Association between Anti-Müllerian Hormone Concentration and Inflammation Markers in Serum during the Peripartum Period in Dairy Cows.

The relationships between changes in anti-Müllerian hormone (AMH) concentration and various traits, including milk somatic cell counts (SCC), were evaluated. Blood samples were collected from 43 Holstein cows 14 days before (D-14) and 10 (D10) and 28 days after (D28) parturition, and vaginal discharge score (VDS) and polymorphonuclear leukocyte (PMNL) percentages were assessed in endometrial samples at D28. Cows were separated into four quartiles (Q1-Q4) based on changes in AMH concentration during the peripartum period (AMH ratio: D28/D-14). Correlations between AMH ratio and each parameter were evaluated and classified into high-AMH (Q4, 1.83 ± 0.12, n = 11) and low-AMH (Q1, 0.83 ± 0.05, n = 11) groups. The AMH ratio was positively correlated with magnesium and non-esterified fatty acids levels, and the albumin/globulin ratio at D10 and D28, but negatively correlated with serum amyloid A (SAA) at D10. SAA and γ-globulin levels were significantly higher in the low-AMH group at D28. There was no significant difference in VDS, PMNL percentage, and milk SCC between the two groups. The decreasing AMH ratio from the prepartum to the postpartum period corresponds to high inflammation biomarker levels. Whether it subsequently affects the reproductive prognosis of postpartum cows needs further investigations.

Effects of Intrauterine Infusion of a Chitosan Solution on Recovery and Subsequent Reproductive Performance of Early Postpartum Dairy Cows with Endometritis: A Pilot Field Trial.

This study investigated the efficacy of intrauterine infusion of a chitosan solution (CHT) on uterine recovery in early postpartum dairy cows with or without endometritis, and their subsequent reproductive performance. In Experiment 1, cows with endometritis at 3 weeks postpartum were administered CHT (n = 5) and prostaglandin F2α (PGF2α) (n = 4). Untreated cows (n = 7) served as the control group. In Experiment 2, 18 cows with a normally recovered uterus at the fresh cow check (mean, 35 days postpartum) were assigned to the CHT (n = 10) and control (n = 8) groups, and intrauterine infusion was conducted in the CHT group. Overall, in Experiment 1, the percentage of polymorphonuclear leukocytes significantly declined in the CHT group (32.3 ± 10.2 to 5.5 ± 2.4, p < 0.05) from week 3 to week 5, but no decline occurred in the PGF2α and control groups. In Experiment 2, the CHT and control groups showed no significant differences in reproductive parameters, suggesting the absence of adverse effects of CHT on fertility. These results suggest that intrauterine infusion of CHT in the early postpartum period effectively accelerates uterine recovery from endometritis and might be a suitable replacement for PGF2α administration.

An overview on how cumulus cells interact with the oocyte in a condition with elevated NEFA levels in dairy cows.

Metabolic stress in humans and animals is associated with impaired fertility. A major characteristic of metabolic stress is elevated levels of free fatty acids (NEFAs) in blood due to mobilization of body fat reserves. Dairy cows undergo a period of metabolic stress during the peri-calving period, the so-called negative energy balance (NEB) in the early weeks postpartum. At the time of NEB, both saturated and unsaturated NEFAs are mobilized to serve as an alternative energy supply for cells, however in particular saturated NEFAs can have a detrimental effect on somatic cells. Circulating NEFAs are also reflected in the follicular fluid of ovarian follicles and hence reach the cumulus-oocyte-complex (COC), which implies a potential risk for the developing oocyte. To this end, the current review focusses on the impact of NEFAs on the quality of the oocyte.

Risk factors associated with reproductive performance in Japanese dairy cows: Vaginal discharge with flecks of pus or calving abnormality extend time to pregnancy.

The present study aimed to determine possible risk factors affecting reproductive performance, especially time taken to establish pregnancy in dairy cows, using Cox's proportional hazard model. The data were collected from 154 Holstein Friesian cows (199 lactations). Cows diagnosed with a vaginal discharge score (VDS) of one or calving abnormality showed significantly delayed pregnancy: hazard ratio (HR)=0.654 (95% confidence interval [CI]: 0.436-0.983; P=0.041) and HR=0.457 (95% CI: 0.270-0.774; P=0.004), respectively. Our study suggested that the occurrence of VDS of one or calving abnormality might be possible risk factors increasing the number of days open and affecting reproductive performance in dairy cows.

An oviduct-on-a-chip provides an enhanced in vitro environment for zygote genome reprogramming.

Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development.

Comparative Effects of Different Dosages of hCG on Follicular Development in Postpartum Dairy Cows With Cystic Ovarian Follicles.

The objective of this study was to determine the effects of different intramuscular dosages of human chorionic gonadotropin (hCG) on ovarian follicular development of dairy cows diagnosed with refractory cystic ovarian follicles (COFs). Cows diagnosed with COFs (≥25 mm in diameter) were allocated to four treatment groups: hCG-1 (n = 3), a single dose of 4,500 IU on day 1; hCG-2 (n = 3), 2,250 IU on days 1 and 3; hCG-3 (n = 3), 1,500 IU on days 1, 3, and 5; and hCG-C (n = 3) received saline on day 1. Blood sampling and ovarian ultrasonographic (US) examinations were performed on days 1, 3, 5, 7, and 14. A progesterone (P4) value < 1 ng/ml was used as an indicator of absence of a functional CL. A significant increase (P < 0.05) in the number of follicles < 4 mm in diameter was observed in the hCG-2 group on day 5. Additionally, there was a significant difference in the number of follicles < 4 mm (P < 0.05) between both the hCG-2 and hCG-3 groups compared to the hCG-C group on day 5, and a tendency (P = 0.08) toward a difference in the number of 5-9 mm follicles in groups hCG-3, hCG-2, and hCG-1, compared with the hCG-C group on day 7. The proportion of cows on days 7 and 14 with P4 > 1 ng/ml was 100% (3/3) and 100% (3/3) in group hCG-1; 100% (3/3) and 67% (2/3) in group hCG-2; 67% (2/3) and 100% (3/3) in group hCG-3; and 33% (1/3) and 33% (1/3) in group hCG-C, respectively. Strong tendencies of P4 increases in group hCG-1 (P = 0.054) and hCG-2 (P = 0.051) were measured after hCG administration. Additionally, P4 values tended to be higher (P = 0.07) for group hCG-1 compared to group hCG-C on day 5. The preliminary findings of this study suggest that multiple smaller doses of hCG might be equally effective as a single large dose of hCG in modulating ovarian follicular development in dairy cows with COFs.

Potential Health and Environmental Risks of Three-Dimensional Engineered Polymers.

Polymer engineering, such as in three-dimensional (3D) printing, is rapidly gaining popularity, not only in the scientific and medical fields but also in the community in general. However, little is known about the toxicity of engineered materials. Therefore, we assessed the toxicity of 3D-printed and molded parts from five different polymers commonly used for prototyping, fabrication of organ-on-a-chip platforms, and medical devices. Toxic effects of PIC100, E-Shell200, E-Shell300, polydimethylsiloxane, and polystyrene (PS) on early bovine embryo development, on the transactivation of estrogen receptors were assessed, and possible polymer-leached components were identified by mass spectrometry. Embryo development beyond the two-cell stage was inhibited by PIC100, E-Shell200, and E-Shell300 and correlated to the released amount of diethyl phthalate and polyethylene glycol. Furthermore, all polymers (except PS) induced estrogen receptor transactivation. The released materials from PIC100 inhibited embryo cleavage across a confluent monolayer culture of oviduct epithelial cells and also inhibited oocyte maturation. These findings highlight the need for cautious use of engineered polymers for household 3D printing and bioengineering of culture and medical devices and the need for the safe disposal of used devices and associated waste.

Cumulus cells protect the oocyte against saturated free fatty acids.

In the cow a major characteristic of metabolic stress is an elevated level of plasma free fatty acid, due to increased lipid mobilization from adipose tissue. Elevated levels of free fatty acids in blood (complexed to albumin) are associated with increased lipotoxicity in non-adipose tissue. An overview is provided on the negative impact of free fatty acids and the metabolic stress imposed on the oocyte and early embryo and thus on bovine fertility. There is increasing evidence that in vitro as well as in vivo the elevated levels of free fatty acids in blood during metabolic stress can severely hamper oocyte and embryo development. However, fatty acids do also form an essential nutrient source for the oocyte and embryo, which indicates that these good and bad effects of fatty acids should be in subtle balance to optimize the developmental competence of the oocyte and embryo.

Effect of diagnosis and treatment of clinical endometritis based on vaginal discharge score grading system in postpartum Holstein cows.

In this study, the prevalence, effectiveness of diagnosis, and treatment based on vaginal discharge score (VDS) of clinical endometritis in cattle were evaluated. To detect clinical endometritis and classify its severity, vaginoscopy was performed during 21 to 60 days postpartum in 164 Holstein cows consisting of 229 lactations. Groups were defined using the 4-point VDS scale. Study groups included the following: non-endometritis (VDS=0; no/clear mucus; NEM group; n=168); mild endometritis, no treatment (VDS=1; mucus containing flecks of white/off-white pus; NTR group; n=30); and severe endometritis, treated with PGF2α (VDS≥2; discharge containing <50% pus; and VDS=3; discharge containing >50% pus, and fluid or uterine horn asymmetry; TEM group; n=31). Cows treated with PGF2α that did not recover (VDS≥1, n=5) received intrauterine procaine penicillin and streptomycin. Prevalence of clinical endometritis (VDS≥1) was 26.6%. The NTR group required significantly more artificial inseminations per pregnancy than NEM and TEM groups (2.8 ± 1.8 vs 2.0 ± 1.3, 1.9 ± 0.8, P<0.05). In survival analysis, the proportion of non-pregnant cows was higher in the NTR group compared to the NEM (P=0.012) and TEM (P=0.076) groups. In the TEM group, calving to first artificial insemination interval tended to be higher in cows treated 41 to 60 days postpartum than cows treated 29 to 40 days postpartum (97.2 ± 27.1 vs 74.4 ± 19.7, P=0.084). Our study suggests that cows with VDS=1 may require treatment to recover fertility. Diagnosis and treatment of clinical endometritis based on a VDS grading system may improve dairy herd reproductive performance.

Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.

Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production.

The oviduct was long considered a largely passive conduit for gametes and embryos. However, an increasing number of studies into oviduct physiology have demonstrated that it specifically and significantly influences gamete interaction, fertilization and early embryo development. While oviduct epithelial cell (OEC) function has been examined during maintenance in conventional tissue culture dishes, cells seeded into these two-dimensional (2-D) conditions suffer a rapid loss of differentiated OEC characteristics, such as ciliation and secretory activity. Recently, three-dimensional (3-D) cell culture systems have been developed that make use of cell inserts to create basolateral and apical medium compartments with a confluent epithelial cell layer at the interface. Using such 3-D culture systems, OECs can be triggered to redevelop typical differentiated cell properties and levels of tissue organization can be developed that are not possible in a 2-D culture. 3-D culture systems can be further refined using new micro-engineering techniques (including microfluidics and 3-D printing) which can be used to produce 'organs-on-chips', i.e. live 3-D cultures that bio-mimic the oviduct. In this review, concepts for designing bio-mimic 3-D oviduct cultures are presented. The increased possibilities and concomitant challenges when trying to more closely investigate oviduct physiology, gamete activation, fertilization and embryo production are discussed.

Free fatty acid levels in fluid of dominant follicles at the preferred insemination time in dairy cows are not affected by early postpartum fatty acid stress.

The fertility of high-yielding dairy cows has declined during the last 3 decades, in association with a more profound negative energy balance (NEB) during the early weeks postpartum. One feature of this NEB is a marked elevation in circulating free fatty acid (FFA) concentrations. During the early postpartum period (≤ d 42), circulatory FFA levels were measured weekly, and progesterone concentrations and the diameter of the dominant follicles were determined thrice weekly. Retrospectively, cows that ovulated within 35 d postpartum were grouped as "normal ovulating" cows (n = 5), and the others were grouped as "delayed ovulating" cows (n = 5). In both groups, high total FFA levels (>500 µM) were evident immediately postpartum. Interestingly, cows with delayed ovulation had higher plasma FFA concentrations in the first weeks postpartum compared with normal ovulating cows. In both cow groups, FFA decreased to control levels of non-NEB cows within 3 wk postpartum. The FFA compositions and concentrations in fluids from the dominant follicles of postpartum cows were not different between the normal and delayed ovulating cows when measured at potential insemination points: d 55, 80, and 105 postpartum. Interestingly, the concentration of monounsaturated oleic acid was higher and that of saturated stearic acid lower in follicular fluids of both groups compared with that in blood. The level of FFA in follicular fluid was correlated with the ratio of 17ß-estradiol (E2) to progesterone (P4) in follicular fluid, with a relatively high level of unsaturated FFA in follicles with a low E2:P4 ratio. Taken together, these results indicate that a more severe NEB early postpartum is related to a delay in the first postpartum ovulation and does not affect FFA composition in follicular fluid at the preferred insemination time. The high FFA level in dominant follicles with a low E2:P4 ratio may be due to a different FFA metabolism in these follicles. The diagnostic value of this observation for selective screening of dominant follicles needs further investigation.

Follicular 17ß-estradiol and progesterone concentrations and degree of cumulus cell expansion as predictors of in vivo-matured oocyte developmental competence in superstimulated heifers.

The quality of an oocyte is crucial for successful generation of offspring, but few selection parameters have been identified that reliably predict oocyte developmental competence. The objective of the present study was to determine whether the developmental competence of in vivo-matured oocytes derived from superstimulated heifers could be predicted by 17ß-estradiol and progesterone concentrations in follicular fluid, degree of cumulus cell expansion, and follicular diameter. Cumulus oocyte complexes were individually collected from follicles ≥8 mm 22 hours after an induced LH peak and individually fertilized and cultured in vitro. Only oocytes that originated from follicles with 17ß-estradiol ≤0.25 µM and progesterone ≥0.26 µM developed into blastocysts. When a combination of these cutoff values was evaluated as a predictor of oocyte competence, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 75%, 49%, and 100%, respectively. Hormone concentrations in follicular fluid were also associated with the degree of cumulus cell expansion and only cumulus oocyte complexes with full expansion developed into blastocysts; sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 71%, 45%, and 100%, respectively, when full expansion was used as the predictive criterion for blastocyst production. Follicular diameter was not a good predictor of oocyte competence. In conclusion, concentrations of 17ß-estradiol and progesterone in the preovulatory follicle and the degree of cumulus cell expansion are predictors of blastocyst production in superstimulated heifers and can be used as selection markers for oocyte developmental competency.

Bovine cumulus cells protect maturing oocytes from increased fatty acid levels by massive intracellular lipid storage.

Metabolic conditions characterized by elevated free fatty acid concentrations in blood and follicular fluid are often associated with impaired female fertility. Especially elevated saturated fatty acid levels can be lipotoxic for several somatic cell types. The aim of this study was to determine the impact of elevated free fatty acid concentrations in follicular fluid on neutral lipids (fatty acids stored in lipid droplets) inside cumulus cells and oocytes and their developmental competence. To this end, cows were exposed to a short-term fasting period during final oocyte maturation. This resulted in elevated, but distinct, free fatty acid concentrations in blood and follicular fluid and a rise in the concentrations of in particular fatty acids with a chain length of 14-18 carbon atoms. Interestingly, elevated free fatty acid concentrations in follicular fluid resulted in a massive increase in the level of neutral lipids in cumulus cells, whereas the level of neutral lipid in oocytes was hardly affected. Furthermore, competence of oocytes to develop to the blastocyst stage after fertilization and culture of cumulus-oocyte-complexes of the experimental and control group was not different. In conclusion these data suggest that short-term elevated free fatty acid concentrations in follicular fluid do not harm oocyte developmental competence. We propose that the involvement of high levels of mobilized oleic acid in follicular fluid in combination with the induced lipid storage in cumulus cells serves to prevent harmful saturated fatty acid exposure to the oocyte.

Oleic acid prevents detrimental effects of saturated fatty acids on bovine oocyte developmental competence.

Mobilization of fatty acids from adipose tissue during metabolic stress will increase the amount of free fatty acids in blood and follicular fluid and, thus, may affect oocyte quality. In this in vitro study, the three predominant fatty acids in follicular fluid (saturated palmitic and stearic acid and unsaturated oleic acid) were presented to maturing oocytes to test whether fatty acids can affect lipid storage of the oocyte and developmental competence postfertilization. Palmitic and stearic acid had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets and a concomitant detrimental effect on oocyte developmental competence. Oleic acid, in contrast, had the opposite effect, causing an increase of lipid storage in lipid droplets and an improvement of oocyte developmental competence. Remarkably, the adverse effects of palmitic and stearic acid could be counteracted by oleic acid. These results suggest that the ratio and amount of saturated and unsaturated fatty acid is relevant for lipid storage in the maturing oocyte and that this relates to the developmental competence of maturing oocytes.

Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex.

Frozen-thawed ovarian cortical fragments (1 mm(3)) were autotransplanted to the uterus of completely ovariectomized goats. The grafts developed preovulatory follicles, accompanied by estrous behavior and a rise in plasma E(2) levels, demonstrating successful cryopreservation and transplantation.

Sperm binding properties and secretory activity of the bovine oviduct immediately before and after ovulation.

The possibility that differences in hormonal regimes between the two oviducts in the cow around ovulation affects secretory activity of the oviduct epithelial cells and/or sperm-oviduct binding was studied. Oviducts were collected immediately after slaughter at 6 hr before to 5 hr after timed ovulation of 14 normally cyclic cows that had been inseminated (n = 6) or not (n = 8) and material obtained from the same cows was processed in three ways. First, in vivo, after artificial insemination of the cows, low numbers of sperm cells (approx. 15 per oviduct) were found within the entire oviducts as observed by scanning electron microscopy (SEM). Almost all sperm were located in the isthmus and then only on ciliated cells and showed without exception fully matured, intact morphology. Secretory activity of noninseminated oviduct epithelia was induced after ovulation which was most predominant in the pockets of the ipsi-lateral ampulla compared to the contra-lateral ampulla (P < 0.01). Second, ex vivo, explants dissected from oviducts of the noniseminated cows were incubated with sperm. In all cases, the sperm bound to the explants in a similar pattern as observed in vivo and this binding was strictly fucose-dependent. The main difference with in vivo experiments was the high numbers of sperm bound at any site of the oviduct ( approximately 3,000 cells per mm(2)) indicating the high sperm binding capacity of the oviduct epithelia. Ovulation induced a striking drop in sperm binding capacity in the oviducts and was most pronounced in the isthmus ( approximately 1,300 cells per mm(2); P < 0.001) and to a lesser extent in the ampulla ( approximately 2,000 cells per mm(2), P < 0.01). Third, in vitro, pieces of tissue dissected from oviducts of the noninseminated cows were cultured to mono-layers. Culturing epithelial cells resulted in loss of their normal morphological appearance. In all cases, the sperm binding capacity in monolayers was very low (<50 cells per mm(2)) when compared to corresponding explants (P < 0.0001). Sperm binding to monolayers originating from the isthmus (<25 cells per mm(2)) was lower than in those from the ampulla (40-50 cells per mm(2); P < 0.01) and remained similar after ovulation. In all three approaches, no significant differences were found in sperm-oviduct binding characteristics and sperm-distribution in the ipsi- versus contra-lateral oviducts. This indicates, that systemic endocrine changes around ovulation rather than specific oviduct changes at the ipsi-lateral oviduct induce secretion in oviduct epithelial cells, and thus induce sperm release.

Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos.

Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activation of the embryonic genome. In the present study, ribosomal RNA (rRNA) transcription was investigated by visualization of the rRNA by fluorescent in situ hybridization, and subsequent visualization of the argyrophilic nucleolar proteins by silver staining. A total of 145 in vivo developed and 200 in vitro produced bovine embryos were investigated to allow comparison of transcription initiation. Signs of active transcription of rRNA were observed in the third cell cycle in 29% of the in vitro produced embryos (n = 35) and in 58% of the in vivo developed embryos (n = 11). Signs of active transcription of rRNA were not apparent in the early phase of the fourth cell cycle but restarted later on. All embryos in the fifth or later cell cycles were all transcribing rRNA. The signs of rRNA synthesis during the third and fourth embryonic cell cycle could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. In conclusion, rRNA transcription is initiated during the third cell cycle at a low level in both in vivo developed and in vitro produced bovine embryos. Transcription seems to be interrupted during the G1 phase of the fourth cell cycle, but reinitiates in the late half of the cycle and persists during subsequent cell cycles.