Verotoxigenic Escherichia coli: costs of illness in Canada, including long-term health outcomes.

The main objective of this study was to provide cost estimates of human Escherichia coli O157 infection to facilitate future assessment of the benefits and costs of alternative preventive strategies to reduce illness. We investigated the costs of illness to Canadians from primary human infection by verotoxigenic E. coli O157 (also called Shiga toxin-producing E. coli O157) using data from the National Notifiable Diseases Registry. We used relative risk information from peer-reviewed publications to estimate the burden of illness and associated costs for eight long-term health outcomes. National estimates of the number of cases (mean and 5th and 95th percentiles), associated costs, and a rank correlation test to identify which outcomes were associated with the highest per capita costs were calculated. An estimated 22,344 cases of primary infections occur in Canada annually, costing $26.7 million. There are 37,867 additional on-going long-term health outcomes costing $377.2 million each year. Our analysis indicated that the annual cost for primary and long-term illness is $403.9 million. The analysis supports evaluation of alternative control and prevention measures and the development and implementation of policy and practices aimed at safe food production.

Effect of FSH and cell localization on dimeric inhibin-A secretion from bovine granulosa cells in culture.

We tested the hypotheses that the secretion of dimeric inhibin-A from cultured bovine granulosa cells is stimulated by FSH, and that antral cells secrete more inhibin-A than do mural cells. Cells from the antral or mural compartment of follicles were cultured in defined medium in two culture systems, and dimeric inhibin-A was measured by two-site ELISA or by Western immunoblotting. In the first culture system, dimeric inhibin-A secretion declined with time in culture, but was significantly (P<0.05) higher from antral than from mural cells (as was total inhibin-alpha measured by RIA). The secretion of dimeric inhibin-A and inhibin-alpha from antral but not mural cells was responsive to FSH. In the second culture system, dimeric inhibin-A secretion increased with time in culture, and was significantly stimulated by FSH, but FSH responsiveness was dependent on the concentrations of insulin in the culture medium. The major forms of inhibin-A secreted had molecular masses of approximately 58, 62, 103-116 and >116 kDa; the 32 kDa form was barely detectable. These different forms were all stimulated by FSH, but the >116 and 62 kDa forms were most responsive to FSH. We conclude that (i) FSH stimulates dimeric inhibin-A secretion from bovine granulosa cells, (ii) the 62 kDa form of inhibin-A may be more responsive to FSH than the 58 kDa form, and (iii) the spatial differentiation of granulosa cell function within the follicle previously observed for oestradiol secretion was also observed for inhibin-alpha and dimeric inhibin-A secretion.

Intracellular regulation of estradiol and progesterone production by cultured bovine granulosa cells.

The objective of the present study was to investigate the implication of protein kinase A (PKA), protein kinase C (PKC), and receptor protein tyrosine kinase (R-PTK) pathways in the regulation of estradiol (E2) and progesterone (P4) production by bovine granulosa cells. Cells were harvested from bovine follicles (8-15 mm diameter) and cultured without serum for an initial 3 days (37 degrees C; 5% CO(2) in air; D1-D3). On the fourth day of culture (D4), E2 and P4 production were stimulated with FSH (1-6 ng/ml) or forskolin (FSK) in the presence or absence of intracellular effectors of PKA, PKC, and R-PTK. Culture medium was collected and replaced each day. Stimulation of granulosa cell adenylate cyclase activity with FSK (0.06-3.75 microM) mimicked FSH, inducing a quadratic increase (P < 0.001) of E2 production and a continuous elevation of P4 (P < 0.01). Inhibition of R-PTK activity with genistein (25-50 microM) increased the sensitivity of cells to FSH as demonstrated by a leftward shift in the dose response curve (P < 0.001). Treatment with transforming growth factor-alpha (TGFalpha; 0. 1 ng/ml) abolished the FSH-induced E2 production (P < 0.001) and this effect was not reversed (P < 0.001) by FSK or by genistein. Furthermore, the inhibitory effect of TGFalpha on FSH-induced E2 production was reproduced by phorbol 12-myristate 13-acetate (PMA; 1. 25-2.5 microM), a PKC activator (P < 0.001). Interestingly, genistein inhibited P4 production (P < 0.05). From these results, we conclude that E2 production by bovine granulosa cells is mediated by intracellular factors and can be stimulated downstream from the FSH receptor. The results also suggest that stimulation of R-PTK and/or PKC activities, as probably occurs with TGFalpha, negatively affects the PKA pathway, thus decreasing E2 production. Furthermore, inhibition of R-PTK leads to an increase production of E2 and may limit luteinization of bovine granulosa cells.

Effects of superovulation on endogenous LH secretion in cattle, and consequences for embryo production.

Stimulation of follicular growth during superovulation is achieved by the injection of FSH or compounds with high FSH-bioactivities. However, some LH-activity is required for follicle maturation. It is of relevance to evaluate, therefore, the effect of superovulatory treatments on endogenous LH secretion. Luteinizing hormone is secreted in discrete pulses, and the pattern of pulsatile LH secretion during superovulation is reviewed. Four of five published studies have shown that LH pulse frequency is significantly reduced by injection of eCG or FSH preparations. This suppression appears within 8 h of treatment Effects of superovulation on LH pulse amplitude are less consistent. The reasons for the decrease in pulse frequency have been investigated, and although the answer is not definitive, it would seem that increased follicular estradiol, acting perhaps in synergism with progesterone, may play a role. Changes in plasma progesterone concentrations are not related to changes in LH pulse frequency. What is the significance of decreased LH pulse frequency? We attempted to investigate this by inducing LH pulses during superovulation, but the result was a major reduction in ovulation rate. More research is required to determine if modification of endogenous LH secretion can improve superovulatory responses.

Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: development of an in vitro model to study estradiol production.

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P < 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells.

Effect of bovine follicular fluid from healthy and atretic follicles on follicle-stimulating hormone-induced production of estradiol by bovine granulosa cells cultured in vitro.

We studied the effects of active factors present in bovine follicular fluid (bFF) from large healthy or atretic follicles on steroidogenic capability of cultured bovine granulosa cells. Pools of bFF were collected from follicles (> 10 mm; abattoir material) and classified individually as being healthy (bFF-healthy) or atretic (bFF-atretic). Pools of jugular plasma were used as controls and were from heifers bled during the growing (plasma-growing) or the regressing (plasma-regressing) phase of follicular dominance. Granulosa cells were cultured in serum-free conditions and under minimal FSH support (.5 ng/mL) for the first 3 d in order to maintain their physiological estradiol production in response to FSH. Effects of addition of bFF and plasma at final concentrations of 0, 1, or 5% on estradiol and progesterone production and on the percentage of apoptotic cells were determined on d 4 of culture following stimulation of granulosa cells with either 2 or 6 ng/mL FSH. In a parallel experiment, evaluation of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity was measured following addition of bFF. In contrast to plasma, addition of bFF pools decreased (P < .001) the FSH-induced estradiol production. Such suppression occurred in a dose-related manner (P < .05) and to a greater extent (P < .001) following addition of bFF from atretic than from healthy follicles. The FSH-induced progesterone production was not affected (P > . 1) by addition of bFF but was stimulated (P < .05) by that of plasma. Follicle-stimulating hormone decreased (P < .001) the percentage of apoptotic granulosa cells and this effect was further enhanced (P < .001) by addition of 1 or 5% bFF. The source of bFF did not affect (P > . 1) the percentage of apoptotic cells measured at the end of the culture period. On d 4, treatment with bFF increased (P < .001) granulosa cell androstenedione conversion into testosterone. Results of the present study indicate that factors contained in bFF can suppress granulosa cell estradiol production, and the suppressive effect varies according to the degree of atresia of the follicle from which the fluid has been harvested.

Alteration of follicular dynamics and superovulatory responses by gonadotropin releasing hormone and follicular puncture in cattle: a field trial.

A field experiment was conducted to determine the influence of follicular alteration on superovulatory responses. Ultrasonography was performed once daily over 4 d prior to gonadotropin treatment (Day 0), on the day of estrus during superstimulation, and on the day of embryo collection to monitor follicular development. Animals were superstimulated between Days 8 and 12 of the estrous cycle. Follicular status was altered 2 d prior to initiation of superstimulation (Day 0) with GnRH (Cystorelin, 200 micrograms i.m.) administered with (GnRH-puncture group, n = 31) or without (GnRH-no puncture group, n = 52) concomitant removal of the largest follicle by follicular aspiration. Responses were compared with those of an untreated control group superovulated 8 to 12 d after estrus (n = 102). The proportion of animals with a high number (> or = 2) of large follicles (> = 7 mm) on Day 0 was lower (P < 0.001) in the 2 GnRH-treated groups than in the control group, while the increase in the number of medium size follicles (4 to 6 mm) on Day 0 was greater (P < 0.02) in the GnRH-puncture group. During superstimulation, the proportion of superovulatory cycles with a high follicular (> or = 10 follicles) response was similar in the control and GnRH-no puncture groups. Within the GnRH-treated animals, follicular and ovulatory responses were greater in the GnRH-puncture than in the GnRH-no puncture group (P < 0.001 to P < 0.02). Despite these changes in follicular and ovulatory responses, however, the mean number of embryos produced did not differ (P < 0.1) among treatments (4.3 +/- 0.4, 3.7 +/- 0.7, and 5.4 +/- 0.8 in control, GnRH-no puncture, and GnRH-puncture groups, respectively). This was due primarily to an increase in the mean numbers of unfertilized ova (P < 0.005) and in degenerated embryos (P < 0.06) in the GnRH-puncture group. Results indicate that the beneficial effects of treatment with GnRH and follicular puncture 2 d prior to superstimulation on follicular and ovulatory responses were limited by an increase in the number of unfertilized ova and degenerated embryos.

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle.

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.

Immunoneutralization of transforming growth factor alpha present in bovine follicular fluid prevents the suppression of the follicle-stimulating hormone-induced production of estradiol by bovine granulosa cells cultured in vitro.

Growth factors such as transforming growth factors alpha (TGF alpha) and beta (TGFbeta) secreted by theca cells and present in bovine follicular fluid (bFF) have been implicated in granulosa cell growth and differentiation. We investigated these phenomena using two complementary approaches to evaluate the physiological contribution of TGF alpha and TGFbeta in the control of the FSH-induced estradiol production in bovine granulosa cells from large follicles. Granulosa cells (3 x 10(5) viable cells/cm2) harvested from eCG-treated prepubertal calves were cultured (serum free) in wells containing 500 microl/cm2 of defined Ham's F-12 medium supplemented with 0.5 ng/ml FSH for the first 3 days (37 degrees C; 95% air:5% CO2). In the first approach, the effects of individual addition of TGF alpha and TGFbeta at final concentrations of 1 x 10(-4) to 10 ng/ml were determined on Day 4 of culture after stimulation of granulosa cell estradiol production with 6 ng/ml FSH. In a second approach, TGF alpha or TGFbeta was removed specifically from bFF (from large follicles > 10 mm) by immunoneutralization. Thereafter, effects of immunoneutralization of TGF alpha or TGFbeta (0, 0.1, 1, 10, and 100 microg/ml anti-TGF neutralizing antibody) present in bFF (2%) were determined on Day 4 of culture following stimulation of granulosa cell estradiol production with 6 ng/ml FSH. On Day 4, FSH increased (p < 0.001) granulosa cell estradiol production (0 vs. 6 ng/ml FSH). Addition of TGF alpha decreased the granulosa cell estradiol production after 6 ng/ml FSH stimulation in a dose-dependent manner (p < 0.001). In contrast, addition of TGFbeta had no effect on the granulosa cell estradiol production (p > 0.1) after the addition of 6 ng/ml FSH. Addition of bFF (2%) decreased (p < 0.0001) the FSH-induced estradiol production by bovine granulosa cells. After immunoneutralization of TGF alpha in bFF, however, this suppressed FSH-induced estradiol production was restored to levels obtained in the absence of bFF, and this occurred in a dose-dependent manner (p < 0.05). Immunoneutralization of TGFbeta in bFF did not prevent (p > 0.1) the suppressive effect of bFF on FSH-induced estradiol production. These results suggest that TGF alpha produced in vivo by large bovine follicles can act locally (auto/paracrine manner) to suppress granulosa cell estradiol production.

In vitro production of bovine embryos: developmental competence is acquired before maturation.

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.

The time interval between FSH-P administration and slaughter can influence the developmental competence of beef heifer oocytes.

Superovulation alone may not be enough to result in developmentally competent oocytes. The objective of this study was to determine if a time interval between FSH administration and slaughter and between slaughter and oocyte recovery could increase the percentage of embryos. Beef heifers (n = 20) were superovulated with 1 bolus injection of 25 mg, im FSH-P diluted in saline and then slaughtered at 24, 48 or 72 h after FSH injection and the ovaries transported to the laboratory at 30 degrees C. For 6 of the heifers that received FSH-P and were then culled at 48 h post treatment, oocytes were recovered 1 to 2 h post slaughter from the first ovary and 4 to 5 h from the second ovary. Ovaries from untreated cows were collected and served as controls. The results indicated that FSH-P and culling at 48 h produced 35% >/= 32-cell embryos, significantly more than FSH-P and culling at 24 and 72 h (19 and 14%, respectively; P < 0.05). Furthermore, FSH-P and culling at 48 h produced 25% >/= 64-cell embryos, significantly more than FSH-P and culling at 24 and 72 h and the nontreatment control group (5, 7 and 15%, respectively; P < 0.05). The FSH-P group culled at 48 h produced more >/= 32-cell embryos, with an average of 84 +/- 5 cells/embryo, than the treated groups culled at 24 and 72 h and the untreated group (52 +/- 6, 60 +/- 5 and 63 +/- 3, respectively; P < 0.01). Finally, oocytes left in the postmortem ovaries for 4 to 5 h resulted in higher rates (51% and 41%) of >/= 32- and >/= 64-cell embryos, respectively, compared with that of the untreated control animals (29 and 18%; P < 0.05), but these rates were not different from oocytes left in ovaries for 1 to 2 h (33 and 24%). It is concluded that culling at 48 h after FSH treatment, as well as the conditioning effect on oocytes in warm postmortem ovaries for 4 to 5 h, increases the number of competent oocytes.

Follicle-stimulating hormone-induced estradiol and progesterone production by bovine antral and mural granulosa cells cultured in vitro in a completely defined medium.

Functional subpopulations of granulosa cells exist in bovine follicles. This study was designed to compare the in vitro steroid production by cultured bovine antral and mural granulosa cells in response to various amounts of FSH. Antral and mural granulosa cells (600,000 viable cells/well) harvested from ovaries of PMSG-treated prepuberal calves were cultured in serum-free conditions for 4 d in wells containing 1 mL of defined Ham's F-12 medium, supplemented with 0, 2, or 10 ng/mL of FSH. Culture medium was collected and replaced each day. The mean concentration of estradiol in culture media of bovine granulosa cells decreased from d 1 to d 4 (P < .001). Granulosa cell production of estradiol increased in antral cells following addition of 2 ng/mL FSH (P < .001) but decreased following addition of 10 ng/mL FSH (P < .001) as determined on d 4 by RIA and thin layer chromatography. In contrast, there was no response to FSH stimulation in mural granulosa cells. Progesterone production increased (P < .01) in a dose-dependent manner following stimulation with 2 or 10 ng/mL FSH and was consistently higher (P < .001) in antral than in mural granulosa cells. Addition of LH on d 4 stimulated estradiol and progesterone production in antral (P < .01) but not in mural cells (P > .10). This suggests that FSH- and LH-induced estradiol and progesterone productions are considerably lower in mural than in antral bovine granulosa cells. This suggests that functional differences between these two cell compartments need to be considered in studies involving in vitro cultures of bovine granulosa cells.

Superovulation can reduce the developmental competence of bovine embryos.

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.

Changes in morphological appearance and functional capacity of recruited follicles in cows treated with FSH in the presence or absence of a dominant follicle.

This study was designed to determine the effect of the presence of a dominant follicle at the beginning of FSH stimulation on the morphological appearance and functional capacity of recruited follicles during FSH stimulation in cattle. Synchronized nonlactating dairy cows were assigned to 1 of 2 groups and treated with FSH in the presence (n = 5) or absence (n = 6) of a dominant follicle between Days 7 and 12 of the estrous cycle (Day 0 = estrus) to stimulate follicular growth. Dominant follicles were identified by daily ultrasonographic observations, beginning on Day 3 of the estrous cycle. Dominant follicle had an ultrasonographic diameter > or = 10 mm and were in a growing phase, or maintaining a constant diameter (> or = 10 mm) for less than 4 d. Ovaries were collected at slaughter on the morning of the third day following initiation of the FSH stimulation. All follicles > 2 mm were dissected, classified according to diameter (Class 1: 2 to 4.4 mm; Class 2: 4.5 to 7.9 mm; Class 3: > 8 mm), and incubated individually for 90 min in medium M-199 (37 degrees C, 5% CO2). Following incubation, integrity of each follicle was evaluated histologically to assess the level of atresia and biochemically to determine the in vitro release of estradiol (E2) and androstenedione in culture media. On Day 3 of the FSH treatment, mean number of follicles in each class was similar (P > 0.1) between the 2 groups. The percentage of atretic follicles in Classes 1 and 3 on Day 3 of the FSH stimulation did not differ (P > 0.1) between the 2 groups. However, the percentage of atretic follicles in Class 2 was higher (P < 0.005) in cows treated with FSH in presence than in absence of a dominant follicle (60.8 vs 38.2%). The release of E2 in culture media by small Class 1 atretic or healthy follicles, by Class 2 atretic and by Class 3 healthy follicles was not affected (P > 0.1) by the ovarian status. However (P < 0.001), the release of E2 in culture media of Class 2 healthy and Class 3 atretic follicles was less for follicles harvested from cows bearing than from those not bearing a dominant follicle. Within each follicular class, concentrations of androstenedione in the culture media did not differ between the 2 groups (P > 0.1). These results suggest that the presence of a dominant follicle at the beginning of FSH stimulation alters the population of follicles recruited FSH stimulation. This may be associated with the reported decrease of the superovulatory response in cows superovulated in presence of a dominant follicle.

Synchronization of ovarian follicular waves with a gonadotropin-releasing hormone agonist to increase the precision of estrus in cattle: a review.

Treatment with GnRH and PGF2 alpha is a practical method for controlling ovarian follicular and luteal functions and increasing the precision of estrus synchronization in cyclic and acyclic postpartum cows and heifers. This method reduces considerably the period of time needed for estrus detection; it synchronizes the estrous cycle of 70 to 80% of the cyclic cows to within a 4-d interval without any detrimental effect on the fertility rate (65 to 85%). Moreover, resumption of ovarian activity and normal fertility in acyclic cows in favored. Administration of GnRH eliminates the large follicles by ovulation or atresia and induces emergence of a new follicular wave within 3 to 4 d after treatment at any stage of the estrous cycle, but it limits further growth of these emerging follicles by increasing atresia. The precision of estrus and the unaltered fertility rate is due to the synchronized selection of a new larger growing follicle, which becomes the ovulatory follicle after PGF(2 alpha)-induced luteolysis 6 d after GnRH treatment. Also, fixed-time AI programs without the need for estrus detection may be possible using a second injection of GnRH in a GnRH-PGF(2 alpha)-GnRH protocol to ovulate the selected follicle at a precise time. We describe a physiological model to explain how the precision of estrus is improved following PGF(2 alpha)-induced luteolysis, via the effect of pretreatment with GnRH on follicular development and luteal functions in cattle. Application of this model to the development of reliable methods of fixed-time insemination is also explored.

Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses.

To understand the causes for poor response to superovulation in mature cows of high genetic potential, endocrine and follicular events during and after superovulation were compared in heifers (<2 yr old) yielding large numbers of embryos and cows (9 to 13 yr old) known to be poor embryo donors. Follicular development was monitored by daily ultrasonography. Blood samples were taken 2 to 3 times a day for the measurements of P4, E2, FSH and LH by RIA. Intensive blood collections at 15-min intervals for 6 h were also performed during preovulatory and luteal phases. The number of embryos produced in the heifers (15.2 +/- 2; mean +/- SEM) and the cows (0.6 +/- 0.4), was similar to the number of ovulatory follicles derived from ultrasonographic observations in the heifers (16.2 +/- 3.7), but not in the cows (7.8 +/- 2.8). Contrary to that observations in heifers, there was no increase in the number of 4- to 5-mm follicles in cows during superovulation. The number of larger follicles (>5 mm) increased during superovulation in both cattle groups, but it was significantly lower in cows than in heifers. During superovulation, the maximal E2 concentration was greater (P < 0.0001) in heifers than in cows. One cow showed delayed luteolysis during superovulation, while another had abnormally high FSH (>10 ng/ml) and LH (>3 ng/ml) concentrations following superovulation. All the cows had a postovulatory FSH rise which was not detected in the heifers. The results showed that attempts to improve superovulatory response in mature genetically valuable cows are hampered by a number of reproductive disorders that are not predictable from the study of the unstimulated cycle.

Follicular development and reproductive endocrinology during a synchronized estrous cycle in heifers and mature cows displaying contrasting superovulatory responses.

Ovarian follicular development and plasma concentrations of progesterone (P4), estradiol-17 beta (E2), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared during a synchronized estrous cycle between heifers and mature cows displaying contrasting superovulatory responses. Six heifers < 2 years old with a history of good responses to superovulatory (SOV) treatment and six cows 9 to 13 years old with poor responses to SOV treatments were used. Follicular development was monitored by daily ultrasonography. Blood samples were collected two to three times daily for P4 and E2 and thrice daily for LH and FSH analysis. Intensive sampling (samples every 15 min for 6 hr) was performed at critical periods of follicular development to analyze the pulsatile secretion of gonadotropins. In both cattle groups, a transient increase (P = 0.0001) in E2 occurred 4 to 5.7 d after the preovulatory LH surge or 2.3 d before the dominant follicle reached its maximum size. FSH concentrations increased (P = 0.006) before the emergence of the second cohort of follicles and then decreased despite no change in the concentration of E2. Contrary to our expectation and despite differences between groups in terms of age, number of previous SOV treatments, and divergent responses to superovulation, follicular development was similar in both groups. However, during the luteal phase, concentrations of E2 and FSH and LH pulse amplitudes were less (P < or = 0.05) in cows than in heifers. Therefore, follicular development monitored by ultrasonography and endocrine profiles during a synchronized estrous cycle are of limited value to predict quality of embryo donors.

Buserelin alters the development of the corpora lutea in cyclic and early postpartum cows.

Changes in proportions of luteal cells after buserelin treatment were studied in the corpus luteum (CL) that was present at the time of treatment (CLP) and in the buserelin-induced CL (CLI) of cyclic and acyclic postpartum cows. On d 0 of the experimental period, eight cyclic (Cyc-sal) cows were injected with saline, whereas eight cyclic (Cyc-bus) cows and eight acyclic (Acyc-bus) cows were treated i.m. with 8 micrograms of buserelin. On d 3 or 6, ovaries were collected and stained using histological techniques. As determined with the point-counting method, the number of nuclei in the CLP was similar on d 3 in Cyc-bus and Cyc-sal groups but was lower (treatment x day, P < .001) in the Cyc-bus group than in the Cyc-sal group on d 6. The volume of the CLP was similar on d 3 in the two groups but was greater (treatment x day, P < .04) in the Cyc-bus than in the Cyc-sal group on d 6. The number of nuclei in the CLI was greater (P < .001) in cyclic than in acyclic cows on d 3 and 6 after treatment. The volume of the CLI was lower (P < .001) in cyclic than in acyclic cows on d 3 and 6. Buserelin did not change the profile of progesterone in cyclic cows from d 0 to 6, but concentrations of progesterone increased (P < .01) in acyclic cows 3 to 6 d after buserelin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects of human growth hormone-releasing hormone and photoperiod on hormone release and puberty in dairy heifers.

Forty-eight Holstein dairy heifers (98.9 kg BW; 3 mo old) were subjected for 246 d to twice-daily s.c. injections of saline (CTL) or human growth hormone-releasing hormone (GRH; 5 micrograms/kg BW) and to photoperiods of 8 h of light (L): 16 h of dark (D) or 16L:8D according to a 2 x 2 factorial arrangement of treatments. Jugular blood samples were collected from 16 heifers at 3, 4, 8, and 11 mo of age to monitor prolactin, growth hormone, and estradiol-17 beta. Plasma progesterone concentrations were monitored weekly in all heifers as an index of puberty (> 1 ng/mL). Growth hormone release was induced by GRH (P < .001) throughout the trial; area under the GH curve (AUC) averaged 1,582 vs 3,643 ng.min-1.mL-1 in CTL vs GRH heifers. However, GRH-induced GH response was less (P < .05) after the second daily injection. There was also an interaction (P = .08) between GRH, photoperiod, and days of treatment on GRH-induced GH response; AUC was greater in GRH-16L:8D than in GRH-8L:16D heifers at 3 mo but less at 8 mo of age. The PRL concentrations were similar for both photoperiods at 3 mo (36.4 vs 41.7 ng/mL) and 8 mo (16.2 vs 12.8 ng/mL) of age but were greater in 16L:8D vs 8L:16D heifers at 4 mo (18.4 vs 39.3 ng/mL) and 11 mo (26.3 vs 44.1 ng/mL) of age (photoperiod x day interaction, P < .001). Photoperiod of 16L:8D vs 8L:16D reduced (P < .01) weight at puberty in CTL heifers (251 vs 303 kg BW) and to a lesser extent in GRH-treated heifers (271 vs 284 kg BW; GRH x photoperiod interaction, P = .10). In conclusion, GH response is maintained throughout 8 mo of GRH treatment, and a 16L:8D photoperiod will reduce age and weight at puberty in heifers. Furthermore, refractoriness to photoperiod-induced PRL changes was detected.

Ovarian follicular development and endocrine responses in follicular-fluid-treated and hemi-ovariectomized heifers.

The effects of charcoal-extracted bovine follicular fluid (BFF) on endocrine profiles and follicular development in intact and hemiovariectomized postpubertal heifers were examined. Oestrus-synchronized heifers received Norgestomet implants on day 1 and 7 of treatment and were then injected s.c. with 11 ml saline (control) or 11 ml BFF twice a day for 12 days. The ovary bearing the largest follicle (OV1) was removed on day 7 and the remaining ovary (OV2) was collected on day 13. Follicles were observed by daily ultrasonography and were classified according to diameter (size 1: 2-3 mm; size 2: 4-6 mm; size 3: 7-10 mm; size 4: > 10 mm). After ovariectomy they were classified by diameter and histologically as normal or atretic. Intact control heifers had increased numbers of size 4 follicles on OV1 on days 6 and 7; no increase was observed in BFF-treated heifers (P < 0.03). In BFF-treated heifers, the mean basal LH concentration was higher (P < 0.05) and that of FSH was lower (P < 0.04) than in controls. FSH concentrations in BFF-treated heifers decreased from 0.60 +/- 0.08 ng ml-1 (day 1) to 0.22 +/- 0.05 ng ml-1 (day 7; P < 0.04). The concentration of oestradiol increased in control heifers, but not in BFF-treated heifers (P < 0.001). After hemicastration, OV2 underwent compensatory hypertrophy in control heifers, with an increase in the number of size 2, 3 and 4 follicles (P < 0.05), whereas BFF-treated heifers did not. Thus, total follicular volume was much lower in BFF-treated than in control heifers on day 13 (92.2 +/- 15.4 versus 1393.8 +/- 276.6 mm3; P < 0.0002). A transient increase in FSH (P < 0.006) and oestradiol (P < 0.01) concentrations occurred after hemiovariectomy in control but not in BFF-treated animals. In control heifers, an analysis of temporal relationships showed negative correlations between the volume of size 3 and size 4 follicles, and between FSH concentrations and the volume of size 3 and 4 follicles. A positive correlation was found between the mean diameter of the largest follicle and the concentration of oestradiol, whereas negative relationships were found between the concentrations of FSH and oestradiol, and between FSH and the mean diameter of the largest follicle. Analysis of the histological data showed that the number and volume of follicles > 8.57 mm was lower in the BFF-treated OV1 ovary, whereas no differences were found for follicles < or = 8.57 mm.(ABSTRACT TRUNCATED AT 250 WORDS)