Summary. Occupied and unoccupied LH receptors in corpora lutea, and LH and progesterone concentrations in circulating plasma, were measured in non-pregnant gilts that had been treated with oestradiol-17β benzoate to prolong luteal function. Oestradiol benzoate (5 mg, administered on Day 12 after oestrus) delayed luteal regression and the decline in LH receptor levels at luteolysis and raised unoccupied receptor levels from 11·8 ± 1·14 fmol/mg protein on Days 10–15 after oestrus to 31·8 ± 3·26 fmol/mg protein on Days 15–21. There was no simultaneous rise in occupied receptor levels and occupancy decreased from 29·8 ± 3·01 to 11·5 ± 1·26%. Basal plasma LH concentrations were unchanged by oestradiol, but mean corpus luteum weight and plasma progesterone concentrations were slightly reduced. Oestradiol benzoate on Day 12 caused a similar increase in unoccupied receptor levels in gilts hysterectomized on Days 6–9 after oestrus, from 17·0 ± 5·83 to 34·5 ±6·00 fmol/mg protein, determined on Days 15–18. Plasma concentrations of LH and progesterone were unchanged by oestradiol. Unoccupied receptor levels in corpora lutea and plasma LH and progesterone were unaltered by hysterectomy in untreated gilts. Occupied receptor levels were not influenced by hysterectomy or oestradiol. It is concluded that oestradiol-17β raises luteal LH receptor levels by a mechanism independent of the uterus.

Summary. Mature beef cows were actively immunized pre partum (N = 5) or post partum (N = 10) against a PGF-2α–ovalbumin conjugate or against ovalbumin alone (control; N = 5). All cows in the control group exhibited first oestrous cycles which were of short duration (⩽ 12 days). Mean specific serum binding to [³H]PGF-2α in the control group was consistently < 1%. In the pre-partum PGF-2α-immunized cows, lifespan and progesterone secretion of the first corpus luteum formed post partum was maintained for > 39 days. Specific serum binding to [³H]PGF-2α in pre-partum and post-partum PGF-2α-immunized cows was elevated. Lifespan of the first corpus luteum formed in post-partum PGF-2α-immunized cows was short (<10 days; N = 1), normal (mean = 22 days; N = 4) or maintained (>31 days; N = 5). Luteal lifespan was dependent upon serum PGF-2α antibody titres, with cows exhibiting higher titres frequently having prolonged luteal lifespans after first ovulation. We conclude that active immunization of beef cows against PGF-2α extends the lifespan and progesterone secretion of corpora lutea anticipated to be short-lived. These results support the concept that the shorter lifespan of some corpora lutea in post-partum cows is due to a premature release of PGF-2α from the uterus.

Keywords: active immunization; PGF-2α; corpus luteum; cattle

Although the decrease of progesterone in serum and in luteal tissue during luteal regression is well characterized, relatively little is known about changes in proteins produced by the corpus luteum during this time. The first objective was to examine changes in patterns of protein secretion that might be associated with functional and structural luteal regression. The second objective was to characterize the expression of two major secretory products of regressing corpora lutea. Thirty normally cyclic heifers were randomly assigned at day 15–16 of the oestrous cycle (oestrus = day 0) to be ovariectomized at 0 h (no PGF_2α; n = 5) or at 4, 8, 12, 24 or 48 h after PGF_2α-induced luteal regression (n = 5 per time point). Total cellular RNA was isolated from tissue frozen at the time of ovariectomy. Thin slices (< 1 mm) of tissue were placed in methionine-deficient minimum essential media with [³⁵S]methionine and placed in a humidified CO₂ incubator at 38°C. Media and tissues were collected 6 h later. Changes in profiles of secreted proteins were analysed by one-dimensional SDS-PAGE. A number of proteins (relative molecular mass ranging from 14 300 to 200 000) were produced by luteal tissue at each time point (0–48 h). The major secretory proteins had relative molecular masses of approximately 21 500, 28 200, 43 700 and 46 000. Secretion of the relative molecular mass 46 000 protein(s) increased (P < 0.05) between 4 and 24 h after PGF_2α injection compared with the 0 h group. Western blot analyses with either tissue inhibitor of metalloproteinase-1 or tissue inhibitor of metalloproteinase-2 antisera detected immunoreactive proteins of relative molecular mass 28 200 and 21 500, respectively. Concentrations of mRNA encoding tissue inhibitor of metalloproteinases-1 increased (P < 0.01) by 8 h after PGF_2α injection, remained stable (P > 0.20) through 24 h and decreased (P < 0.05) by 48 h after PGF_2α. Concentrations of tissue inhibitor of metalloproteinases-2 mRNA were highest (P < 0.05) at 8 h after PGF_2α injection and lowest (P < 0.05) 48 h following induction of luteolysis. In summary, the profile of luteal protein production changed during luteolysis and two secretory products (tissue inhibitor of metalloproteinases-1 and -2) were identified. Metalloproteinase inhibitors may have an important role in tissue remodelling during structural luteolysis.

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

Summary. This study was conducted to determine whether intrauterine infusion of recombinant bovine interferon-α_I1 (rboIFN-α_I1), which has 70% sequence identity to bovine trophoblast protein-1, will prevent regression of corpora lutea anticipated to have a short lifespan.

Twenty-six beef cows in good body condition were allotted to four treatment groups at parturition in a 2 × 2 factorial design. Treatments were: group 1, saline; group 2, rboIFN-α_I1; group 3, norgestomet–saline; and group 4, norgestomet–rboIFN-αI1. Norgestomet implants were inserted on days 21–24 postpartum and removed 9 days later (before injection of human chorionic gonadotrophin (hCG)). Ovulation was induced 30 to 33 days postpartum with 5000 or 10 000 iu hCG. Groups 1 (n = 7) and 3 (n = 5) were given intrauterine infusions (rectocervical approach) twice daily with saline on days 1–12 or 13–24 after hCG injection, respectively. Cows allotted to groups 2 (n = 8) and 4 (n = 6) were given intrauterine infusions (rectocervical approach) of 2 mg rboIFN-α_I1 twice daily on days 1–12 or 13–24 after hCG injection, respectively.

Treatment with both norgestomet and rboIFN-αI1 delayed (P < 0·01) luteolysis. Lengths of luteal phases (days; mean ± sem) were 8·4 ± 0·7 (group 1, saline), 12·1 ± 1·0 (group 2, rboIFN-α_I1), 18·6 ± 1·3 (group 3, norgestomet–saline and 20·8 ± 1·2 (group 4, norgestomet–rboIFN-α_I1). Concentration of progesterone in serum was similar among all groups the first 6 days following hCG-induced ovulation, but differed (P < 0·01) thereafter. Mean concentration of progesterone was lower (P < 0·01) in saline-treated cows (group 1) from days 8 to 13 than in cows infused with rboIFN-α_I1 or pretreated with norgestomet. In summary, intrauterine infusion of rboIFN-α_I1 delayed luteolysis of corpora lutea anticipated to have a short lifespan.

Keywords: corpus luteum; progesterone; norgestomet; recombinant bovine interferon-αI1; cow

A study was conducted to examine the effects of gonadotropins on ovarian follicular development and differentiation in GnRH agonist (GnRHa)-treated cattle. Holstein cows were allotted into two pre-treatment groups: controls (n = 5) and GnRHa-treated (n = 9). Ovaries were removed from control cows on day 5 following a synchronized estrus. Treatment with GnRHa resulted in follicular arrest at <5 mm. Following follicular arrest, GnRHa-treated cows received a constant infusion of FSH for 96 h (GnRHa/FSH), with a randomly selected subset receiving hourly pulses of LH in addition to FSH during the last 48 h of infusion (GnRHa/FSH + LH). At the end of infusion, ovaries were removed, follicles were counted and measured, and follicular fluid samples were collected from large follicles (>10 mm). Differences in expression of mRNA for LH receptor, FSH receptor, cytochrome P450 side-chain cleavage, 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase (P450c17) and cytochrome P450 aromatase were determined in large follicles using in situ hybridization. The number of large follicles did not differ between GnRHa/FSH-treated and GnRHa/FSH + LH-treated cows (P = 0.64), but was greater than control animals (P ≤ 0.004). Follicular fluid concentrations of estradiol-17β and androstenedione were highest in GnRHa/FSH + LH-treated cows (P ≤ 0.04), intermediate in control cows, and lowest in GnRHa/FSH-treated cows. Hybridization intensity of P450c17 was greater in GnRHa/FSH + LH-treated versus control or GnRHa/FSH-treated cows (P ≤ 0.03). These results indicate that while FSH can support bovine follicular growth >10 mm, LH increases androgen production and expression of P450c17.

Summary. Non-lactating, multiparous dairy cows diagnosed as having cysts by palpation per rectum were used. Cysts were induced with oestradiol-17β (15 mg) and progesterone (37·5 mg) dissolved in ethanol and injected s.c. twice daily for 7 days. Following initial diagnosis of cysts, ovaries were exposed by midventral laparotomy, and the perimeter of the base of each cyst was marked with subepithelial injections of charcoal. Ovaries were removed from cows by transvaginal incision at 10 days (Group 1; N = 8), 20 days (Group 2; N = 8), or 40 days (Group 3; N = 7) after marking of cysts. Ovaries were examined for structures present and their relationship to the marked site. Corpora lutea with ovulation papilla were present in 7/23 cows (1/8, 4/8 and 2/7, for Groups 1, 2 and 3, respectively). In these 7 cows, corpora lutea were at a site different from the original structure that was marked. Marked structures persisted for the duration of the experimental period in 1 and 2 cows, in Groups 1 and 3, respectively. In the remaining 13 cows, new large follicular structures (cysts) were present at a site other than that marked with charcoal. These structures developed on the ovary contralateral to the one originally marked in 9 of 13 cows. Cysts are therefore dynamic in nature and may persist or may be replaced by others.

Keywords: ovary; follicular cysts; turnover rate; dairy cattle

Summary. Two experiments were conducted to (1) investigate developmental endocrinology of ovarian follicular cysts (cysts) in cattle and (2) evaluate effects of cysts on hypothalamic and hypophysial characteristics. Cysts were induced with oestradiol-17β (15 mg) and progesterone (37·5 mg) dissolved in alcohol and injected s.c. twice daily for 7 days. Cysts were defined as the presence of follicular structures (which may or may not have been the same structure) of 2·0 cm in diameter or greater that were present for 10 days without ovulation and corpus luteum development.

In Exp. 1, 22 non-lactating, non-pregnant Holstein cows were allocated to 3 groups. Beginning on Day 5 (oestrus = Day 0) of the oestrous cycle, 7 cows (Controls) were treated with twice daily s.c. injections of ethanol (2 ml/injection) for 7 days. Luteolysis was then induced with PGF-2α and blood samples were collected daily every 15 min for 6 h from the morning after the PGF-2α injection (Day 13) until oestrus. Steroids to induce cysts were injected as previously described into the remaining cows (N = 15). Three blood samples were collected at 15-min intervals every 12 h throughout the experimental period. Additional blood samples were collected every 15 min for 6 h on a twice weekly basis. After steroid injections, follicular and luteal structures on ovaries were not detected via rectal palpation for a period of 36 ± 4 days (static phase). Then follicles developed which ovulated within 3–7 days (non-cystic; N = 7) or increased in size with follicular structures present for 10 days (cystic; N = 8). Mean (± s.e.m.) concentrations of LH, FSH, oestradiol-17β and progesterone in serum remained low and were not different during the static phase between cows that subsequently developed cysts or ovulated. During the follicular phase, mean serum concentration of LH (ng/ml) was higher (P < 0·1) in cows with cysts (2·9 ± 0·2) than in cows without cysts (1·1 ± 0·1) or control cows (1·4 ± 0·2). In addition, LH pulse frequency (pulses/6 h) and amplitude (ng/ml) were higher (P < 0·1) in cows with cysts (3·6 ± 0·3 and 2·2 ± 0·3, respectively) than in non-cystic (2·3 ± 0·2 and 1·0 ± 0·2, respectively) and control (1·8 ± 0·1 and 1·1 ± 0·2, respectively) groups during the follicular phase.

In Exp. 2, 20 non-lactating, non-pregnant dairy cows were used: 15 cows received exogenous steroids as previously described. Hypothalamic and hypophysial tissues were collected after diagnosis of cystic structures in 11 cows (cystic group). The remaining 4 cows in the steroid-treated group ovulated and were assigned to the control group in addition to 5 non-steroid treated cows. Hypothalamic and hypophysial tissues were collected during the late-luteal phase (Days 16–18) from these control cows (N = 9). Anterior pituitary concentrations (μg/g) of LH (60·5 ± 11·0, 44·6 ± 11·7), FSH (30·2 ± 4·0, 22·1 ± 4·6) and receptors for GnRH (17·2 ± 2·2, 23·4 ± 2·6 m × 10⁻¹⁰/mg protein) did not differ between cows with cysts and control cows, respectively. Content of GnRH (ng) in the combined preoptic area and hypothalamus proper was higher (P < 0·05) in control cows (37·7 ± 6·6) than cows with cysts (18·6 ± 6·1). In the pituitary stalk median eminence, GnRH content (ng) tended to be higher (P ≥ 0·1) in cows with cysts (38·5 ± 9·6) compared with control (21·1 ± 15·2) cows.

Secretory patterns (mean concentration, pulse frequency and amplitude) of LH were therefore increased during the follicular phase in cows which developed cysts compared to cows which subsequently ovulated. In addition, hypothalamic GnRH content, but not pituitary characteristics, appeared to be altered in cows with cysts.

Keywords: ovary; follicular cysts; dairy cattle; gonadotrophins; hypothalamus; pituitary

Luteal lifespan is short after first postpartum ovulation in early-weaned beef cows unless cows are pretreated with a progestogen. Regression of the short-lived corpus luteum in the postpartum beef cow is due to a premature release of prostaglandin F_2α (PGF_2α) from the uterus. The premature release of PGF_2α may be mediated through lower concentrations of receptors for progesterone, higher concentrations of oxytocin receptors, or both, in the endometrium. Thirty-one beef cows were randomly assigned to four groups at parturition. Calves from cows assigned to the short cycle group (n = 6; control) and the short cycle/endometrium group (n = 10) were weaned at 30–32 days post partum. Cows in the normal cycle group (n = 5; control) and the normal cycle/endometrium group (n = 10) received norgestomet implants for 9 days beginning 21–23 days post partum and calves were weaned at implant insertion. Duration of oestrous cycle (x ± sem; P < 0.01) following first postpartum ovulation for the short cycle group was 11.5 ± 1.9 days compared with 18.8 ± 0.6 days for the normal cycle group. On day 5 following first postpartum ovulation, cows in the short cycle/endometrium and the normal cycle/endometrium groups were hysterectomized and endometrial tissue collected for measurement of progesterone and oxytocin receptors. Mean number of total progesterone receptors per cell was lower (P < 0.05) in the short cycle/endometrium group than in the normal cycle/endometrium group. Mean concentration of oxytocin receptors (fmol mg⁻¹ protein) in the short cycle/endometrium group was higher (P < 0.05) than that in the normal cycle/endometrium group. In conclusion, uterine receptor populations for progesterone and oxytocin may influence the timing of PGF_2α secretion during short oestrous cycles.