Quantitative analyses of insulin-like peptide 3 and sex steroid hormones in dominant follicles and corpora lutea during the estrus cycle and in follicular cysts in beef heifers

Insulin-like peptide 3 (INSL3) and sex steroids were measured in bovine dominant follicles and corpora lutea during the estrus cycle and in follicular cysts. Paired ovaries from beef heifers (n = 47) were classified, by their morphological features, either into four stages of the estrus cycle (Day 1 = day of ovulation, Day 20 = day of estrus) as Stage I (Days 1–4; n = 8), Stage II (Days 5–10; n = 10), Stage III (Days 11–17; n = 8), and Stage IV (Days 18–20; n = 11) or follicular cystic (n = 10). Cysts (n = 15) were subdivided into estrogen-active (n = 7) and estrogen-inactive (n = 8) cysts. INSL3, testosterone, and estradiol-17β concentrations in the dominant follicular fluid of Stage IV were higher than those in Stages II and III (P < 0.05). INSL3 concentrations in the cystic fluid were similar to those in dominant follicles at Stage IV, whereas testosterone and estradiol-17β concentrations were lower in cysts (P < 0.05). INSL3 content per estrogen-inactive cyst was higher than that of Stage IV (P < 0.05). INSL3 and progesterone concentrations in luteal tissue and contents per corpus luteum were higher in Stages II and III (P < 0.05). In conclusion, INSL3 secretion in bovine dominant follicles increased with maturation. Follicular cysts may retain the production of INSL3 during their formation but tend to lose the capacity for testosterone secretion. Estrogen-inactive cysts subjected to advanced atresia may accumulate more INSL3. INSL3 production in bovine corpora lutea is enhanced during maturation.     

Plasma concentrations of inhibin A in cattle with follicular cysts: relationships with turnover of follicular waves and plasma levels of gonadotropins and steroid hormones

We investigated the profiles of circulating levels of inhibin A and total inhibin in beef cows with follicular cysts in relation to the patterns of follicular development and circulating gonadotropins and steroid hormones. Turnover of follicular waves was monitored in five cows every 2 days for 70 days from 10 days after detection of estrus without ovulation. The mean interwave intervals were 19.6 ± 1.0 days (n = 18 waves with cysts from the five cows). Circulating levels of inhibin A were approximately 170 pg/ml before emergence of follicular waves with cysts and increased (P < 0.05) concomitantly with follicle emergence. High concentrations of inhibin A (greater than 300 pg/ml) were noted for 7 days during the growth phase of cystic follicles, but inhibin A levels decreased gradually when development of the cysts reached a plateau. This profile of inhibin A was similar to those of total inhibin and estradiol, but was inversely related to the changes in plasma FSH concentrations. LH pulse frequency and mean concentrations of LH in cows with cysts were higher than those observed in the luteal phase of normal cyclic cows. These results indicate that the capacity to secrete inhibin, as well as estradiol, is maintained in cystic follicles, the growth of which is extended by LH secretion at levels greater than those seen in the normal luteal phase. Inhibin A plays an important role in the extension of interwave intervals by suppressing recruitment of a new cohort of follicles.     

Changes in the peripheral concentrations of inhibin, follicle-stimulating hormone, luteinizing hormone, progesterone and estradiol-17beta during turnover of cystic follicles in dairy cows with spontaneous follicular cysts

Several studies have clarified that the follicular cysts degenerate and are replaced by newly growing follicles that develop into new follicular cysts without ovulation, i.e., turnover of ovarian follicular cysts in cows. However, the relativity of endocrinological changes, including the inhibin profile during turnover of spontaneous follicular cysts in dairy cows, is still unclear. In the present study, the relationship between turnover of follicular cysts and changes in the peripheral blood concentrations of progesterone (P), estradiol-17beta (E(2)), luteinizing hormone (LH), follicle stimulating hormone (FSH) and inhibin were examined in lactating dairy cows. Five cows diagnosed with follicular cysts (follicles of more than 25 mm in diameter in the absence of a corpus luteum) were investigated. Their ovarian dynamics were monitored using ultrasonography, and blood samples were collected at 2- or 3- day intervals throughout the experiment. The day when a follicle fated to become a follicular cyst reached more than 8 mm in diameter was defined as the start of a cystic follicular wave. Four of the 5 cows exhibited a similar patterns of cystic follicular changes and hormone profiles. The data from the 4 cows was used for analysis of the relationships between turnover of cystic follicles and the hormone profiles. Two or three new cystic follicular waves occurred in each cow during the experimental period. The mean diameter of the cystic follicles was more than 25 mm 13 to 15 days after the start of the cystic follicular wave, and it began to decrease 1 to 6 days before the start of the subsequent cystic follicular wave. The levels of E(2) and inhibin tended to decrease for 7 to 9 days before the start of a new cystic follicular wave and to increase concomitantly with new follicular cyst growth. The levels of FSH rose for 1 to 3 days before the start of a new cystic follicular wave. The present study clarified the relationship between FSH and inhibin during turnover of spontaneous follicular cysts in dairy cows and found that it was very similar to previous results for cows. The present results suggest that an increase in FSH secretion following a reduction in inhibin secretion triggers turnover of cystic follicles in cows with spontaneous follicular cysts.     

Relationship between ovarian ultrasonographic findings on the seventh post-estrus day and plasma progesterone concentration,nutritional metabolic factors,and pregnancy outcome in dairy cows

To improve the accuracy of ultrasonographic assessment of luteal function, we investigated the relationship between ovarian ultrasonographic findings on Day 7 (Day 1 = ovulation) and plasma progesterone (P₄) concentration, nutritional metabolic factors, and pregnancy outcome. A total of 47 spontaneous estrus events were investigated in 38 lactating Holstein cows (artificial insemination, n = 31; embryo transfer, n = 16). Transrectal ultrasonography was performed on Days 0 and 7 to measure the pre-ovulatory follicle area on Day 0 and the luteal tissue area (LTA), luteal blood flow area (LBF), relative LBF (rLBF) (= LBF/LTA), and dominant follicle area (DFA) on Day 7. Blood samples were collected on Day 7 to measure plasma P₄, insulin-like growth factor-I (IGF-I), insulin, and metabolites. Plasma P₄ concentration was positively correlated with LTA but was not associated with LBF or rLBF. Plasma P₄ concentration was positively correlated with blood glucose and IGF-I and negatively correlated with blood urea nitrogen and free fatty acid, and no significant relationship was found between the ultrasonographic findings of the corpus luteum (CL) and these blood metabolites. Pregnant cows had smaller DFA than non-pregnant cows. In conclusion, LTA measurement can help predict plasma P₄ concentration, but it was difficult to detect variations in plasma P₄ concentration in relation to changes in energy status by evaluating the CL ultrasonographically. A combined assessment of CL and first-wave dominant follicle may be important in evaluating fertility.     

Comparison of bacterial endotoxin lipopolysaccharide concentrations in the blood,ovarian follicular fluid and uterine fluid: a clinical case of bovine metritis

We investigated the concentration of the bacterial endotoxin lipopolysaccharide (LPS) in the blood, ovarian follicular fluid and uterine fluid of a clinical case of bovine metritis. A 2-year-old lactating Holstein cow exhibited continuous fever >39.5°C for more than 2 weeks after normal calving. The cow produced a fetid, watery, red-brown uterine discharge from the vagina and was diagnosed with metritis. The LPS concentrations in plasma and uterine fluid were 0.94 and 6.34 endotoxin units (EU)/ml, respectively. One of seven follicles showed an extremely high level of LPS (12.40 EU/ml) compared to the other follicles (0.62–0.97 EU/ml). These results might suggest the presence of high concentration of LPS in follicles in cows with postpartum metritis.     

Synovial fluid and plasma concentrations of ceftiofur after regional intravenous perfusion in the horse

OBJECTIVE: To determine radiocarpal (RC) joint synovial fluid and plasma ceftiofur concentrations after regional intravenous perfusion (RIP) and systemic intravenous (IV) administration. STUDY DESIGN: Experimental cross-over study. ANIMALS: Five normal adult horses. METHODS: One RC joint was randomly selected for RIP and the contralateral RC joint was sampled to determine intrasynovial ceftiofur concentrations after IV administration. Wash-out between IV and RIP was > or = 14 days. After surgical introduction of an intraarticular catheter, ceftiofur (2 g) was administered under general anesthesia either IV or by RIP after tourniquet application. Plasma and synovial fluid were collected over 24 hours. Samples were analyzed using high-performance liquid chromatography with ultraviolet detection and the results were statistically analyzed using a linear mixed effect model. RESULTS: Mean synovial fluid ceftiofur concentrations were consistently higher after RIP than after IV administration and were > 1 mug/mL (minimal inhibitory concentration [MIC] for common pathogens) for >24 hours. Mean synovial fluid peak concentration of ceftiofur after RIP and IV administration was 392.7+/-103.29 microg/mL at 0.5 hours postinjection (HPI) and 2.72+/-0.31 mug/mL at 1 HPI, respectively. Large variations in synovial fluid and plasma ceftiofur concentrations were observed between horses regardless of administration technique. RIP did not cause adverse effects. CONCLUSIONS: Under the present experimental conditions RIP with ceftiofur (2 g) induced significantly higher intraarticular antibiotic concentrations in the RC joint in comparison with IV administration. Moreover, after RIP, synovial fluid ceftiofur concentrations remain above the MIC for common pathogens (1 microg/mL) for > 24 hours. No adverse effects from the technique or the antibiotic were observed. CLINICAL RELEVANCE: RIP with high doses of ceftiofur may be a beneficial adjunctive therapy when treating equine synovial infections which are caused by cephalosporin susceptible microorganisms.     

Changes of leukocyte counts and expression of pro‐ and anti‐inflammatory cytokines in peripheral leukocytes in periparturient dairy cows with retained fetal membranes

In dairy cows, retained fetal membranes (RFM) affect reproductive performance. The aim of this study was to examine the leukocyte counts and the gene expression of tumour necrosis factor α (TNFα), interleukin 1β (IL‐1β), IL‐8, and IL‐10 in polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) in cows with (n = 5) or without (n = 5) RFM during the peripartum period. The lymphocyte counts in RFM cows were higher than those in control cows throughout the experiment (p < .05). The expression of IL‐8 in PMNs of control cows was higher (p < .05) compared with that of RFM cows postpartum. In cows with RFM, IL‐1β expression was higher (p < .05) in PMNs at 6 weeks postpartum whereas the expression of IL‐1β was lower (p < .05) in PBMCs at 4 weeks postpartum. The expression of IL‐10 in PBMCs of control cows was higher (p < .05) than that of RFM cows at 2 weeks prepartum and 4 weeks postpartum. Taken together, our data indicate that changes of gene expression of pro‐ and anti‐inflammatory cytokines in RFM cows might be associated with the delayed placental separation and development of uterine inflammation in RFM cows.     

Amino acids profile within peripheral blood and follicular fluid based on high‐performance liquid chromatography methods may explain differences in folliculogenesis between short‐term under/over‐fed treatments during luteal phase of Hu sheep

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed‐supplemented group (S, 1.5 × M, N = 12) and feed‐restricted group (R, 0.5 × M, N = 12) on days 6–12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB‐to‐FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5–3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P₄) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short‐term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.     

Changes in the C-reactive protein and 13,14-dihydro-15-keto-prostaglandin F2α concentrations of uterine lavage samples after the administration of povidone-iodine in cows

Changes in the C-reactive protein (CRP) and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) concentrations of uterine lavage fluid were examined in cows given an intrauterine povidone-iodine (PI) infusion. The mean polymorphonuclear leukocyte (PMN) ratios (the ratio of PMN to total cells) and CRP concentration of uterine lavage fluid on the day after the treatment were significantly (P<0.05) greater in the PI infusion group (PMN: 53.0 ± 32.7%, CRP: 50.2 ± 32.3 ng/mL) than in the non-treatment control group (PMN: 7.9 ± 21.9%, CRP: 17.2 ± 5.9 ng/mL), whereas there was no significant difference in the mean PGFM concentration between the two groups. The present findings suggest that the uterine CRP level is a useful biomarker of local uterine inflammation in cows.     

Resumption of ovary activity and normal estrus cycles in postpartum cows after inclusion of placenta‐containing additives in the diet

Food additives made of placenta tissues of healthy cattle, included in the diet of cows 23–38 days before calving and/or in different intervals of the postpartum period, stimulated the resumption of ovary activity and the manifestation of normal estrus cycles (with ovulation) in cows. The cow fertilization rates during 80 days after calving was 50–80%. These values increased more than twofold in comparison with the fertility level of cows fed a normal diet. The level of fertilization after inclusion of placenta‐containing additives in the diet of cows 5 days before ovulation, in estrus cycles that took place during 6 months after calving, increased 38 % on average, in comparison with that of cows fed a normal diet.     

Changes in the predicted function of the rumen bacterial community of Japanese Black beef cattle during the fattening stages according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses

We investigated changes in the predicted functions of the rumen bacterial community in Japanese Black beef cattle during fattening. Nine cattle were fed a high-concentrate diet during the early, middle, and late fattening stages consecutively (10–14, 15–22, and 23–30 months of age, respectively). The rumen fluid and solid samples collected at each stage were subjected to sequencing analyses. The sequencing results were clustered and classified into operational taxonomic units (OTUs). Representative sequences and a raw counting table for each OTU were submitted to the Piphillin website. The predicted functions were revealed by the Kyoto Encyclopedia of Genes and Genomes database as the ratio of the total sequence. In the early stage, “Biosynthesis of secondary metabolites” was significantly higher in the fluid fraction than in the solid fraction. “Two-component system” in the middle stage was significantly lower and “Purine metabolism” in the late stage was significantly higher in the fluid fraction than those in the solid fraction. The fluid fraction was significantly correlated with acetic acid, propionic acid, and bacterial metabolism, such as “Biosynthesis of secondary metabolites” and “Sugar metabolism.” Moreover, the solid fraction was correlated with “Purine metabolism” and “Biosynthesis of secondary metabolism”. These results suggest that the rumen bacterial community in Japanese Black beef cattle adapts to changes in rumen conditions by altering their functions in response to a long-term high-grain diet.