Pituitary and ovarian function in postpartum beef cows. I. Effect of suckling on serum and follicular fluid hormones and follicular gonadotropin receptors

The effect of suckling on serum and follicular fluid hormones and on follicular gonadotropin receptors was studied. Sixteen anestrous postpartum cows were assigned to 1 of 2 groups: suckled (S) or weaned (W). All calves were allowed to suckle ad libitum from parturition to 21 days postpartum when calves from W cows were weaned. All cows were ovariectomized on Day 25 postpartum. W cows had more (P less than 0.01) pulses of LH during the 96-h period from weaning until ovariectomy than S cows (6.3 vs. 1.3 pulses). Serum concentrations of prolactin (Prl), estrone (E1), estradiol-17 beta (E2) and progesterone (P) were not different (P greater than 0.10) between groups. Furthermore, there were n differences (P greater than 0.10) in follicular in contents of luteinizing hormone (LH), E1, E2 and P between the treatment groups. However, follicular fluid content of Prl was greater (P less than 0.05) in the W cows than in the S cows (123 vs. 65.1 ng/cow). The number of follicular LH receptors was greater (P less than 0.05) in the W cows than in the S cows (71.1 vs. 48.3 fmoles/mg protein) although the number of follicular follicle-stimulating hormone (FSH) receptors was not different (P greater than 0.10) between W cows and S cows (1531 vs. 1862 fmoles/mg protein). There were no correlation between serum hormone concentrations and follicular fluid hormone content; however, the numbers of follicular LH receptors and follicular fluid Prl content were highly correlated in the W cows (r = 0.85; P less than 0.05). It is concluded that removal of the suckling stimulus increases pulsatile LH release and the accumulation of Prl in the follicular fluid. These factors, either together or separately, may at least in part be responsible for the increase in follicular LH receptor concentrations that were observed in the W cows.     

Macro- and trace element concentrations in blood plasma and cerebrospinal fluid of dairy cows exposed to electric and magnetic fields.

Eight multiparous, nonlactating pregnant Holstein cows (at 198 +/- 35 days of gestation and weighing 608 + 24 kg) and seven nonlactating nonpregnant ovariectomized heifers (weighing 370 + 29 kg) were confined to wooden metabolism crates in an electric and magnetic field chamber. Subarachnoidal catheters were inserted before the activation of the electric and magnetic fields. For 30 days, cows and heifers were continuously exposed in separate trials to electric and magnetic fields (60 Hz, 10 kV/m, and 30 microT). Blood plasma and cerebrospinal fluid samples were collected for 3 consecutive days before the exposure period, the last 3 days of the exposure period, and for 3 days starting 5 days after the exposure period. Concentrations of Ca, Mg, Cu, Zn, Fe, Mn, Na, P, and K in blood plasma and cerebrospinal fluid were determined. Exposure to electric and magnetic fields resulted in decreased concentrations of Mg in blood plasma and in increased concentrations of Ca and P and decreased concentrations of Fe and Mn in cerebrospinal fluid.     

Milk yield, energy balance, hormone, follicular and oocyte measures in early and mid-lactation Holstein cows

The purpose of the study was to determine the influence of energy status on metabolic and endocrine measures, follicular development, and the quality of oocytes obtained from cows during early and mid-lactation (ML). We selected Holstein cows at calving to be assigned to the early lactation (EL) group (n = 8), while we assigned cows at about day 90 postpartum to the ML group (n = 7). We obtained blood samples twice weekly from 4 weeks before aspiration to the aspiration periods for metabolite and hormone determinations. We performed ultrasound-guided transvaginal follicular aspiration (TVFA) twice weekly on all cows for a 10-week period. We obtained follicular fluid from the largest follicle > 10 mm in diameter for hormone determinations. We analyzed data by ANOVA, using the general linear model (GLM) procedures. Energy balance was positive (2.43 +/- 0.32 Mcal/kg) for ML cows and negative (-1.55 +/- 0.33 Mcal/kg) for EL cows. Serum progesterone (P4) for ML cows decreased rapidly from the first aspiration session (2.7 +/- 0.1 ng/ml) and reached a nadir at Week 8 (0.33 +/- 0.1 ng/ml), while follicular fluid P4 increased from 0.9 +/- 0.5 to 5.6 +/- 0.05 ng/ml. Serum and follicular fluid P4 remained relatively constant over the entire aspiration period for EL cows. Follicular fluid insulin-like growth factor I (TGF-I) concentrations increased linearly for EL and ML cows, but the increase was more rapid (159 +/- 36 to 200 +/- 36 ng/ml) for ML cows than for EL cows (145 +/- 36 to 164 +/- 36 ng/ml). Serum IGF-I followed the same pattern for ML cows but declined for EL cows. Early lactation cows experienced a rapid decrease in serum nonesterified fatty acids (NEFA; 0.32 +/- 0.2 to 0.22 +/- 0.2 meq/l), while serum NEFA concentrations were relatively stable (0.19 +/- 0.2 to 0.22 +/- 0.2 meq/l) for ML cows over the aspiration period. The number of follicles obtained from the twice weekly aspiration sessions increased linearly for both EL and ML cows (P < 0.05) over the 10-week period. However, the number of follicles increased from 14.2 +/- 0.5 (Day 119) to 18.1 +/- 0.5 (Day 190) in the ML cows, compared to the changes from 14.9 +/- 0.3 (Day 32) to 15.7 +/- 0.5 (Day 90) for the EL cows. These results indicate that cows are physiologically under more production stress during EL, but increasing follicular fluid and serum IGF-I throughout ML may reflect potential differences in follicle and oocyte measures, compared to cows in EL.     

Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress

It is known that prolactin (PRL) is the third pituitary hormone serving gonadotropic function in mammals. However, its role in the regulation of ovarian folliculogenesis and, in particular, its relationship to follicular atresia as well as the mechanism of its influence on follicular cells are poorly understood. We investigated PRL levels in follicular fluids (FFs) and intracellular store calcium ([Ca2+]is) in cell walls of bovine ovarian follicles with diameters of 10 to 20 mm and their relationship to follicular atresia. Ovarian follicles were categorized on the basis of macroscopic criteria and of microscopic examination of granulosa cell (GC) smears. Prolactin concentrations in FFs were measured by RIA and levels of [Ca2+]is in follicular cells were determined by using the fluorophore chlortetracycline. Compared to atretic follicles, morphologically normal follicles were characterized by higher concentrations of PRL in FFs (P < 0.001) and lower contents of [Ca2+]is in follicular cells (P < 0.01). Furthermore, follicles containing no more than 20% of pycnotic GCs had higher levels of PRL in their fluids than those containing over 40% of pycnotic GCs (P < 0.05). Finally, the direct effect of PRL on [Ca2+]is content in follicular cells was studied in vitro. Compared to control, PRL decreased (P < 0.001) the levels of [Ca2+]is in the cells after 24 h culture of follicular walls from morphologically normal follicles in TCM 199 supplemented by 10% fetal calf serum. Our findings suggest that the decline of PRL concentrations in FFs and the rise of [Ca2+]is contents in follicular cells are related to atresia of large bovine follicles and that there appears to be a relationship between the two biochemical parameters.     

Concentrations of somatotropin and prolactin in the follicular fluid and blood serum of cows in different phases of the estrous cycle

Comparative investigations of somatotropin and prolactin contents in the fluid of antral follicles and blood serum of cows in different phases of the oestrous cycle were performed. The somatotropin concentration in the fluid was shown to rise with increasing the follicle diameters from 3-5 to 6-10 mm in the follicular phase and to decrease in follicles of diameter 11-20 mm in the luteal phase. The prolactin concentration was higher in the fluid of follicles 11-20 mm in diameter than in those of 3-5 mm in diameter in the follicular phase and did not depend on the follicle size in the luteal phase. Concurrently, the prolactin content in follicles 3-5 mm in diameter was higher in the luteal than follicular phase of the cycle. As compared to the follicular phase, an increase in the prolactin concentration in the bovine blood serum during the luteal phase was also found. The data obtained indicate that changes in the somatotropin and prolactin contents in the follicular fluid are related to processes regulating growth and development of antral follicles depending on the phase of oestrous cycle and to changes in the blood hormone concentrations as well.     

Elevated inhibin concentration in the follicular fluid of dairy cows with chronic cystic ovarian disease

This study compared serum and follicular fluid inhibin and gonadotropin profiles between chronic cystic ovarian diseased (CCOD) and normal cyclic dairy cows. Blood samples and follicular fluid were collected from CCOD cows (n=15) and cyclic cows in the follicular phase of the estrous cycle (control, n=6) and analyzed for inhibin, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations. There was a significant increase in inhibin and a decrease in FSH and LH concentrations in the follicular fluid of CCOD cows compared with those of cyclic cows (P < 0.05). Mean serum inhibin, FSH and LH concentrations between CCOD and cyclic cows were not differnt (P > 0.05), however, there was a tendency for serum inhibin to be higher and FSH to be lower in CCOD cows compared to cyclic animals (P < 0.1). The FSH pulse frequency also was lower in CCOD cows than in cyclic cows (P < 0.05). These data suggest that increased production of inhibin from cystic follicles of CCOD cows alters pituitary FSH secretion and subsequently reduces the concentration of FSH in follicular fluid. As a result, decreased FSH stimulation at the ovarian level could ultimately lead to the reduction in follicular LH and FSH receptor concentrations, resulting in abnormal follicular steroidogenesis in CCOD dairy cows.     

Ovarian hydrobursitis in female camels (Camelus dromedaries): biochemical, bacterial and protozoal evaluation

The aim of this study was to evaluate female camels affected with ovarian hydrobursitis (n = 31) for hematological and biochemical findings and for bacterial and protozoal infections. Blood samples were obtained and surgical ablation of the affected bursa was performed. Bursal fluid, follicular fluid, and serum were subjected to hormonal and biochemical analyses. Bursal fluids were cultured and colonies were identified using BioMérieux Vitek two compact system. Passive haemagglutination test was used for detection of Trypanosoma evansi. Indirect ELISA technique was carried out for detection of anti-Hydatid cysts anti-bodies. Neutrophilia was found in the affected animals (P = 0.01) with tendencies for monocytosis (P = 0.06) and eosinophelia (P = 0.05). Bursal fluid had a tendency for high estradiol-17β concentration compared to blood serum (P = 0.07). Progesterone and cholesterol concentrations were similar in bursal fluid, follicular fluid and serum. Total protein, phosphorus, and magnesium concentrations were greater (P < 0.05) in the bursal fluid than in serum. Oligella urethralis, Alloiococcus otitis, Granulicatella adicens, Escherichia coli, Sphingobacterium thalpophilum, Streptococcus sanguinis, Aeromonas salmonicida, Pseudomonas stutzeri, Staphylococcus warneri, Staphylococcus hominis, and Rhizobium radiobacter were isolated from 46.7% of bursal fluids. T. evansi was positive in 9.7% of cases. None were positive for hydatid cyst. Accordingly, we suggest that the ovarian hydrobursitis syndrome is initially an inflammatory process and the accumulated bursal fluid is partially originated from follicular fluid.     

Immunological study of ovarian inhibin

Antisera to purified porcine follicular fluid inhibin of 32 K protein (pFF 32 K inhibin) were raised in rabbits. Increasing doses of an antiserum with high titer could neutralize the maximal suppression of FSH secretion caused by 10 ng of pFF 32 K inhibin from rat anterior pituitary cells in culture. A radioimmunoassay was developed using the antiserum and 125I-labelled pFF 32 K inhibin. Specificity of the antiserum was examined by comparing immunological and biological potencies of various inhibin preparations. Cross-reactivity tests revealed that the antiserum almost recognizes rat ovarian inhibin preparations. The antiserum also recognizes purified bovine follicular fluid inhibin of 32 K protein (bFF 32 K inhibin), but with a cross-reactivity of approximately 20%. Cross-reactivity of human follicular fluid to the antiserum was less than 10%. The antiserum also recognizes inhibin forms of higher molecular weights, 100 K, 80 K, and 55 K proteins, which have previously been identified by gel filtration or SDS-PAGE of crude pFF inhibin preparations under protein-dissociation conditions, indicating that these inhibin forms have common or closely related immunodetermining sites.     

Depression of follicle stimulating hormone in bulls injected with follicular fluid

Eight bulls were divided into two groups and injected with either charcoal-extracted steer blood serum or charcoal-extracted bovine follicular fluid (bFF). Ten-milliliter injections were given subcutaneous every 12 h for 4 wk. Jugular blood collected before, during and after the injection period was analyzed for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by radioimmunoassay. All bulls were exposed to restrained, estrual heifers for 15 min every 2 wk for 16 wk starting 4 wk before the first injection. The number of mounts and services by each bull was recorded. Semen was collected with an artificial vagina and evaluated on alternate weeks during the same period. The concentration of FSH in serum decreased (P < 0.05) by 12 h after the first injection and remained 61% lower than that of serum-injected bulls during the injection period. The concentration of FSH increased (P < 0.05) by 3 d after the last injection. Injections of bFF did not affect the concentration of LH in serum. Bovine follicular fluid injections significantly depressed FSH; however, libido, serving capacity, and semen characteristics were unchanged.     

Association between blood plasma urea nitrogen levels and reproductive fluid urea nitrogen and ammonia concentrations in early lactation dairy cows

Two experiments were conducted to study the relationship of blood plasma urea nitrogen (PUN) concentrations with NH3, urea nitrogen, K, Mg, P, Ca, and Na concentrations in fluid of preovulatory follicles (experiment 1) and the relationships of PUN concentration and stage of estrus cycle with ammonia and urea nitrogen concentrations in uterine fluids (experiment 2) in early lactation dairy cows. Mean PUN levels were used to distribute cows into two groups: cows with PUN>or=20 mg/dl (HPUN), and cows with PUN<20 mg/dl (LPUN). In experiment 1, blood and follicular fluids from preovulatory follicles of 38 early lactation dairy cows were collected on the day of estrus (day 0) 4h after feed was offered. Follicular fluid NH3 was higher (P<0.01) in HPUN cows (339.0 micromol/L+/-72.2) compared to LPUN cows (93.9 micromol/L+/-13.1). Follicular fluid urea N was higher (P<0.001) in HPUN cows (22.4 mg/dl+/-0.4) compared to LPUN cows (17.0 mg/dl+/-0.3). PUN and follicular fluid urea N were correlated (r2=0.86) within cows. In experiment 2, blood and uterine fluids were collected from 30 cows on day 0 and on day 7. Uterine fluid NH3 was higher (P=0.05) in HPUN cows (1562 micromol/L+/-202) than in LPUN cows (1082 micromol/L+/-202) on day 7, but not on day 0. Uterine fluid urea N was higher (P<0.001) in HPUN cows than in LPUN cows on day 0 (26.9 mg/dl+/-1.3 and 20.4 mg/dl+/-0.7) and day 7 (26.5 mg/dl+/-1.1 and 21.4 mg/dl+/-1.1). There was a correlation (r2=0.17) between PUN and uterine fluid urea N within cows. The results of this study indicate that high PUN concentrations were associated with elevated NH3 and urea N concentrations in the preovulatory follicular fluids on the day of estrus and in the uterine fluid during the luteal phase of the estrous cycle in early lactation dairy cows. Elevated NH3 or urea N concentrations in the reproductive fluids may contribute to reproductive inefficiency in dairy cows with elevated plasma urea nitrogen due to embryo toxicity.     

Direct effects of ovine follicular fluid on ovarian steroid secretion and expression of markers of cellular differentiation in sheep

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin-activin beta B subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.     

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 × 0.47 or 5 × 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.     

The influence of serum separators on biochemical values in experimental animals]

The influence of three serum separators, A, B and C, on biochemical values was examined. With A or B, changes in the values of ChE, LAP, UN, K, P, Cl and Ca in rat serum; ChE, UN, Cl and Ca in dog serum; and K and P in monkey serum took place over a period of 20 min after blood collection. Therefore, the biochemical values of whole blood were considered to be stable after 20 min. Thus, biochemical tests were conducted on serum from the three serum separators after allowing the blood to stand 20 min. The values obtained for each separator were not markedly different from those of the control. These results suggest that a serum separator is useful for separation of serum of experimental animals under the proper conditions.     

Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase Delta(4), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P4), oestradiol-17beta (E2) and androstenedione (A4) concentrations. Frozen sections (14 microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3beta-HSD.     

Follicular fluid effects on progesterone secretion are not due to follicle-stimulating hormone or steroids

An antiserum raised against porcine follicle-stimulating hormone (FSH) was unable to eliminate the stimulatory action of fluid from large antral porcine follicles on progesterone secretion by granulosa cells from small antral porcine follicles. The same titers of the antiserum were completely effective at eliminating the effect of 2 micrograms of NIH-FSH-P12, whereas maximal stimulation of progesterone secretion was observed with 0.5 micrograms FSH/ml. The androgen and estrogen concentrations measured in charcoal-treated inhibitory follicular fluid from small porcine antral follicles and from stimulatory follicular fluid from large follicles were added separately and together to culture media supplemented with serum to determine if these low concentrations (5 X 10(-11) to 5 X 10(-10) M) of steroids could mimic the actions of follicular fluid on progesterone secretion. Neither the inhibitory nor the stimulatory actions of the follicular fluids could be mimicked by these low concentrations of steroids. Higher concentration of steroids (10(-8) to 10(-7) M range) did stimulate progesterone secretion as reported by others. Our data indicate that the actions of charcoal-treated follicular fluids on granulosa cell progesterone secretion cannot be explained by difference in FSH or steroid contents between the inhibitory and stimulatory fluids and serum.     

Calcium measurements in primates during and after hypokinesia in establishing calcium deficiency during prolonged hypokinesia

Hypokinesia (diminished movement) induces significant calcium (Ca) changes, but little is known about the effect of hypokinesia (HK) on Ca deficiency. Measuring Ca changes during and after HK the aim of this study was to determine Ca deficiency during prolonged HK. Studies were done on 12 male Macaca mulatta (rhesus monkeys) aged 3–5 yr (5.58–6.42 kg) during a 90-d pre-HK period, a 90-d HK period, and a 15-d post-HK period. Monkeys were equally divided into two groups: vivarium control monkeys (VCM) and hypokinetic monkeys (HKM). Hypokinetic monkeys were kept in small individual cages that restricted their movements in all directions without hindering food and water intakes. Urinary, fecal, and serum Ca, urinary and serum magnesium (Mg) and phosphate (P), serum intact parathyroid hormone (iPTH), and calcitonin (CT) concentration, body weight, food intake, fluid consumed and eliminated in urine were measured. During the HK period, fecal Ca loss, urinary Ca, P, and Mg excretion, fluid elimination, and serum P, Ca, and Mg concentration increased significantly (p≤0.01), whereas serum iPTH and CT concentration, food and fluid intakes, and body weight decreased significantly (p≤0.01) in the HKM group when compared with the VCM group. During the initial days of the post-HK period, serum Ca, Mg, and P concentration, fecal Ca loss, urinary Ca, Mg, and P excretion, and fluid elimination decreased significantly (p≤0.01), whereas fluid intake increased significantly (p≤0.01) in the HKM group when compared with the VCM group. Food intake, body weight, and serum iPTH and CT concentrations remained significantly (p≤0.01) depressed in the HKP group when compared with the VCM; however, they increased as the duration of the post-HK period increased. By contrast, the corresponding parameters remained stable in the VCM group when compared with the baseline control values. It was shown that fecal and urinary Ca loss and serum Ca concentration increases significantly during HK, whereas during post-HK fecal, urinary, and serum Ca decreases significantly. It was concluded that significant decrease of serum, urinary, and fecal Ca during post-HK may suggest the presence of Ca deficiency during prolonged HK.     

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.     

Effects of the presence of a dominant follicle and exogenous oestradiol on the duration of the luteal phase of the bovine oestrous cycle.

The presence of a developing dominant follicle may be a factor in the control of the luteolytic cascade mechanism and the number of follicular waves during the bovine oestrous cycle. In this study, ovaries of all animals were examined once a day by transrectal ultrasonography. It was expected that heifers (n = 18) would have two follicular waves if the second wave occurred later than day 10 after oestrus (Expt 1) and that cows (n = 14) would have three waves if the second wave occurred on or before day 10 (Expt 2). The objective of Expt 1 was to determine if absence of a large follicle late in the luteal phase delays luteal regression in heifers that are expected to have two follicular waves. Nine heifers were injected i.v. with 10 ml charcoal-treated bovine follicular fluid three times a day for 4 days, starting on the day after initiation of the second follicular wave, to delay growth of the second wave dominant follicle. Nine heifers were injected with 0.9% NaCl as controls. The duration of the luteal phase (calculated as the number of days that serum progesterone was > 0.5 ng ml-1) was greater (P < 0.01) in the follicular fluid-treated group compared with the controls (18.7 versus 14.1 days). FSH and follicular growth were suppressed during the period of injection of follicular fluid (P < 0.01 and 0.03, respectively). The objective of Expt 2 was to determine the effect of increased oestradiol on the duration of the luteal phase in cows that were expected to have three follicular waves. Seven cows were injected i.m. three times a day for 4 days with 1 ml oestradiol (100 micrograms ml-1 in corn oil) and seven cows were similarly injected three times a day with 1 ml 0.9% NaCl (control) starting the day after cessation of growth of the second wave dominant follicle. Luteal phase duration was shorter in oestradiol-treated animals than in the controls (14.0 versus 19.0 days; P < 0.04). Serum oestradiol concentrations were higher in the oestradiol-treated group during the period of injection (P < 0.01). In summary, luteolysis was delayed when follicular growth was suppressed with follicular fluid (Expt 1). Exogenous oestradiol administration during the development of uterine oestradiol responsiveness initiated luteolysis earlier compared with control animals (Expt 2).     

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows

Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows (n=30) and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6–8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, β-hydroxybutyrate (β-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, β-OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P<0.05). Significant correlations were observed for chloride (r=0.40), glucose (r=0.56), β-OHB (r=0.85), urea (r=0.95) and total protein (r=0.60) for all three follicle size classes and for triglycerides (r=0.43), NEFA (r=0.50) and total cholesterol (r=0.42) for large follicles (P<0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.