Acute dietary restriction in heifers alters expression of genes regulating exposure and response to gonadotrophins and IGF in dominant follicles

Dietary restriction in growing cattle and severe negative energy balance in lactating cows have been associated with altered gonadotropin secretion, reduced follicle diameter, reduced circulating oestradiol concentrations and anovulation. Therefore, we hypothesised that acute dietary restriction would influence the fate and function of the dominant follicle by altering the expression for genes regulating gonadotrophin and IGF response in ovarian follicles. Newly selected dominant follicles were collected 7-8 days after prostaglandin F(2α) (PGF) administration from heifers (n=25) that were individually fed a diet supplying 1.2 maintenance (M; control, n=8) or 0.4 M (restricted, n=17) for a total duration of 18-19 days. Heifers within 0.4 M were ovulatory (n=11) or anovulatory (n=6) depending on whether the dominant follicle present at PGF ovulated or became atretic following luteolysis. Control animals were all ovulatory. Acute dietary restriction decreased IGF-I (P<0.001) and insulin (P<0.05) in circulation; oestradiol (P<0.01) and IGF-I (P<0.01) in follicular fluid; and mRNA for FSHR (P<0.01) in granulosa cells but increased mRNA for IGFBP2 (P<0.05) in theca cells of the newly selected dominant follicle. However, this only led to anovulation when dietary restriction also decreased mRNA for CYP19A1 (P<0.05), IGF2 (P<0.01) and IGF1R (P<0.05) in granulosa cells and LHCGR (P<0.05) in theca cells of follicles collected from heifers fed 0.4 M. These results suggest that the catabolic environment induced by dietary restriction may ultimately cause anovulation by reducing oestradiol synthesis, FSH-responsiveness and IGF signaling in granulosa, and LH-responsiveness in theca cells of dominant follicles.     

Effect of days after calving on insulin-like growth factor-I, insulin-like growth factor binding proteins, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant follicles of postpartum beef cows

The effect of days after calving on IGF-I, IGFBP, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant ovarian follicles (DF) was evaluated in Angus × Hereford cows. Growth of DF (>9 mm) was monitored daily by ultrasonography and fluid from DF was collected in vivo at either 30 ± 2 d or 47 ± 2 d postpartum. Follicular fluid (FF) was also aspirated from DF of contemporary ovulatory cows at proestrus. Estrous behavior was monitored continuously using the HeatWatch system, and progesterone in plasma collected twice weekly was used to assess luteal activity. Anovulatory DF aspirated 30 and 47 d postpartum had similar concentrations of IGF-I, IGFBP, progesterone, estradiol and androstenedione in FF and IGF-I and IGFBP in plasma. The intervals from aspiration to estrus were similar for cows aspirated 30 and 47 d postpartum. Proestrous follicles had greater (P < 0.01) estradiol (435 ± 79 ng/mL) than DF at 30 d (107 ± 63 ng/mL) or 47 d (68 ± 53 ng/mL) after calving. Concentrations of androstenedione in FF were also greater (P < 0.01) in proestrous follicles than in DF aspirated at 30 or 47 d after calving. Concentrations of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding proteins (IGFBP) in FF and plasma, and aromatase mRNA in granulosa cells were similar for anovulatory and proestrous cows. In conclusion, estradiol production by DF of postpartum anovulatory cows may be limited by inadequate production of androstenedione during the postpartum anovulatory interval and this may influence follicular maturation. Concentrations of IGF-I and IGFBP were similar in anovulatory and proestrous cows, an indication that alterations in the IGF-I system in the DF at 30–47 d after calving are not associated with delayed follicular development in postpartum beef cows.     

Changes in ovarian function in mature beef cows grazing endophyte infected tall fescue

The objective was to examine follicular and luteal development and function in mature, lactating beef cows grazing endophyte free (E-) or endophyte infected (E+) tall fescue during the early postpartum period. Angus, Hereford, and Angus x Hereford cows were exposed to pasture for 37-39 days before synchronized estrus. Serum concentrations of prolactin were evaluated during the luteal phase before the synchronized estrus. Every Monday, Wednesday, and Friday for one estrous cycle ovaries were monitored by transrectal ultrasonography and blood was collected for determination of serum concentrations of progesterone and estradiol in cows that responded to synchronization. Signs of fescue toxicosis in E+ cows included decreased serum concentrations of prolactin (84.9+/-13.6 pg/ml versus 32.3+/-12.0 pg/ml; P < 0.009) measured during the luteal phase (day 37 of grazing) and decreased body condition of cows and weight of cows and calves (P < 0.001). Neither serum concentrations of progesterone or estradiol, nor diameter of the CL differed between treatments. Diameter of the largest follicle tended to be smaller for cows grazing E+ fescue, especially between days 8 and 12 of the estrous cycle (P < 0.08). Numbers of class 1 (3-5 mm) and class 3 (>10 mm) follicles were similar (P > 0.05) between treatments, but number of class 2 (6-9 mm) follicles was reduced in E+ cows for most of the cycle (days 10 through 20; P < 0.03). Length of synchronized estrous cycle, days open, calving interval, and pregnancy rate at 30, 45, 60, and 90 days post-breeding was similar (P > 0.05) among treatment groups. Even though follicular dynamics (diameter of the largest follicle and number of class 2 follicles) were altered in cows grazing E+ tall fescue, follicular function was apparently not affected by ergot alkaloids.     

Pattern of follicular growth and resumption of ovarian activity in post-partum beef suckler cows

The ovaries of 18 post-partum beef suckler cows were examined daily, using ultrasound, from Day 5 post partum until a normal oestrous cycle was completed. Periods of growth and regression of medium-sized (5-9 mm) follicles were identified before one medium follicle became dominant (single large follicle greater than or equal to 10 mm). The mean (+/- s.e.m.) number of days from parturition to detection of the first post-partum dominant follicle was 10.2 +/- 0.5. The first post-partum dominant follicle ovulated in 2/18 (11%) cows. The interval from calving to first ovulation (mean +/- s.e.m. = 35.9 +/- 3.3 days) was characterized by the growth and regression of a variable number (mean = 3.2 +/- 0.2; range 1-6) of dominant follicles. The maximum diameter of the dominant follicle increased as the cows approached first ovulation (P less than 0.05). Behavioural oestrus was not detected in 16/18 (89%) cows at first ovulation. Following first ovulation, the length of the subsequent cycle was short (mean = 9.7 +/- 0.5 days; range 8-15 days) in 14/18 (78%) cows and was characterized by the development and ovulation of a single dominant follicle. During oestrous cycles of normal length (mean = 20.6 +/- 0.5 days; range 18-23 days) one (N = 2), two (N = 7) or three (N = 8) dominant follicles were identified. The growth rate, maximum diameter or persistence of non-ovulatory dominant follicles before first ovulation or during oestrous cycles were not different (P greater than 0.05). These data show that, in beef suckler cows, follicular development and formation of a dominant follicle occur early after parturition and the incidence of ovulation of the first dominant follicle is low. The number of dominant follicles that develop before first ovulation is variable; first ovulation is rarely associated with oestrus and short cycles are common after first ovulation. It is concluded that prolonged anoestrus in post-partum beef suckler cows is due to lack of ovulation of a dominant follicle rather than delayed development of dominant follicles.     

Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle

Nutrition is a major factor affecting cow reproductive efficiency. Long-term moderate or chronic dietary restriction results in a gradual reduction in dominant follicle (DF) growth rate, maximum diameter and persistence. Animals become anoestrus when they lose on average 22-24% of their initial body weight. There is evidence of significant animal-to-animal variation in the interval from the imposition of dietary restriction to onset of anoestrus and from the recommencement of re-alimentation to resumption of ovulation. In contrast, acute dietary restriction to 40% of maintenance requirements rapidly reduces dominant follicle growth rate and maximum diameter and induces anoestrus in a high proportion (60%) of heifers within 13-15 days of dietary restriction. In lactating dairy and beef cows negative energy balance or reduced dietary intake in the early post-partum period, while not affecting the population of small-to-medium size follicles, adversely affects the size and ovulatory fate of the dominant follicle. Re-alimentation of nutritionally induced anoestrous heifers results in an initial gradual increase in dominant follicle growth rate and maximum diameter, followed by a more accelerated increase in dominant follicle growth rate and maximum diameter as the time of resumption of ovulation approaches. Increased dominant follicle growth rate and maximum diameter are associated with increased peripheral concentrations of IGF-I, pulsatile LH and oestradiol. Direct nutritional effects on ovarian function appear to operate through hepatic rather than follicular regulation of IGF-I, and on systemic concentrations of IGF-I BPs and insulin; cumulatively reducing follicular responsiveness to LH and ultimately shutting down follicular oestradiol production. Indirect nutritional effects are apparently mediated through altering the GnRH pulse generator and in-turn selectively reducing pulsatile LH secretion without any apparent adverse effect on FSH secretory patterns. Endogenous opioid peptides, NPY and glucose appear to play a role in the nutritional regulation of GnRH release and in turn pulsatile LH secretion.     

Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers

A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (approximately 1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.     

Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.     

Effects of suckling frequency and breed on productive performance,follicular dynamics and postpartum interval in beef cows

One experiment was conducted to determine the influence of suckling frequency (restricted suckling (RS) versus ad libitum suckling (AS)) and breed (Brown Swiss versus Pirenaica) on productive performance, follicular dynamics and postpartum cyclicity resumption in beef cows. Twenty-four spring-calving cows (12 Brown Swiss and 12 Pirenaica) were used. After calving cows were assigned, within breed, to the two suckling frequencies (RS versus AS). During the postpartum period, cows were fed 100% of the daily energy estimated requirements. The study was conducted from calving to either day 90 postpartum or to detection of the second ovulation whenever it came later. The pattern of follicular development was monitored daily by transrectal ultrasonography, and blood samples were collected three times a week to analyse progesterone concentrations by RIA. Body condition score at calving was 2.5 (from 1 to 5). Productive parameters were not significantly affected by suckling frequency. Pirenaica cows gained more weight (P<0.001), while Brown Swiss cows had higher milk yield (P<0.001) and thus their calves grew faster (P<0.05). In the present moderate conditions of nutrition, either suckling frequency or breed did not affect follicular dynamics (on average there were 6.5 follicles in emergence, growth rate of dominant follicle (DF) was 1.3mm per day and maximum diameter of dominant follicle was 13.9mm). There were gradual increases in maximum diameter and growth rate of dominant follicle related to the proximity of the ovulatory waves, and they were defined by third order polynomial equations. Postpartum anoestrous interval (PPI) was markedly affected by the interaction between suckling frequency and breed (P<0.001): ad libitum suckling prolonged significantly the interval from calving to first ovulation in Brown Swiss cows (31.8 day versus 82.7 day, in RS and AS groups, respectively, P<0.05), but not in Pirenaica cows (43.5 day versus 32.6 day, in RS and AS groups, respectively, not statistically significant). It is concluded that in moderate pre- and postpartum nutritional conditions Brown Swiss cows were more susceptible to the suppressor effect of ad libitum suckling on postpartum anoestrous interval than Pirenaica cows, although this breed difference did not seem to be mediated by differences in energy balance or in ovarian follicular dynamics.     

A potential role for insulin-like growth factor binding protein-4 proteolysis in the establishment of ovarian follicular dominance in cattle.

A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and growth of dominant ovarian follicles. We have investigated whether a component of the insulin-like growth factor (IGF) system, namely IGFBP-4 protease, is associated with the establishment of follicular dominance in cattle. IGFBP proteases degrade IGFBPs, freeing IGFs to interact with their receptors. In experiment 1, follicular fluid from preovulatory follicles (n = 4) degraded about 80% of the added recombinant human (rh) IGFBP-4 within 18 h of incubation. The IGFBP-4 protease exhibited optimal activity at neutral/basic pH and its sensitivity to various protease inhibitors suggested a metalloprotease. The decline in the intensity of the band corresponding to intact rhIGFBP-4 was accompanied by the appearance of immunoreactive fragments of molecular weights approximately 18 and 14 kDa, which were not detectable by ligand blot analysis. In experiment 2, follicular fluid samples were collected from dominant and subordinate follicles on Day 2 or 3 of the first follicular wave, after ovariectomy (experiment 2a, n = 3/day) or by ultrasound-guided follicular aspiration (experiment 2b, n = 4-5/day). Estradiol concentrations in follicular fluid from dominant vs. subordinate follicles confirmed their identities and indicated that the dominant follicle had been selected by Day 2 of the follicular wave. In both experiments 2a and 2b, IGFBP-4 proteolytic activity was 2- to 3.5-fold (P < 0.05) and 5-fold (P < 0.01) higher in follicular fluid from dominant than subordinate follicles on Days 2 and 3 of the follicular wave, respectively. The finding that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave, strongly suggests a role for IGFBP-4 protease in the establishment of ovarian follicular dominance.     

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.     

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.     

Presence of an aromatase inhibitor, possibly heat shock protein 90, in dominant follicles of cattle.

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 +/- 0.09%) than in the culture medium (1.3 +/- 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp 90 to bovine follicles in vitro depressed aromatase activity by altering the K(m) value (and possibly the Vmax value) of the enzyme. It is proposed that hsp 90 is a functional regulator of follicular maturation through its action on aromatase.     

Effect of environmental heat stress on follicular development and steroidogenesis in lactating Holstein cows

Lactating Holstein cows were utilized over two replicate periods (July and September, 1990) to examine the effect of summer heat stress on follicular growth and steroidogenesis. On day of synchronized ovulations, cows were assigned to shade (n=11) or no shade (n=12) management systems. Follicular development was monitored daily by ultrasonography until ovariectomy on Day 8 post estrus. At time of ovariectomy, dominant and second largest follicles were dissected from the ovary. Aromatase activity and steroid concentrations in dominant and subordinate follicles were measured. Acute heat stress had no effects on patterns of growth of first wave dominant and subordinate follicles between Days 1 and 7 of the cycle. Compared with shaded cows, the heat stressed cows did not have suppression of medium size (6 to 9 mm) follicles between Days 5 and 7. A treatment x follicle interaction was detected (P<0.01) for follicular diameter and fluid volume at Day 8. Dominant follicles in shade were bigger (16.4>14.5 mm) and contained more fluid (1.9>1.1 ml) than dominant follicles in no shade. Conversely, subordinate follicles in no shade were bigger (10.1>7.9 mm) and contained more fluid (0.4>0.2 ml) than subordinate follicles in shade. Concentrations of estradiol in plasma and follicular fluid were higher (P<0.01) in July than in September. Heat stress appears to alter the efficiency of follicular selection and dominance, and to have adverse effects on the quality of ovarian follicles.     

Immediate and delayed effects of heat stress on follicular development and its association with plasma FSH and inhibin concentration in cows

The aim of this study was to characterize the immediate effects of heat stress on plasma FSH and inhibin concentrations, and its involvement in follicular dynamics during a complete oestrous cycle, and to examine a possible delayed effect of heat stress on follicular development. Holstein dairy cows were oestrous synchronized and randomly assigned to either cooled (n = 7) or heat-stressed (n = 6) treatment groups. During a complete oestrous cycle, control cows, which were cooled, maintained normothermia, whereas heat-stressed cows, which were exposed to direct solar radiation, developed hyperthermia. At the end of this oestrous cycle (treated cycle), both groups were cooled and maintained normothermia for the first 10 days of the subsequent oestrous cycle. Throughout this period, follicular development was examined by ultrasonography, and plasma samples were collected. During the second follicular wave of the treated oestrous cycle, a significantly larger cohort of medium sized follicles (6-9 mm) was found in heat-stressed cows than in cooled cows (P < 0.05). The enhanced growth of follicles in this wave in heat-stressed cows was associated with a higher plasma FSH increase which lasted 4 more days (days 8-13 of the oestrous cycle; P < 0.05), and coincided with a decrease in the plasma concentration of immunoreactive inhibin (days 5-18 of the oestrous cycle; P < 0.05). During the follicular phase (days 17-20 of the treated cycle), heat-stressed cows showed an increase in the number of large follicles (>/= 10 mm), and the preovulatory plasma FSH surge was significantly higher in heat-stressed cows than in cooled cows (P < 0.01). The effect of heat stress was also observed during the first follicular wave of the subsequent cycle: the postovulatory plasma FSH concentration was higher (P < 0.01), but fewer medium follicles developed, and the first follicular wave decreased at a slower rate in previously heat-stressed cows than in cooled cows (0.40 and 0.71 follicles per day, respectively). This study shows both immediate and delayed effects of heat stress on follicular dynamics, which were associated with high FSH and low inhibin concentrations in plasma. These alterations may have physiological significance that could be associated with low fertility of cattle during the summer and autumn.     

Effect of active immunization against androstenedione on ovarian function in dairy cows and prepubertal gilts

Two experiments were performed to assess the effects of immunization against androstenedione on ovarian function in cows during the first 100 days after calving and in gilts at the onset of puberty.Seven Holstein-Friesian cows were immunized against androstenedione-11α-human serum albumin (AND-HSA) at 3 weeks before and at 3 weekly intervals after calving and compared with ten cows immunized against HSA. Titres against androstenedione were significant but low and variable (1: 280-1: 4). Two cows with the highest titres exhibited abnormal ovarian function which could be attributable to treatment. One cow developed a large follicular cyst and nymphomania. The other cow exhibited mild super ovulation in her first cycle after calving and multiple ovulations during later cycles. Other cows in the AND-HSA group also had a tendency to develop larger than normal follicles and follicular cysts.Groups of twelve prepubertal gilts were immunized against either androstenedione 11α-hemisuccinyl bovine serum albumin (AND-BSA) or BSA at 100, 121 and 142 days of age. Titres against AND were significant but were low and variable (1: 2500-1: 10). There was no effect of immunization against AND on onset of ovulation, puberty or ovulation rate in the first or second ovulatory cycles.     

Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

It was hypothesized that growth divergence of dominant and subordinate follicles during Wave 1 and growth termination of the dominant follicle would be associated with changes in the number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ significantly over Days 2, 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid decreased (P < 0.05) from Days 2 to 10 (373 +/- 150 to 42 +/- 12 ng/ml) and concentrations of progesterone in follicular fluid increased (P < 0.05) from Days 2 to 10 (12.2 +/- 2.3 to 24.4 +/- 4.8 ng/ml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased (P < 0.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated greater (P < 0.05) estradiol concentrations in the dominant follicle on Day 2, but the number of gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of follicular fluid estradiol, but not numbers of granulosa cell gonadotropin receptors, were associated with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual growth termination of the dominant follicle (Day 10). Late divergence (Day 4) was associated with higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than in the subordinate follicles. These results indicate that an increase in estradiol productivity of the selected dominant follicle occurred before an increase in the number of gonadotropin receptors.     

Differential abundance of IGF1, bile acids,and the genes involved in their signaling in the dominant follicle microenvironment of lactating cows and nulliparous heifers

It is well documented that incidence of fertility problems is high in lactating cows but not in heifers of the same genetic merit. Understanding the metabolic and molecular differences between fertile heifers and relatively infertile lactating cows will help us understand the pathogenesis of infertility in dairy cows. Follicular waves in lactating cows (30–50 days in milk; n = 12) and heifers (n = 10) were synchronized by ultrasound-guided follicle ablation. Follicular fluid and granulosa cells of the dominant follicle were collected by ultrasound-guided aspiration along with blood sampling on Day 6 after synchronization. Dominant and subordinate follicles were larger in lactating cows than in heifers. Metabolic stress in lactating cows was evidenced by lower glucose and higher ß-hydroxy butyric acid compared with heifers. Insulin-like growth factor 1 signaling was reduced in the dominant follicle in lactating cows through reduced insulin-like growth factor 1 concentrations in plasma and follicular fluid of the dominant follicle, and reduced expression of pregnancy-associated plasma protein A (PAPPA) in their granulosa cells. We also found increased levels of total bile acids in the follicular fluid of the dominant follicle of lactating cows compared with heifers. Granulosa cells of the dominant follicle had higher expression of SLC10A2 and GPBAR1 (bile acid transporter and receptor, respectively) in lactating cows. These novel data are indicative of increased bile acid signaling within the dominant follicles of lactating cows compared with heifers. Overall, we demonstrate in the present study the metabolic, endocrine, and molecular differences within the microenvironment of the dominant follicles in lactating cows and heifers. These differences in follicular microenvironment may contribute toward abnormal ovarian function in lactating dairy cows.     

Influence of parity on follicular dynamics and resumption of ovarian cycle in postpartum dairy cows

The present study examined ovarian changes preceding the resumption of the ovarian cycle in postpartum dairy cows with different parities under similar body nutritional conditions. In postpartum primi- (n=6), bi- (n=4), and multiparous (n=6) Holstein dairy cows, ovarian ultrasonographic observations starting at 7 days after calving were performed every other day and then daily after the confirmation of clinical signs of oestrus for the detection of postpartum first ovulation. Blood samples were collected at the same time as ultrasonography and analyzed for oestradiol and progesterone to monitor ovarian activity. To evaluate the nutritional condition of the cows, body weight and body condition score (BCS, 1=emaciated to 5=obese) were measured weekly and blood samples for the analysis of glucose, insulin, and non-esterified fatty acid (NEFA) were collected at the same time until postpartum second ovulation. Dominant follicles (>8mm in diameter) of the first follicular wave were detected at 7 days after calving in all cows. The first wave follicle ovulated in five of six multiparous cows, whereas no first wave follicle ovulated in any of the primiparous cows. The days to first ovulation after calving in primiparous cows (31.8+/-8.3 days) were significantly greater (p<0.05) than those in multiparous cows (17.3+/-6.3 days), but were not significantly different from biparous cows (28.8+/-8.6 days). There was a significant relationship between parity and days to first ovulation after calving (p<0.05). BCS was maintained at a level of more than 2.5 during the postpartum period in all cows and there was no influence of parity on postpartum changes in BCS, glucose, insulin, or NEFA throughout the experiment. The present study demonstrated a negative relationship between parity and number of days from calving to first ovulation in dairy cows under similar body nutritional conditions. It is possible that the influence of parity on the resumption of ovarian cycle is modulated by the factors different from the nutrition-related changes during the postpartum period in dairy cows.     

Effect of exogenous LH pulses on the fate of the first dominant follicle in postpartum beef cows nursing calves

Prolonged postpartum anoestrus in beef cows is due to failure of early dominant follicles to ovulate. It is hypothesized that this failure to ovulate is due to inadequate LH pulse frequency. The objective of this study was to determine whether administration of hourly LH pulses would cause the first dominant follicle to ovulate. In Expt 1, 16 cows received either saline (n = 8) or porcine LH (pLH; 50 micrograms h-1; n = 8) as hourly pulses for 3-5 days from the second day of dominance of the first dominant follicle (day 0). In Expt 2, 21 cows received either saline (n = 7), or 50 micrograms pLH (n = 7) or 100 micrograms pLH (n = 7) as hourly pulses for 3 days. Appropriate ovarian scanning and assays of blood samples were carried out. In Expt 1, the number of dominant follicles that underwent atresia was not affected by increasing the number of LH pulses, but the duration of dominance (days) of the first and second dominant follicles and maximum size (mm) of the second dominant follicle were increased (P < 0.05). Oestradiol concentrations were higher (P < 0.05) in cows given hourly pLH pulses (3.1 +/- 1.2 pg ml-1) compared with controls (1.2 +/- 0.2 pg ml-1). Four of eight treated cows had an anovulatory LH surge. The number of follicle waves to first ovulation was not different (P < 0.05) between control (4.6 +/- 0.9) and pLH treated cows (3.9 +/- 0.5). In Expt 2, four of seven cows given pulses of 100 micrograms pLH h-1 ovulated the first dominant follicle, and the interval from calving to first ovulation was decreased (P < 0.05). In the remaining three cows, the duration of dominance of the first dominant follicle was increased (P < 0.005), the maximum size of the first dominant follicle was greater (P < 0.05), and the interval (days) from the start of infusion to new wave emergence was greater (P < 0.05) compared with cows that failed to ovulate in either the 50 micrograms pLH h-1 or control treatments. In conclusion, hourly pulses of pLH from day 1 after dominance of the first dominant follicle in postpartum beef cows can either prolong dominance or induce it to ovulate. This finding supports the hypothesis that LH pulse frequency is a key determinant of the fate of the dominant follicle in the early postpartum period.