSA), fibre/mm 2 and Pax7 were measured by immunohistology. Satellite cells were isolated at each time point and proliferation rates were measured by EdU incorporation. Calves fed a HPN had greater (p < 0.05) BW, ADG and hip height than those fed a LPN for 2, 4 or 8 weeks. HPN calves contained a greater (p < 0.05) percentage of fat tissue than LPN calves. Liver, spleen and thymus weights were less (p < 0.05) in LPN calves than HPN animals. Calves fed HPN had larger (p < 0.05) LM CSA at 8 weeks than LPN fed animals with no differences between the groups in numbers of satellite cells per fibre. Proliferation rates of satellite cells isolated from HPN fed calves were greater (p < 0.05) at 2 weeks than LPN fed animals, which exhibited greater (p < 0.05) proliferation rates at 4 weeks than HPN fed calves. We conclude a LPN diet reduces body growth and organ size and metabolically reprograms satellite cell activity., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

Caloric restriction decreases skeletal muscle mass in mammals, principally due to a reduction in fiber size. The effect of suboptimal nutrient intake on skeletal muscle metabolic properties in neonatal calves was examined. The longissimus muscle (LM) was collected after a control (CON) or caloric restricted (CR) diet was cosnumed for 8 wk and muscle fiber size, gene expression, and metabolic signal transduction activity were measured. Results revealed that CR animals had smaller ( P < 0.05) LM fiber cross-sectional area than CON, as expected. Western blot analysis detected equivalent amounts of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) but reduced ( P < 0.05) amounts of the splice-variant, PGC1α-4 in CR LM. Expression of IGF-1 , a PGC1α-4 target gene, was 40% less ( P < 0.05) in CR than CON. Downstream mediators of autocrine IGF-1 signaling also are attenuated in CR by comparison with CON. The amount of phosphorylated AKT1 was less ( P < 0.05) in CR than CON. The ratio of p4EBP1 T37/46 to total 4EBP1, a downstream mediator of AKT1, did not differ between CON and CR. By contrast, protein lysates from CR LM contained less ( P < 0.05) total glycogen synthase kinase-3β (GSK3β) and phosphorylated GSK3β than CON LM, suggesting blunted protein synthesis. Smaller CR LM fiber size associates with increased ( P < 0.05) calpain 1 (CAPN1) activity coupled with lower ( P < 0.05) expression of calpastatin , the endogenous inhibitor of CAPN1. Atrogin-1 and MuRF expression and autophagy components were unaffected by CR. Thus CR suppresses the hypertrophic PGC1α-4/IGF-1/AKT1 pathway while promoting activation of the calpain system., (Copyright © 2017 the American Physiological Society.)

The objective of this experiment was to determine whether uterine or ovarian vascular dynamics could be used to identify cows at risk for pregnancy loss. Our hypothesis was that cows that subsequently lose their pregnancy will have decreased corpus luteal (CL) perfusion, or an increased resistance index (RI; reduced blood flow), or both, at d 34 of pregnancy. Day 34 was chosen because it is a common time for dairy cattle to be checked for pregnancy. This experiment was performed in 2 replicates from November 2011 to April 2012 (n = 69) and from November 2012 to April 2013 (n = 53). Cows were bred via timed artificial insemination using Ovsynch-56 and checked for pregnancy on d 32 after artificial insemination. At d 34, cows confirmed pregnant were examined via transrectal Doppler ultrasonography. Blood samples collected via coccygeal vein were used to measure circulating plasma progesterone concentrations. Diameter of the corpus luteum and crown-rump length were measured. Color power Doppler ultrasonography was used to determine vascular perfusion to the CL, and RI was measured for the uterine arteries just after branching from the umbilical artery. Records were later examined to identify pregnancy status of cows after reconfirmation. Abortion rate did not differ between replicates (11.6% in replicate 1, 9.4% in replicate 2). Mean crown-rump length of embryos that were carried to term was greater on d 34 than that in cows that aborted (14.23 ± 0.27 vs. 13.21 ± 0.53 mm). Circulating progesterone concentration at d 34 was greater for cows that carried pregnancies to term than for those that aborted (9.1 ± 0.7 vs. 7.5 ± 1.0 ng/mL). The final logistic regression model consisted of crown-rump length, progesterone concentration, and RI of the uterine artery contralateral to pregnancy. Decreased crown-rump length and progesterone concentration tended to be associated with increased odds ratio for pregnancy loss, whereas CL perfusion and uterine blood flow were not associated with increased odds ratio of pregnancy loss. In conclusion, examining CL perfusion and RI of the uterine arteries on d 34 of pregnancy does not offer a method to identify lactating Dairy cattle at risk for pregnancy loss after d 34., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Uterine secretions are crucial for conceptus development in mammals. This is especially important for species that undergo extended preimplantation development, like cattle and other ungulates. The present study examined cooperative interactions for epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) on the proliferation of the bovine trophoblast cell line CT1 and bovine embryo development. Proliferation of CT1 cells increased after supplementation of the culture medium with 10ngmL -1 EGF, 10ngmL -1 FGF2 or 50ngmL -1 IGF1, as well as with any combination of two factors. Greater increases in CT1 cell proliferation were detected when the growth medium was supplemented with all three factors. Supplementing the culture medium with individual or multiple factors during bovine embryo culture resulted in several positive outcomes, including increased blastocyst development, expansion, and hatching to varying degrees depending on the particular factor or combination of factors. Supplementation of the culture medium with all three factors increased embryonic trophoblast cell numbers on Day 8, as well as hatching rates and blastocyst diameter on Day 12 after fertilisation. Western blot analyses and the use of pharmacological inhibitors suggest that EGF and IGF1 affect CT1 proliferation by activating mitogen-activated protein kinase 3/1, whereas FGF2 activates AKT. In conclusion, the findings of the present study indicate that there are cooperative interactions among EGF, FGF2 and IGF1 that enhance trophoblast cell development during early embryogenesis.

Leptin is involved in various reproductive processes in humans and rodents, including placental development and function. The specific ways that leptin influences placental development and function in cattle are poorly understood. This work was completed to explore how leptin regulates hormone, cytokine and metalloprotease transcript abundance, and cell proliferation in cultured bovine trophoblast cells. In the first set of studies, cells were cultured in the presence of graded recombinant bovine leptin concentrations (0, 10, 50, 250 ng/mL) for 6 or 24 h. Transcript profiles were examined from extracted RNA. Leptin supplementation did not affect abundance of the maternal recognition of pregnancy factor, interferon-tau (IFNT), but leptin increased (P < 0.05) abundance of chorionic somatomammotropin hormone 2 (CSH2; ie, placental lactogen) at both 6 and 24 h at each concentration tested. At 24 h, the greatest CSH2 abundance (P < 0.05) was detected in cells supplemented with 50 ng/mL leptin. Transcript abundance of the remodeling factor, metalloprotease 2 (MMP2), was greater (P < 0.05) in leptin-treated cells at 24 h but not at 6 h. The 24 h MMP2 response was greatest (P < 0.05) at 250 ng/mL. Transcript abundance for MMP9 was not altered by leptin treatment. In a separate set of studies, cell proliferation assays were completed. Leptin supplementation did not affect bovine trophoblast cell line proliferation at any dose tested. In conclusion, leptin supplementation did not affect bovine trophoblast cell proliferation or IFNT expression, but leptin increases CSH2 and MMP2 transcript abundance. Both of these factors are involved with peri-implantation and postimplantation placental development and function, and this implicates leptin as a potential mediator of early placental development and function in cattle., (Copyright © 2016 Elsevier Inc. All rights reserved.)