Effects of monoclonal antibody against PMSG administered shortly after the preovulatory LH surge on time and number of ovulations in PMSG/PG-treated cows

Normally cyclic heifers received 2500 i.u. PMSG i.m. at Day 10 of the oestrous cycle and 15 mg prostaglandin (PG) i.m. 48 h later. From 30 h after PG the LH concentration in the peripheral blood was estimated every hour using a rapid RIA method which allowed the LH concentration to be known within 4 h. Monoclonal antibody against PMSG was injected in the jugular vein of 29 heifers at 4.8 h after the maximum of the preovulatory LH peak; 28 heifers were not treated with anti-PMSG (controls). Peripheral blood concentrations of PMSG, LH, progesterone and oestradiol were compared. Ovaries were collected by ovariectomy at fixed times, 22-30 h after the LH peak, and numbers were counted of small (2-10 mm), large (greater than 10 mm) and ovulated follicles, and of follicles with a stigma. In anti-PMSG-treated cows, the PMSG concentration fell sharply to non-detectable levels within 2 h of the treatment, indicating that PMSG was neutralized in these cows at the onset of final follicular maturation. In all cows, the concentration of oestradiol showed a significant decrease at about 8 h after the LH peak. After anti-PMSG treatment ovulations took place from 24 until 30 h after the LH peak, whereas in control cows follicles had already ovulated at or before 22 h and ovulations continued until 30 h. At 30 h 90% of the follicles had ovulated in anti-PMSG-treated cows vs 72% in the controls, resulting in 15 and 8 ovulations per cow respectively (P less than 0.05). Also, administration of monoclonal antibody against PMSG synchronized final follicular maturation and shortened the period of multiple ovulations. In conclusion, neutralization of PMSG shortly after the preovulatory LH peak suppresses adverse effects of PMSG on final follicular maturation, leading to an almost 2-fold increase of the ovulation rate.     

Improved embryo yield and condition of donor ovaries in cows after PMSG superovulation with monoclonal anti-PMSG administered shortly after the preovulatory LH peak

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted. No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.     

Follicular and oocyte maturation in cows treated for superovulation

The maturational stage of oocytes and their follicles was assessed at 24 26 h after the preovulatory luteinizing hormone (LH) peak by means of morphological criteria. Follicles were obtained from cows treated for superovulation (PMSG/PG) with additional anti-PMSG to neutralize the residual PMSG. Follicular fluid was also recovered and analyzed for progesterone and estradiol levels. Seventy-two percent of the oocytes were at the Metaphase II (M(II)) stage of meiosis, whereas only 28% of the follicular walls were at the proper maturational stage; assessed on morphological characteristics, 78% of the follicles were progesterone-dominated. Earlier maturational stages of oocytes and follicles were also present, including those that are restricted to periods shortly after the LH peak in the normally cyclic cow. It is concluded that upon treatment for superovulation not all oocytes and follicles mature synchronously, and that not all oocytes mature in harmony with their follicles.     

Progesterone-synthesizing ability of preovulatory follicles of cows relative to the peak of LH

Preovulatory cow follicles (n = 34) were collected at different times after the onset of oestrus until shortly before ovulation. In-vitro conversion of tritiated pregnenolone in the presence of NAD+ by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) a moderate conversion into progesterone occurred, but it was subsidiary to that into 17 alpha-hydroxypregnenolone and other unidentified steroids. During phases 1 (0-6 h after the LH peak), 2A (6-14 h) and 2B (14-20 h) the production of progesterone and 17 alpha-hydroxypregnenolone remained constant; at phase 2B the percentage of remaining pregnenolone was higher than in the preceding phases. In phase 3 (20 h after the LH peak until ovulation) conversion into progesterone had increased about 4-fold to the highest levels observed (97% after 2 h incubation), and production of 17 alpha-hydroxypregnenolone and unidentified steroids was low. In an additional experiment, homogenates of the wall of 3 follicles at phase 3 were also incubated with tritated progesterone in the presence of NADPH. The percentage of remaining progesterone was high, and a moderate conversion into 17 alpha-hydroxyprogesterone occurred. In the main experiments, however, production of this steroid was not observed. The results indicate that steroid synthesis in the preovulatory follicle of the cow changes to the production of progesterone shortly before ovulation.     

Bovine model of reproductive aging: response to ovarian synchronization and superstimulation

The responsiveness of the hypothalamo-pituitary axis to steroid treatments for ovarian synchronization and the ovarian superstimulatory response to exogenous FSH was compared in 13-14 year old cows and their 1-4 year old young daughters. We tested the hypotheses that aging in cattle is associated with: (1) decreased follicular wave synchrony after estradiol and progesterone treatment; (2) delayed LH surge and ovulation in response to exogenous preovulatory estradiol treatment; (3) reduced superstimulatory response to exogenous FSH. Higher plasma FSH concentrations (P<0.01), and a tendency (P=0.07) for fewer 4-5 mm follicles at wave emergence were observed in old cows (n=10) than in young cows (n=9). The suppressive effect of estradiol/progesterone treatment on FSH was similar between old and young cows. Although the preovulatory LH surge in response to estradiol treatment was delayed in old than young cows (P=0.01), detected ovulation times were not different. No difference in ovarian superstimulatory response was detected between age groups, but old cows (n=8) tended (P=0.10) to have fewer large follicles (>or=9 mm) 12 h after last FSH treatment than in young cows (n=7). We concluded that pituitary and ovarian responsiveness to estradiol/progesterone synchronization treatment was similar between old and young cows, but aging was associated with a delayed preovulatory LH surge subsequent to estradiol treatment. Old cows tended to have fewer large follicles after superstimulatory treatment than young cows.     

Formation of cyclic adenosine monophosphate (cAMP) in the preovulatory rabbit follicle: role of prostaglandins and steroids

The preovulatory increase in follicular prostaglandins (PG) stimulated by luteinizing hormone (LH) is dependent upon 3'-5'-cyclic adenosine monophosphate (cAMP) and is essential for ovulation. It has been proposed that follicular PG stimulate a second rise in cAMP, independent of LH. This study examined the temporal relationships among PGE2, PGF2 alpha 6-keto-PGF1 alpha, estradiol-17 beta, progesterone, testosterone, androstenedione and the biphasic increases of cAMP in follicles of rabbits. Does received indomethacin (IN, 20 mg/kg, i.v.; n = 30) or phosphate buffer (C; n = 30), 0.5 h before 50 ug of LH. At laparotomy at 0, 0.5, 1, 2, 4 or 8 h after LH, blood was collected from each ovarian vein and two follicles per ovary were aspirated of fluid and excised. Plasma and follicular tissue and fluid were assayed for PG and steroids. Tissue and fluid were assayed for cAMP. In C does, cAMP (pmol/follicle) in tissue increased from 11.3 at 0 h to 14.2 at 0.5 h, decreased at 1 h (5.4) and increased linearly through 8 h to 14.5. In IN-treated does, cAMP remained high from 0.5 (13.2) to 2 h (16.3), decreased at 4 h (7.9) then increased again by 8 h (15.5). Indomethacin decreased all PG in follicular tissue but 6-keto-PGF1 alpha rose after 2 h, whereas PGE2 and PGF2 alpha did not. Estradiol-17 beta, progesterone, and androstenedione did not vary with treatment; testosterone was increased (P less than .05) by IN. PGE2 or PGF2 alpha may terminate the first phase of cAMP production, rather than initiate the second phase.     

Length of progesterone exposure needed to resolve large follicle anovular condition in dairy cows

Hypothalamic unresponsiveness to an estradiol surge appears to be an underlying cause of large follicle anovular condition (follicular cysts), but progesterone exposure for 7 days resolves this condition. In this study, dairy cows with induced (Experiment 1) or naturally occurring (Experiment 2) follicular cysts were treated for different times with progesterone. In Experiment 1, 16 of 26 cows (62%) were induced into anovulation by causing a GnRH/LH surge when no ovulatory follicle was on the ovary. Anovular cows (n = 16) were assigned to one of four treatment groups ( 0, 1, 3, or 7 days of progesterone treatment) using an intravaginal, progesterone-releasing implant (CIDR). All anovular cows had low circulating progesterone concentrations before controlled internal drug releasing (CIDR) and greater concentrations that reached steady state (1.3 +/- 0.1 ng/mL progesterone) by 3 h after CIDR insertion. Circulating progesterone decreased to basal concentrations by 4 h after CIDR removal. Cows were treated with 5mg estradiol benzoate (EB) 12 h after CIDR removal. None (n = 4) of the control cows (0 day) had an LH surge after EB. All of the 3 days (5/5) and 7 days (4/4) CIDR-treated cows had an LH surge following EB, but only one of the 1 day (1/3) CIDR-treated cows. Magnitude of the LH peak was similar in the 3 and 7 days cows. All cows treated for 7 days ovulated (4/4), whereas, ovulation occurred in only 3/5, 1/3, and 0/4 of the cows treated for 3, 1, and 0 day, respectively. The two cows in the 3 days group that did not ovulate had a normal LH surge, but these two cows had a smaller maximal follicle size than cows that ovulated. In Experiment 2, naturally anovular lactating dairy cows (24 of 248) were identified using weekly ultrasonography. All anovular cows grew follicles to >12 mm, with 54% (13 of 24) having follicles larger than ovular size (15-24 mm) and 33% (8 of 24) having follicles that would be considered cystic (>25 mm). Anovular cows were randomly assigned to CIDR treatment for 0, 1, or 3 days. All (7/7) of 3 days, 33% (3/9) of 1 day, and 25% (2/8) of control (0 day) cows ovulated by 1 week after CIDR removal. Thus, 3 days but not 1 day of progesterone exposure appears to be sufficient to reinitiate estradiol responsiveness of the hypothalamus.     

Perturbation of estradiol-feedback control of luteinizing hormone secretion by immunoneutralization induces development of follicular cysts in cattle

We used immunoneutralization of endogenous estradiol to investigate deficiencies in the estradiol-feedback regulation of LH secretion as a primary cause of follicular cysts in cattle. Twenty-one cows in the prostaglandin (PG) F(2alpha)-induced follicular phase were assigned to receive either 100 ml of estradiol antiserum produced in a castrated male goat (n = 11, immunized group) or the same amount of castrated male goat serum (n = 10, control group). The time of injection of the sera was designated as 0 h and Day 0. Five cows in each group were assigned to subgroups in which we determined the effects of estradiol immunization on LH secretion and follicular growth during the periovulatory period. The remaining six estradiol-immunized cows were subjected to long-term analyses of follicular growth and hormonal profiles, including evaluation of pulsatile secretion of LH. The remaining five control cows were used to determine pulsatile secretion of LH on Day 0 (follicular phase) and Day 14 (midluteal phase). The control cows exhibited a preovulatory LH surge within 48 h after injection of the control serum, followed by ovulation of the dominant follicle that had developed during the PGF(2alpha)-induced follicular phase. In contrast, the LH surge was not detected after treatment with estradiol antiserum. None of the 11 estradiol-immunized cows had ovulation of the dominant follicle, which had emerged before estradiol immunization and enlarged to more than 20 mm in diameter by Day 10. Long-term observation of the six immunized cows revealed that five had multiple follicular waves, with maximum follicular sizes of 20-45 mm at 10- to 30-day intervals for more than 50 days. The sixth cow experienced twin ovulations of the initial persistent follicles on Day 18. The LH pulse frequency in the five immunized cows that showed the long-term turnover of cystic follicles ranged from 0.81 +/- 0.13 to 0.97 +/- 0.09 pulses/h during the experiment, significantly (P < 0.05) higher than that in the midluteal phase of the control cows (0.23 +/- 0.07). The mean LH concentration in the immunized cows was also generally higher than that in the luteal phase of the control cows. However, the LH pulse and mean concentration of LH after immunization were similar to those in the follicular phase of the control cows. Plasma concentrations of total inhibin increased (P < 0.01) concomitant with the emergence of cystic follicles and remained high during the growth of cystic follicles, whereas FSH concentrations were inversely correlated with total inhibin concentrations. In conclusion, neutralization of endogenous estradiol resulted in suppression of the preovulatory LH surge but a normal range of basal LH secretion, and this circumstance led to an anovulatory situation similar to that observed with naturally occurring follicular cysts. These findings provide evidence that lack of LH surge because of dysfunction in the positive-feedback regulation of LH secretion by estradiol can be the initial factor inducing formation of follicular cysts.     

Hormonal and ovarian responses to a 5-day progesterone treatment in anoestrous dairy cows in the third week post-partum

Primiparous cows with low body condition at calving have an extended anovulatory period. Induction of ovulation and oestrus is possible with progesterone treatment but the response to this treatment differs between Friesian and Jersey breeds. The objective of this study was to describe changes in pulsatile LH secretion and the synchrony of developing ovarian follicles that occur during a progesterone treatment period of 5 days in primiparous anovulatory cows. The experimental model compared the progesterone treatment with spontaneous post-partum changes as well as a breed comparison in a factorial design.Thirty-six cows (Jersey n=19 and Friesian n=17) were managed to calve with a low body condition score (BCS<4. 5). Daily changes in ovarian follicle size were observed with transrectal ultrasonography in each cow from 8 days post-partum. Thirty of these cows were diagnosed to be anovulatory at 12-18 days post-partum (day 0) and allocated to a treatment (n=16) or a control group (n=14), balanced for breed. Each treated cow had a progesterone-releasing controlled internal drug-releasing (CIDR) device inserted vaginally for 5 days while control cows were left untreated. Changes in plasma LH concentrations were measured with intensive blood sampling over 8 h on days -1, 1, and 4. Blood samples were also collected daily (06:00 h) for determination of plasma progesterone as well as oestradiol concentrations on days 6 and 8.Treatment with progesterone was associated with a transient initial decrease (day 1) in both LH pulse frequency and mean LH concentrations after device insertion, but both had returned to pre-treatment levels by day 4. Jersey cows had a greater pulse frequency, but there was no breed difference in mean LH concentrations. Patterns of ovarian follicle growth were affected by progesterone treatment with an increase in diameter of the dominant follicle (DF) identified after treatment initiation. This followed an earlier emergence of a new DF after device insertion. Follicular response to progesterone was dependent on the diameter of the DF present at treatment initiation. Those follicles >/=9 mm were replaced by a new DF during treatment such that the DF observed at the time of device removal was large (>/=9 mm) and growing in 13/16 cases.Progesterone was not effective for the induction of an LH surge, ovulation and oestrus in anovulatory cows with a low BCS. However, treatment was associated with synchronous development of a DF so that it was large and growing at the end of the treatment period in most cases. This synchronous development may be due to the transient suppression of LH and the presence of an LH-dependent DF.     

Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-17 beta, progesterone, luteinizing hormone, and follicle-stimulating hormone associated with follicular development in cows induced to superovulate with equine chorionic gonadotropin.

Changes in concentrations of bioactive and immunoreactive (ir-) inhibin, estradiol-17 beta, progesterone, LH, and FSH in peripheral blood were determined in cows induced to superovulate with eCG. The pattern of follicular growth was also characterized by daily ultrasonographic examination. Hormonal profiles and follicular development during the intact estrous cycle of the same animals before eCG treatment served as controls. Equine CG increased the number of follicles of various sizes (small, greater than or equal to 4 less than 7, medium, greater than or equal to 7 less than 10; large, greater than or equal to 10 mm in diameter) by 4 days after administration. The second growth of large follicles occurred within 1 day after superovulation. Inhibin bioactivity in jugular vein blood was detectable 48 h after eCG injection (44 h before LH peak), whereas it was not detected before administration of eCG or during control cycles. Circulating levels of bioactive inhibin further increased during the two waves of growth of large follicles. The highest activity of inhibin was noted at the time of the preovulatory LH peak (0 h). Thereafter, bioactivity of inhibin in peripheral plasma dropped from 0 to 24 h after the LH peak, and the activity increased again at 72 h compared to the value at -44 h. Plasma levels of ir-inhibin showed a pattern similar to changes in bioactive inhibin in the eCG-treated cows. Plasma concentrations of estradiol-17 beta also increased concomitantly with two waves of growth of large follicles. There was no correlation between plasma levels of progesterone and inhibin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive hormones and follicular growth during development of one or multiple dominant follicles in cattle

The mechanisms regulating ovulation rate under natural conditions are not yet defined, particularly for monovular species. In the present study, we evaluated ovarian structures (every 12 h by ultrasonography) and circulating hormones (every 6 h) to determine the differences between cows that developed one (single dominant; n = 16), two (double dominant; n = 8), or three (triple dominant; n = 3) dominant follicles. The four largest follicles were tracked retrospectively, and the data were normalized to the time of expected follicular deviation (F1 >/= 8.5 mm; hour 0). Follicular dynamics from emergence to deviation were similar, whereas after deviation, expected subordinate follicles continued to grow at a rate similar to the dominant follicle. Triple dominants had greater FSH than double dominants (hour -24 to hour -12) and single dominants (hour -42 to hour -6), and double dominants had greater FSH than single dominants (hour -24 to hour -12). Increased circulating estradiol but lower inhibin were observed in cows that developed multiple follicles. In addition, double dominants had greater LH than single dominants (hour -42 to hour -24 and hour -6 to hour 0) and lower progesterone than single dominants (hour -12 and hour -6). Luteal volume was similar between groups, but milk production was greater for codominant than for single-dominant cows. Thus, selection of multiple dominant follicles during high milk production is related to a transient increase in circulating FSH and LH during the 24 h before follicular selection, producing continued postdeviation growth of follicles that ordinarily would have regressed. Increased FSH and LH probably result from decreased circulating inhibin and progesterone in cows that develop codominant follicles.     

Influence of intrauterine infections and follicular development on the response to GnRH administration in postpartum dairy cows

The effects of intrauterine infections and prior follicular development on the response to gonadotropin releasing hormone (GnRH) administration in postpartum dairy cows were studied. Fifty lactating Holstein cows were assigned at random to one of two groups after calving. Group I (control) consisted of 25 cows given a single intramuscular injection of saline on Day 15 postpartum. Group II (treated) consisted of 25 herdmates given a single i.m. injection of 100 mug of GnRH on Day 15 postpartum. Palpation per rectum and real-time ultrasonography were used to monitor ovarian activity, and endometrial swabs were cultured to determine the presence of uterine infection. Blood samples were collected for progesterone (P(4)) and luteinizing hormone (LH) analysis. Fourteen cows (control, n = 5; treated, n = 9) did not ovulate during the first 60 d postpartum. Ovaries in these cows contained 4 to 8-mm size follicles and both P(4) and LH remained at basal concentrations. Fourteen other cows (control, n = 6; treated, n = 8) ovulated by Day 15 postpartum. Follicles >/= 10 mm were demonstrable in the ovaries of these cows before or by Day 12 postpartum. GnRH treatment had no effect on the lifespan of the existing corpus luteum in these cows. In the remaining cows, 7 of 14 Control and all 8 Treated cows ovulated within 3 d of treatment. All cows ovulating within this period were free of uterine infection and the ovaries contained follicles 相似文献   

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.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular dynamics, plasma metabolites, hormones and insulin-like growth factor I (IGF-I) in lactating cows with positive or negative energy balance during the preovulatory period.

The effect of dietary energy balance (EB) on growth of ovarian follicles was tested. Cows (n = 9) were fed a high energy diet (HE diet; positive EB; n = 4) or switched to a low energy diet (LE diet; negative EB; n = 5) during the preovulatory period. Non-esterified fatty acids (NEFA) were greater in cows fed the LE diet. Concentrations of luteinizing hormone (LH) were similar in HE and LE cows. However, the growth of preovulatory follicles in cows fed the LE diet was 50% that of cows fed the HE diet. Insulin-like growth factor-I (IGF-I) in plasma was less in LE-fed cows compared with HE-fed cows, and plasma IGF-I was positively correlated to estrogen: progesterone ratio in follicular fluid of dominant follicles. In summary, slower follicular growth in cows fed an LE diet occurred despite normal plasma LH and coincided with reduced IGF-I and elevated NEFA in plasma.     

An alteration in the hypothalamic action of estradiol due to lack of progesterone exposure can cause follicular cysts in cattle

Many mammals, including cattle, can develop ovarian follicular cysts, but the physiological mechanisms leading to this condition remain undefined. We hypothesized that follicular cysts can develop because estradiol will induce a GnRH/LH surge on one occasion but progesterone exposure is required before another GnRH/LH surge can be induced by estradiol. In experiment 1, 14 cows were synchronized with an intravaginal progesterone insert (IPI) for 7 days, and prostaglandin F(2alpha) was given on the day of IPI removal. Estradiol benzoate (EB; 5 mg i.m.) was given 3 days before IPI removal to induce atresia of follicles. Cows were given a second EB treatment 1 day after IPI removal to induce a GnRH/LH surge in the absence of an ovulatory follicle. All cows had an LH surge following the second EB treatment, and 10 of 14 cows developed a large-follicle anovulatory condition (LFAC) that resembled follicular cysts. These LFAC cows were given a third EB treatment 15 days later, and none of the cows had an LH surge or ovulation. Cows were then either not treated (control, n = 5) or treated for 7 days with an IPI (n = 5) starting 7 days after the third EB injection. Cows were treated for a fourth time with 5 mg of EB 12 h after IPI removal. All IPI-treated, but no control, cows had an LH surge and ovulated in response to the estradiol challenge. In experiment 2, cows were induced to LFAC as in experiment 1 and were then randomly assigned to one of four treatments 1) IPI + EB, 2) IPI + GnRH (100 microg), 3) control + EB, and 4) control + GnRH. Control and IPI-treated cows had a similar LH surge and ovulation when treated with GnRH. In contrast, only IPI-treated cows had an LH surge following EB treatment. Thus, an initial GnRH/LH surge can be induced with high estradiol, but estradiol induction of a subsequent GnRH/LH surge requires exposure to progesterone. This effect is mediated by the hypothalamus, as evidenced by similar LH release in response to exogenous GnRH. This may represent the physiological condition that underlies ovarian follicular cysts.     

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.     

Restoring ovulation in beef donor cows with ovarian cysts by progesterone-releasing intravaginal silastic devices.

The objective of this study was to determine the efficacy of a progesterone-releasing intravaginal silastic device (Controlled Internal Drug Release: CIDR) for inducing ovulation in beef cows with persistent ovarian cysts. Fifteen cows with cysts and abnormal cycles for over 40 days were randomly assigned to receive either a single CIDR (CIDR group, n=9), or a CIDR containing no progesterone (blank CIDR) (BLANK group, n=6) for about 14 days. Determination of plasma progesterone levels at the beginning of CIDR treatment indicated 4 of 6 BLANK cows with non-luteinized cysts and 5 of 9 CIDR cows with non-luteinized cysts. In 5 of 6 BLANK cows, one follicular wave appeared and newly emerged dominant follicles increased in size up to 20 mm in diameter and persisted during the experiment, while one cow experienced estrus with spontaneous ovulation. In contrast, during CIDR treatment, 2 or 3 waves, in which dominant follicles were from 7 to 15 mm in diameter, appeared approximately at 7-day intervals. Within 3 days after CIDR removal, estrous behavior was detected followed by ovulation of the dominant follicle in the last wave. All CIDR cows resumed normal cyclicity with 2 follicular waves for over 2 months. Insertion of a CIDR caused a rapid increase of about 2 ng/mL in plasma progesterone. The levels were greater than 1.3 ng/mL until removal of a CIDR, then dropped under 0.3 ng/mL. Concentrations of plasma estradiol in BLANK cows increased during growth of the cystic follicles, with high levels greater than 10 pg/mL for over 10 days. In 4 of 5 cows with non-luteinized cysts, with high plasma estradiol on the day of CIDR insertion, CIDR treatment resulted in rapid decline of estradiol levels. During placement of the CIDR, estradiol levels showed no increase in the growth phase of a newly appeared dominant follicle. After CIDR removal, however, estradiol significantly increased associated with the growth of ovulatory follicles in all 9 cows. A transient increase in plasma FSH levels preceded detection of each follicular or cyst wave in both BLANK and CIDR cows. Pulse frequency and mean concentration of LH in cows with non-luteinized cysts showed values corresponding to those in normal follicular phase. However, throughout CIDR treatment, these parameters reduced to levels found in the normal luteal phase. In cows with luteinized cysts, parameters of LH secretion were as low as in the normal luteal phase before and during CIDR treatment, then increased significantly after CIDR removal. Present results indicate that treatment with CIDR proved effective in restoring ovulation and reestablishing normal cyclicity in beef donor cows with cysts persistent for a long period. The CIDR reduced and maintained LH secretion at normal luteal levels, thereby, inducing atresia of estrogen-active cysts and preventing formation of cysts from the newly emerged follicles.     

Expression of the bovine oestrogen receptor-beta (bERbeta) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERbeta mRNA of second wave follicles after LH infusion into cows

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.     

Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotrophin

Eighteen lactating Holstein cows were randomly divided into three groups of equal size. Six cows were not superovulated; the remaining cows were superovulated using either FSH-P or PMSG beginning on Day 12 of the estrous cycle (day of ovulation = Day 0). Animals treated with FSH-P were injected intramuscularly (i.m.) with 4 mg FSH-P every 12 h for 5 d. PMSG was administered i.m. as a single injection of 2350 IU. Cloprostenol (PG, 500 ug) was injected i.m. 56 and 72 h after commencement of treatment and at the same time in the cycle of controls. All cows were inseminated 56, 68 and 80 h after the first PG injection. Blood samples (5 ml) were collected daily and every 15 min for a period of 9 h on Days -1, 0, 2, 8 and 10, with continuous blood sampling at 15-min intervals during Days 3 to 6. Ovulation rate was 27.7 +/- 8.22 in animals treated with PMSG, and 8.0 +/- 3.2 embryos per donor were recovered. In the FSH group, ovulation rate was 8.3 +/- 1.48 and 3.0 +/- 1.1 embryos per donor were recovered. Progesterone concentrations were similar in all three groups until the onset of the LH surge, when progesterone concentrations were greater (P<0.05) in animals of the PMSG group. After the preovulatory LH surge, concentrations of progesterone started increasing earlier (44 h) in cows treated with PMSG, followed by FSH-treated cows (76 h) and controls (99 h). The LH surge occurred earlier (P<0.05) in PMSG-treated cows (37 h after first PG treatment), than in animals treated with FSH-P (52 h) or controls (82 h). In animals treated with FSH-P, the magnitude of the preovulatory LH surge (24.2 +/- 1.02 ng/ml) was higher (P<0.05) than in the other two groups (PMSG = 17.1 +/- 2.04 ng/ml; control, 16.7 +/- 1.24 ng/ml). Superovulation with FSH-P or PMSG did not affect either mean basal LH concentration, frequency or amplitude of LH pulses during Days -1, 0, 2, 3, presurge periods, or Days 8 and 10 post-treatment. At ovariectomy, 8 d post-estrus, more follicles > 10 mm diam. were observed in the ovaries after treatment with PMSG (8.5 +/- 5.66) than after treatment with FSH-P (0.7 +/- 0.42) (P<0.05). Maximum concentrations of PMSG were measured 24 h after administration. Following this peak, PMSG levels declined with two slopes, with half-lives of 36 h and 370 h.