Follicle selection in cyclic guinea pigs with active immunization against inhibin alpha-subunit

Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine alpha-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3+/-0.3 to 7.0+/-1.6 per each ovary), and preovulatory sized follicles (from 1.8+/-0.6 to 7.0+/-1.6 per each ovary), and follicles stained positively for inhibin alpha-subunit (from 2.3+/-0.5 to 6.3+/-1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin alpha-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.     

Induction of superovulation by inhibin vaccine in cyclic guinea-pigs

Experiments were conducted to determine whether neutralizing endogenous inhibin affects follicular development and ovulation rate in guinea-pigs. Eighteen female guinea-pigs bearing 4 week progesterone implants were divided into three groups. At 1 week after removal of the progesterone implants, the animals were given a s.c. injection of 1 ml placebo (saline in oil emulsion; control), or 25 or 50 micrograms inhibin vaccine three times at 4 week intervals. Blood samples were collected once a week throughout the experiment for measuring inhibin antibody titres. After the third injection of inhibin vaccine, blood samples and ovaries were collected on the morning of day 8 after the day of oestrus. Inhibin vaccine increased the ovulation rate in a dose-dependent manner (placebo: 4.2 +/- 0.4; 25 micrograms inhibin vaccine: 6.2 +/- 0.9; 50 micrograms inhibin vaccine: 9.8 +/- 0.9) without any effects on the duration of the oestrous cycle. The results also showed that active immunization against inhibin increased the number of atretic follicles of 300-399 microns in diameter on day 8 after ovulation. The present study is the first to show that the active immunization against inhibin may be a useful method for inducing multiple ovulation in guinea-pigs.     

Ovulation and embryo recovery rates following immunization of mares against an inhibin alpha-subunit fragment

Six normally cycling mares were immunized 5 times at 3-week intervals with a synthetic porcine inhibin alpha-subunit fragment which had been conjugated to bovine serum albumin and emulsified in Freund's incomplete adjuvant. Immunized mares ovulated a significantly larger (P < 0.01) number of follicles per estrous cycle (2.8 +/- 1.1; range 1 to 8 ovulations) than 14 nonimmunized control mares (1.1 +/- 0.1; range 1 to 2 ovulations). Day-7 embryo recovery rates tended to be higher (P < 0.1) in immunized mares (1.6 +/- 0.5 embryos per flush) than in control mares (0.7 +/- 0.2 embryos per flush). No differences in interovulatory intervals were found between the 2 groups. These results indicate that immunization against inhibin may be useful in inducing development and ovulation of multiple follicles for embryo transfer in the mare.     

Plasma LH,FSH, testosterone,and age at puberty in ram lambs actively immunized against an inhibin alpha-subunit peptide

Active immunization against inhibin has been shown to advance puberty and increase ovulation rate in ewe lambs; but in ram lambs, effects on puberty and sperm production are equivocal. The objective of the present study was to determine whether active immunization against an inhibin alpha-subunit peptide advances the onset of puberty in ram lambs. St. Croix hair sheep ram lambs were assigned to inhibin-immunized (n = 7) and control (n = 8) treatment groups. Lambs in the inhibin-immunized group were immunized against a synthetic peptide-carrier protein conjugate, alpha-(1-25)-human alpha-globulin (halpha-G), and control lambs were immunized against halpha-G. Lambs were immunized at 3, 7, 13, 19, 25, 31, and 37 weeks of age. On the day of immunization a blood sample was collected and lambs were weighed. Another blood sample was collected 1 week following each immunization. At 20 weeks of age additional blood samples were collected at 20 min intervals for 8h. Beginning at 20 weeks of age and at weekly intervals thereafter, scrotal circumference (SC) was measured and semen was collected using electroejaculation. A subsequent ejaculate was collected 1 week following onset of puberty, which was defined as the week of age when an ejaculate first contained > or =50 x 10(6) sperm cells. In control lambs, plasma alpha-(1-25)-antibody (Ab) was nondetectable. In inhibin-immunized lambs, alpha-(1-25)-Ab titer increased from 7 to 25 weeks of age and then plateaued at a level that varied (P<0.001) among animals. Body weight and SC of control and inhibin-immunized lambs were similar at the onset of puberty. At pubertal onset inhibin-immunized lambs were older than control lambs (31.9+/-0.5 vs. 29.5+/-0.7 weeks of age, P<0.05). Plasma FSH concentrations were similar in control and inhibin-immunized lambs from 3 to 38 weeks of age. Plasma LH levels were lower (P<0.01) in inhibin-immunized than control lambs. During the 8-h blood sampling period at 20 weeks of age, LH and testosterone concentrations were lower (P<0.05) in inhibin-immunized than control ram lambs, and the LH pulse frequency was similar in the two groups of animals. The decreased LH secretion is consistent with the immunoneutralization of a putative inhibin alpha-subunit-related peptide that stimulates LH secretion in ram lambs. Present findings show that active immunization against an inhibin alpha-peptide delays rather than advances puberty in ram lambs.     

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.     

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.     

Systemic and intraovarian effects of dominant follicles on ovine follicular growth

The objective was to study the endocrine activity in sheep with large ovarian follicles and the effects of dominant follicles on other follicles, looking for possible intraovarian differences. Induction of dominant follicles was achieved using controlled exogenous LH pulses every 90 min over 14 days in eight Scottish Blackface ewes. During this period, follicular development was assessed by daily transrectal ultrasonography and jugular venous blood samples were collected every 12 h for FSH, LH inhibin and oestradiol assay. The exogenous LH pulses caused the appearance of large follicles in all the ewes, which reached a maximum mean diameter of 7.2 +/- 0.5 mm on Day 5.5 +/- 2.6 after first detection. In the presence of a dominant follicle, no other follicle grew to a diameter larger than 4 mm and there was a decrease in the number of new growing follicles (P < 0.05) and in the number of smaller follicles (P < 0.01). This effect of dominance was mediated by changes in FSH concentration, since FSH level decreased (P < 0.05) as dominant follicles grew and the decrease in FSH levels was related to a decline in the number of remaining follicles (P < 0.05). However, the greatest decrease in the number of small follicles growing to larger sizes was observed in the ovary ipsilateral to the dominant follicle (P < 0.05). These data confirm that the presence of a large follicle depresses the recruitment and growth of other follicles by systemic factors and provide some evidence of local inhibitors blocking the final development of other putative large follicles.     

Effect of a DNA vaccine harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n = 18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 μg (T₁, T₂ and T₃, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P < 0.05) in T₃ than in either T₁ or T₂ (0.341 ± 0.123, 0.236 ± 0.068, and 0.251 ± 0.077, respectively, mean ± SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P < 0.05) in T₃, and were higher (P < 0.05) in T₁ and T₂ than in control groups. For antibody-positive rats, there was a correlation (P < 0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T₃ group (46.00 ± 4.65) was higher (P < 0.05) than in two control groups (29.25 ± 3.72 and 27.92 ± 3.48), and also higher (P < 0.05) than in T₁ and T₂ (37.17 ± 4.99 and 38.75 ± 7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75 ± 4.23 vs. 35.60 ± 3.38, P < 0.05). Moreover, litter size and number of placentas were increased (P < 0.05) in the pcISI immunization groups, except for the T₁ group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 μg yielded the best immune response.     

Kinase pathways in dominant and subordinate ovarian follicles during the first wave of follicular development in sheep

The mechanism by which one or more dominant ovarian follicles continue development while other subordinate follicles regress is not known. The mitogen activated protein kinases (MAPKs) are a group of kinases that are activated by hormonal factors and form a cascade of processes that regulate cell growth, division and differentiation. The aim of the present experiment was to characterise the presence of the MAPKs, Erk 1/Erk 2 and Akt in healthy dominant follicles and regressing subordinate follicles. Following in vivo monitoring of ovarian follicle development, three ewes were ovariectomised and the follicular fluid and follicle wall (theca and granulosa cells) saved from the dominant and largest subordinate follicle. The dissected diameter and follicular fluid oestradiol concentration of the dominant follicle was larger (P<0.01) than the largest subordinate follicle (6.5+/-0.0mm and 41.3+/-4.9ng/ml versus 4.7+/-0.3mm and 0.6+/-0.4ng/ml). Western blot analyses showed that there was more Akt (202.7+/-6.4 versus 59.6+/-32.7 units; P<0.05) and Erk 1/Erk 2 (104.5+/-10.6 versus 0.3+/-0.2 units; P<0.01) present in follicle wall samples from the dominant compared to the largest subordinate follicles. Phosphorylated forms of Akt and Erk 1/Erk 2 were detected in samples from dominant but not subordinate follicles. We suggest that signal transduction pathways involving Akt and Erk 1/Erk 2 may play an important role in determining the outcome of ovarian follicle growth and development in sheep.     

Inhibin production in vitro by granulosa cells from Booroola ewes which were either homozygous or non-carriers of a fecundity gene influencing their ovulation rate

The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10-13 of the oestrous cycle, from Booroola x Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3-4.5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P less than 0.05) than that by cells from non-atretic 3-4.5 mm diameter ++ follicles, but similar to that of cells from non-atretic greater than or equal to 5 mm diameter ++ follicles. The production of oestradiol-17 beta by cells cultured in the presence of testosterone (1 microgram/ml) followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17 beta production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17 beta synthesis, testosterone generally had a slight, stimulatory effect on inhibin production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of overexpression of inhibin alpha (1-32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.     

Heterologous expression and characterization of Indian Sahiwal cattle (Bos indicus) alpha inhibin

Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T(5) promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25?μg?mL(-1)) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.     

Follicular growth, endocrine response and embryo yields in sheep superovulated with FSH after pretreatment with a single short-acting dose of GnRH antagonist

The objective of this study was to characterize follicular development, onset of oestrus and preovulatory LH surge, and in vivo embryo yields of sheep superovulated after treatment with a single dose of 1.5mg of GnRH antagonist (GnRHa). At first FSH dose, ewes treated with GnRH antagonist (n=12) showed a higher number of gonadotrophin-responsive follicles, 2-3mm, than control ewes (n=9, 13.5+/-3.8 versus 5.3+/-0.3, P<0.05). Administration of FSH increased the number of >or=4mm follicles at sponge removal in both groups (19.3+/-3.8, P<0.0005 for treated ewes and 12.7+/-5.4, P<0.01 for controls). Thereafter, a 25% of the GnRHa-treated sheep did not show oestrous behaviour whilst none control sheep failed (P=0.06). The preovulatory LH surge was detected in an 88.9% of control ewes and 66.7% of GnRHa-treated sheep. A 77.8% of control females showed ovulation with a mean of 9.6+/-0.9 CL and 3.3+/-0.7 viable embryos, while ewes treated with GnRHa and showing an LH surge exhibited a bimodal distribution of response; 50% showed no ovulatory response and 50% superovulated with a mean of 12.2+/-1.1 CL and 7.3+/-1.1 viable embryos. In conclusion, a single dose of GnRHa enhances the number of gonadotrophin-dependent follicles able to grow to preovulatory sizes in response to an FSH supply. However, LH secretion may be altered in some females, which can affect the preovulatory LH surge and/or can weak the terminal maturation of ovulatory follicles.     

Effect of active immunization against inhibin on hormonal concentrations and semen characteristics in Shiba bucks

Active immunization against inhibin increased ovulation rate in females; in males, the effects of active immunization against inhibin on hormonal concentrations and sperm production need more investigation. To test the hypothesis that active immunization against inhibin increases FSH secretion and sperm output, the present study was undertaken to determine the effects of active immunization against inhibin on hormonal profile and sperm production in Shiba bucks. The bucks were actively immunized against inhibin alpha-subunit (immunized group, n=6) or Freund adjuvant (control group, n=5) four times, at 5-weeks intervals. Blood samples were collected twice-weekly and two successive ejaculates of semen were collected (with an artificial vagina) once-weekly. Plasma concentrations of FSH, LH and testosterone were measured by radioimmunoassay (RIA) and sperm motility characteristics were measured by computer-assisted sperm analysis (CASA). All inhibin-immunized bucks produced antibodies against inhibin. Relative to control bucks, in immunized bucks there were significant increases in plasma FSH concentrations and in sperm concentrations from 5 to 9 weeks and from 8 to 11 weeks, respectively, after primary immunization. However, plasma concentrations of LH and testosterone, semen volume, percentage of motile spermatozoa and motility parameters (straight-line velocity, curvilinear velocity and linearity index) were similar in both groups. In conclusion, active immunization against inhibin alpha-subunit increased FSH secretions and enhanced sperm production in bucks, whereas LH and testosterone concentrations, semen volume and sperm motility parameters were unaffected. Active immunization against inhibin could be used to improve fertility in Shiba bucks.     

Measurement of inhibin A and follicular status predict the response of ewes to superovulatory FSH treatments

Variability in superovulatory response to FSH stimulation is common to most mammals and imposes practical problems for assisted reproduction. In sheep, we have studied if this response is related to the ovarian follicular population and activity before the stimulation. During the breeding season, 30 ewes were treated with 40 mg FGA sponges for 14 days and 125 microg cloprostenol injection on Day 12, considering Day 0 as the day of progestagen insertion. Superovulatory response was induced with two different FSH regimes using the same total dose (8.8 mg), administered twice daily from 60 h before to 24 h after progestagen withdrawal. At the first FSH injection, all follicles > or = 2 mm were observed by transrectal ultrasonography and plasma FSH and inhibin A levels were determined. The number of corpora lutea and the number of and viability of recovered embryos in response to the treatment were determined on Day 7 after sponge withdrawal. No significant differences were found between treatments. The total mean number of corpora lutea (11.5 +/- 1.2) and recovered embryos (7.9 +/- 1.1) were positively correlated (P < 0.05 and <0.01, respectively) with the number of small antral follicles (2-3 mm: 9.2 +/- 0.7) and inhibin A concentration (240 +/- 18 pg/ml; P < 0.05 for corpora lutea and P < 0.005 for recovered embryos) observed at the onset of the superovulatory treatment, which was also positively correlated with the number of viable embryos (5.8 +/- 0.9, P < 0.005). In 18 ewes with follicles > or = 6 mm prior to FSH treatment, the ovulation rate was unaffected but the number of embryos (6.1 +/- 0.9 versus 11.6 +/- 2; P < 0.05) and their viability (4.5 +/- 0.8 versus 8.5 +/- 2; P < 0.05) was reduced. The lower number of embryos produced when a large follicle is present suggest that a proportion of the smaller follicles are in early stages of atresia and the developmental competence of their oocyte is compromised.     

The effect of subluteal levels of exogenous progesterone on follicular dynamics and endocrine patterns during early luteal phase of the ewe

Nineteen Corriedale ewes were treated with an im dose of a PGF2alpha during the luteal phase to synchronize estrus. After ovulation had been detected by using ultrasonography (Day 0); the ewes were randomly assigned to 2 different groups. In 11 ewes a CIDR, which had previously been used for 10 d, was inserted on the fourth day after ovulation. The ewes then received a dose of PGF2alpha on Day 5 to induce luteolysis. The CIDR remained in place until the end of the experiment (Day 9). Control ewes (n = 8) received no treatment. Blood samples were taken daily for estradiol, progesterone and FSH determinations. In the untreated ewes, 2 follicular waves were detected in all of the animals throughout the monitoring period, with a mean wave interval of 4.5 d. The total number of follicles which were > or =2 mm decreased from Day 0 to Day 4 (8.8+/-1.0 to 5.3+/-0.6; P< or =0.05) and then increased at Day 7 (7.5+/-0.9; P< or =0.05). The growth profiles of both the largest and the second largest follicles of Wave 1 showed significant divergence, while no divergence was observed in Wave 2. Serum estradiol concentrations decreased significantly from the day before to the day of ovulation and then increased again during the growing phase of the largest follicle of Wave 1. Concentrations of FSH were high on the day of emergence of both waves, but while a significant decline was observed after emergence in Wave 1, the levels remained high in Wave 2. In 8 of the 11 treated ewes, the largest follicle of Wave 1 was still present on the ninth day after ovulation (persistent follicle). In the other 3 ewes, the largest follicle of Wave 1 was already regressing on the day that the treatment was administered, and the largest follicle that was present on Day 9 originated from Wave 2 (nonpersistent follicle). In persistent follicle ewes, the largest follicle of Wave 1 prolonged its lifespan significantly, attaining the maximum diameter (Day 8.1+/-0.8) later than in untreated (Day 3.0+/-0.4) and nonpersisted follicle ewes (Day 2.0+/-0.6). The total number of follicles decreased in persistent follicle ewes between Day 0 and Day 4 (7.9+/-1.5 to 4.5+/-0.5, respectively; P< or =0.05) and remained low until the end of the experiment. Progesterone concentrations (nmol/L) between Days 6 and 9 were significantly different between untreated and persistent follicle ewes (12.8+/-1.0 vs. 9.4+/-1.0, P< or =0.02). The present study confirms that the largest follicle of Wave 1 is dominant in the ewe and that subluteal progesterone concentrations can prolong its lifespan and extend this dominance.     

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.     

Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells

The objective was to determine ovarian follicular fluid concentrations of glucose, lactate, and pyruvate in relation to follicle size in buffalo and sheep. The effect of varying concentrations of these substances on in vitro oocyte maturation, oocyte protein content, and granulosa and cumulus cell growth was also investigated. Follicular fluid was aspirated from various sizes of follicles (from ovaries without a dominant follicle) collected from adult, cycling nonpregnant buffalo (Bubalus bubalis) and sheep (Ovis aries) during the breeding season. Overall, mean (+/-S.E.M.) concentrations (mM) were glucose 2.42+/-0.31 and 1.40+/-0.22, lactate 7.56+/-2.61 and 10.42+/-1.64, and pyruvate 0.02+/-0.01 and 0.002+/-0.00, in buffalo and sheep, respectively. In both species, as follicles became larger, concentrations of glucose significantly increased, lactate significantly decreased, but pyruvate was not affected. Oocyte maturation was higher (P<0.05) in medium containing supra-physiological concentrations of either glucose (5 mM), or pyruvate (10 mM) alone, or physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub- or supra-physiological concentrations of glucose, lactate and pyruvate in combination (both species). The protein content of oocytes was not significantly affected by the concentration of glucose, lactate, and pyruvate in the maturation medium. However, growth of granulosa and cumulus cells was higher (P<0.05) in medium containing supra-physiological concentrations of glucose (5 mM) alone, or pyruvate (10 mM) alone, or physiological, or supra-physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub-physiological concentrations of glucose, lactate and pyruvate in combination (both species). In conclusion, concentrations of glucose, pyruvate and lactate in the medium had cell type-specific effects on oocyte maturation, and on growth of granulosa and cumulus cells. Furthermore, glucose and pyruvate were the principal energy sources for oocytes and follicular somatic cells in buffalo and sheep.     

Ovarian response in sheep superovulated after pretreatment with growth hormone and GnRH antagonists is weakened by failures in oocyte maturation

The administration of growth hormone (GH) or GH plus GnRH antagonists (GnRHa) in sheep allows the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries and may be useful to increase yields obtained in embryo programmes. The objective of the current study was to evaluate the ability of follicles recruited in response to treatment with GH and GnRHa to grow in response to exogenous follicle stimulating hormone (FSH) and the competence of their oocytes to resume meiosis. Seven females were treated with two doses of GnRHa (days 0 and 3) and three doses of 15 mg of GH (days 3, 4 and 5). Thereafter, this group and a second group (n = 7) were treated with three doses of 1.5 ml of FSH 12 h apart. A third group (control; n = 4) did not receive GH/GnRHa or FSH. The mean number of follicles aspirated on day 7 was higher in ewes treated with GH and GnRHa prior to the stimulation with exogenous FSH than in ewes treated with FSH without pretreatment and in untreated control sheep (20.4 +/- 2.6 vs 17.7 +/- 3.9 and 11.5 +/- 0.8, p < 0.05 and p < 0.01, respectively). The number of recovered cumulus-oocyte complexes after follicular aspiration was higher in the GH/GnRHa + FSH group (8.7 +/- 0.9 vs 6.8 +/- 1.3 in FSH group, n.s., and 4.5 +/- 0.8 in control, p < 0.05), but there were no differences found in the resumption of meiosis (63.1 +/- 9.5% for GH/GnRHa + FSH vs 79.5 +/- 6.3% for FSH and 60.0 +/- 8.8% for control). These results indicate that GH and GnRHa would be useful to increase the number of gonadotrophin-responsive follicles in the ovary, but adjustment of later FSH treatment allowing further development of follicles may be necessary prior to its use in superovulatory protocols.     

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.