Plasma follicle stimulating hormone in Merino ewes immunized with an inhibin-enriched fraction from bovine follicular fluid

Plasma FSH concentrations were measured in Merino ewes immunized with either an inhibin-enriched preparation from bovine follicular fluid (bFFI) or bovine serum albumin. When compared during the normal oestrous cycle, ewes reimmunized three times with bFFI and which showed increased ovulation rates before the experiment had significantly elevated plasma FSH concentrations on Day 13–14 and at Day 2 of the subsequent cycle. There was a positive correlation (P < 0.05) between plasma FSH concentration and the ovulation rate of the ewes in previous cycles (during the period of immunization) and in the cycle under investigation. In a larger group of ewes immunized against bFFI, which showed a variable increase in ovulation rate, there was no comparable increase in plasma FSH concentration when compared with control ewes in the follicular phase of the cycle.By contrast, when luteolysis was induced by a prostaglandin analogue the bFFI-immunized ewes had lower plasma FSH concentrations than control ewes immediately before and after the preovulatory LH surge. This decrease was significant in the period 9–21 h after the LH surge (P < 0.05–0.01) so that the onset of the second FSH peak was delayed.When the ewes were ovariectomized, the post-castration rise in plasma FSH concentration (but not LH) was delayed for a period of 24 h in bFFI-immunized ewes relative to controls.These experiments show that immunization of ewes with an inhibin-like fraction of bFF does not lead to consistently elevated plasma FSH. However, such ewes have altered feedback regulation leading to differential responses of FSH to prostaglandin-induced luteolysis and to castration.     

Suppression of plasma FSH concentrations with bovine follicular fluid blocks ovulation in GnRH-treated seasonally anoestrous ewes

The specific requirement for FSH in the final stages of preovulatory follicle development was assessed in seasonally anoestrous ewes given 2-h injections of GnRH (250 ng/injection), with (N = 10) or without (N = 10) concurrent treatment with bovine follicular fluid (bFF: 2 ml given i.v. at 8-h intervals). Treatment with bFF significantly (P less than 0.01) suppressed plasma FSH concentrations, but, at least for the first 30 h of treatment, did not influence the magnitude of GnRH-induced LH episodes (mean max. conc. 3.00 +/- 0.39 and 3.63 +/- 0.51 ng/ml for bFF-treated and control ewes, respectively). Of 10 animals treated with GnRH for 72 h, 5/5 control ewes showed oestrus and ovulated whereas 0/5 bFF-treated ewes showed oestrus or ovulated in response to GnRH treatment. There was, however, a transient (13.2 +/- 1.0 h) increase in plasma LH concentrations in the ewes given bFF (mean max. conc. 4.64 +/- 1.57 ng/ml), which was coincident with the preovulatory LH surge recorded in animals given GnRH alone. In 10 GnRH-treated ewes slaughtered after 32 h of treatment, the mean diameter of the largest antral follicle was significantly (P less than 0.001) greater in control ewes (5.92 +/- 0.17 mm) than in animals that were also given bFF (3.94 +/- 0.14 mm). In addition, the incidence of atresia in the 3 largest antral follicles present at this time was greater in bFF-treated ewes. These results show that, when plasma FSH concentrations are suppressed by administration of bFF, although the magnitude of GnRH-induced LH episodes is unchanged, preovulatory follicular development is impaired and ovulation does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

The secretion of bioactive and immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) throughout the menstrual cycle of the rhesus monkey (Macaca mulatta)

The present study provides the first evaluation of related changes in serum levels of bioactive FSH (Bio FSH) and immunoreactive FSH (iFSH), and concurrent dynamics of LH and FSH bioactivity throughout the menstrual cycle of the rhesus monkey. Mean concentrations of Bio FSH were elevated on days 0 and 1 (n = 7; P < 0.05; day 0 = preovulatory LH surge). Data from individual animals revealed that an average (± SEM) of 1.43 ± 0.29 and 2.71 ± 0.61 discrete surges of Bio FSH occurred in each monkey's follicular and luteal phase, respectively. Analysis of the collective data indicated that periods of increased Bio FSH secretory activity spanned days −1 to 1 and 8 to 10 (P < 0.025). Increases in serum Bio FSH and iFSH concentrations were not precisely correlated on a daily basis (38.9%), although 72.2% of the peaks of Bio FSH and iFSH surges occurred within a day of one another. Similarly, only 36.1% of the Bio FSH surges were accompanied by elevations in bioactive LH (Bio LH). A significant rise in Bio LH, but not Bio FSH, occurred on day −1 (P < 0.01). Concentrations of Bio LH, but not Bio FSH, were elevated in the early luteal phase (P < 0.01). The bioactivity/immunoactivity ratios (Bio/I) of LH and FSH were maximal on the day of the preovulatory surge (P < 0.01). On day −1, LH Bio/I significantly increased (P < 0.05), but no change in FSH Bio/I was detected. The Bio/I of LH, but not FSH, remained elevated in the early luteal phase. In summary: the relative increase in Bio FSH exceeds iFSH during the preovulatory surge. Surges of Bio FSH occur during the follicular and luteal phases which potentially could support follicle selection/maturation. Divergencies between circulating LH and FSH biopotency may reflect a differential regulation of secretion and/or biosynthesis of these hormones. The prolonged early luteal elevation of LH Bio/I is consistent with the idea of a functional role of elevated LH biopotency in the maintenance of the corpus luteum.     

Follicle populations,ovulation rates and plasma profiles of LH,FSH and prolactin in Scottish Blackface ewes in high and low levels of body condition

Scottish Blackface ewes in high body condition (mean score = 2.86) had a higher mean ovulation rate (1.8 v. 0.9; P < 0.05) and more large (⪖ 4 mm diameter) follicles (4.6 v 2.2; P < 0.05) than ewes in low condition (mean score = 1.84) but similar numbers of small (1–4 mm diameter) follicles (6.3 v 6.0; NS). There was little difference in LH profiles with body condition but FSH and prolactin concentrations were significantly greater, during both luteal and follicular phases of the cycle, in ewes in high condition.Despite the relationships between body condition and ovulation rate and between condition and hormone concentrations, within the high condition groups, there was no significant difference in FSH levels with ovulation rate. Prolactin levels were higher in ewes with a single ovulation than in ewes with two or three ovulations. There was a trend towards a higher mean LH pulse frequency in the luteal phase and a higher mean LH pulse amplitude in the follicular phase in ewes with multiple ovulations compared with ewes with a single ovulation. During oestrus, only circulating prolactin concentrations differed with body condition, being significantly higher in ewes in high condition, but mean LH concentrations were higher and FSH concentrations lower in ewes with multiple ovulations. Subsequent luteal function, as measured by circulating progesterone concentrations, was normal in all ewes. It is concluded that body condition affected the size of the large follicle (⪖ 4 mm diameter) population through changes in FSH and possibly pulsatile LH secretion and prolactin secretion during the luteal and follicular phases of the cycle and that the number of follicles that were potentially ovulatory was probably determined during the luteal phase of the cycle. However, their ability to undergo the final stages of development and to ovulate may be related to the amount of LH secreted during the follicular phase.     

Dietary-induced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova

In the first of three experiments, eight ovariectomised Greyface ewes primed with exogenous progesterone were used to provide quantitative data on the effects of two contrasting feeding levels (0.3 vs. 1.4 × maintenance) on plasma progesterone concentrations. Over the 9 day study period, mean (± SEM) daily progesterone concentrations were 4.3 ± 0.13 and 3.3 ± 0.17 μg l⁻¹ for the low and high feeding regimens, respectively (P = 0.06), indicating that high feed intake suppressed circulating progesterone levels. The second experiment examined the effect in superovulated Finn-Dorset ewes of a diet supplying either 0.6 (Group L, n = 8) or 2.3 (Group H, n = 8) times their daily energy needs for maintenance, from 1 day before introduction of exogenous progesterone to the time of insemination, on plasma progesterone concentrations and the viability of ova recovered 4 days after insemination. Mean (± SEM) plasma progesterone concentrations were 4.5 ± 0.17 μg l⁻¹ and 2.8 ± 0.16 μg l⁻¹ for L and H ewes, respectively, during the 12 day priming period (P < 0.001). Eight hours after progesterone withdrawal, levels had fallen to 0.9 ± 0.06 μg l⁻¹ and 0.8 ± 0.07 μg l⁻¹, respectively, then rose to 17.8 ± 3.01 μg l⁻¹ and 12.9 ± 2.50 μg l⁻¹ (P > 0.10) at ovum collection. Intervals (mean ± SEM) to oestrous onset (14.5 ± 0.38 h) and the luteinising hormone (LH) surge (27.1 ± 0.98 h) were unaffected by feed intake. Mean (± SEM) ovulation rates (8.1 ± 1.57 vs. 7.8 ± 1.10) and numbers of ova recovered (5.0 ± 1.39 vs. 4.8 ± 1.11) were also similar for each group. However, the proportions of ova considered viable (over 32 cells) at recovery were 0.53 and 0.22 for L and H groups, respectively (P < 0.005). Following 72 h culture (Tissue Culture Medium-199 (M199) + 10% foetal calf serum (FCS)), 0.55 and 0.27, respectively, had developed to blastocysts (P < 0.025). Of ova assessed as viable at recovery, similar proportions (0.86 vs. 0.75) from L and H treatments developed to blastocysts, with corresponding nuclei counts (mean ± SEM) of 55 ± 5.2 and 55 ± 13.2. The third experiment used 12 superovulated Greyface ewes, each offered a different feed level within the range 0.6–2.5 × maintenance, to determine the nature of the relationship between feeding level, pre-ovulatory progesterone concentrations and ovum development at Day 2 following insemination and subsequently during 7 day co-culture (M199 + FCS). Increases in feeding level were accompanied by linear decreases in plasma progesterone (r² = 0.79, P < 0.001), the interval to oestrous onset (r² = 0.52, P < 0.01) and timing of the LH surge (r² = 0.32, P < 0.06). Although undetectable at ovum collection, and somewhat equivocal after 4 day culture, high feeding levels prior to ovulation reduced the proportion of ova (0.16 vs. 0.58) developing to or beyond the expanding blastocyst stage after 7 day culture. Quantitative indices of cell division and protein synthesis confirmed this. In conclusion, excessive feeding during follicular recruitment and oocyte maturation in superovulated ewes imparts a legacy of embryonic loss and developmental retardation.     

The Effect of oestradiol benzoate on the duration of oestrus and release of LH in ovariectomized Galway ewe lambs and adult ewes

The oestrous and LH responses by ovariectomized adult ewes (N=23) and 8-month-old ewe lambs (N=24) to i.m. injection of 10, 25, 62.5 or 156.25 μg oestradiol benzoate (ODB) were compared. The animals were primed by six daily injections of progesterone and ODB was administered 48 h after the last progesterone injection. The interval between ODB injection and onset of oestrus declined linearly (P<0.01) as the dose of ODB increased and was similar for the two age groups. The mean (±SEM) intervals to oestrus for levels of 10, 25, 62.5 and 156.25 μg ODB were 22.9±1.90, 18.0±1.33, 14.5±1.26 and 13.5±1.32 h, respectively. The duration of oestrus, determined by checking with Finnish Landrace rams at 3-h intervals, increased linearly (P<0.01) as the dose of ODB was raised and was significantly longer for ewe lambs (63.1±2.95 h) than for adult ewes (50.4±3.52 h). The overall mean (±SEM) durations of oestrus for levels of 10,25, 62.5 and 156.25 μg ODB were 16.9±5.91, 37.0±4.13, 75.2±3.94 and 97.8±4.13 h, respectively. A “pre-ovulatory” -type LH surge was observed in 32 of the 47 animals studied. The interval between injection of ODB and the beginning of the LH release declined as the dose of ODB increased (P<0.01) and was shorter (P<0.01) for ewe lambs (19.8±0.74 h) than for adult ewes (23.2±0.90 h). There was no evidence for an effect of either ewe age or dose of ODB on the maximum LH concentration observed, duration of LH discharge or total quantity of LH released. The sensitivity of the two age groups to the negative feedback effects of ODB on LH secretion was similar.     

Plasma FSH concentrations in seasonally anoestrous ewes induced to ovulate with repeated injections or continuous infusion of GnRH

Plasma FSH concentrations were monitored in 34 seasonally anoestrous ewes in which ovulation was induced by the administration of low doses of GnRH, given as either a series of i.v. injections (75, 125, 250 or 500 ng/2 h) or as a continuous i.v. infusion (125 or 250 ng/h). Fifteen of the animals had been pretreated with progesterone for 14 days. Before the start of GnRH treatment, mean FSH concentrations did not differ between progesterone-pretreated and non-pretreated ewes (23 ± 3.0 and 20 ± 2.0 ng/ml, respectively). In a significant (P < 0.01) proportion of animals mean FSH concentrations were elevated for the first 2 h of GnRH treatment, but thereafter they declined progressively and were significantly (P < 0.001) lower than pretreatment levels over the second 12 h of GnRH treatment. These changes in FSH concentrations were not related to dose of GnRH, the mode of administration or to progesterone priming. These results demonstrate that the pattern of FSH secretion associated with GnRH-induced ovulation in the seasonally anoestrous ewe is similar to that observed from the time of luteal regression in the naturally cycling ewe. In addition, although pretreatment with progesterone has a marked effect on subsequent luteal function, this is not mediated through changes in plasma FSH concentrations.     

Effects of phyto-estrogenic alfalfa consumption on plasma LH levels in cycling ewes

Plasma luteinizing hormone (LH) concentrations were determined in five Dorset ewes fed orchard grass hay (Dactylus glomerata) and five ewes fed alfalfa (Medicago sativa). Total phyto-estrogen content ($\text{X}\text{±}\text{SEM}$ genistein equivalents) of the orchard grass hay and alfalfa was 16.9 ± 2.9 and 118 ± 12.3 ppm respectively. LH was determined at regular intervals during the estrous cycles synchronized with progesterone impregnated pessaries and characterized by marker ram and vaginal cytology.Peak LH levels in control ewes (40.1 ± 5.5 ng/ml) were lower (P<0.05) than in ewes fed phyto-estrogenic alfalfa (66.0 ± 16.8 ng/ml). Results also indicate that the LH peak may occur later (P<0.05) in the estrus period of ewes fed phyto-estrogenic alfalfa (15.4 ±4.5 h). These experiments may suggest that peak LH concentrations are elevated and delayed further into the estrus period in ewes fed phyto-estrogenic alfalfa.     

The continuous presence of ewes in estrus in spring influences testicular volume,testicular echogenicity and testosterone concentration,but not LH pulsatility in rams

The continuous presence of active male small ruminants prevents seasonal anestrus in females, but evidence of the same mechanism operating from the females to the males is scarce. This study assessed the effects of the continuous presence of ewes in estrus in spring on ram sexual activity, testicular size and echogenicity, and LH and testosterone concentrations. On 1 March, 20 rams were assigned to two groups (n = 10 each): isolated (ISO) from other sheep, or stimulated (STI) by 12 ewes, which were separated from the rams by an openwork metal barrier, allowing contact between sexes. Each week, four ewes were induced into estrus by intravaginal sponges. Live weight, scrotal circumference, testicular width (TW) and length (TL) were recorded at the beginning and at the end of the experiment, and testicular volume (TV) was calculated; at the same time, testicular ultrasonography and color Doppler scanning were performed. Blood samples (March to May) were collected once per week for testosterone determinations, and at the end of the experiment, blood samples were collected for 6 h at 20-min intervals for LH analysis. Rams were exposed to four estrous ewes in a serving-capacity test. Scrotal circumference, TW and TL were higher in the STI than in the ISO rams (P < 0.05) in May, and TV was higher (P < 0.05) in the STI (391 ± 17 cm³) than in the ISO rams (354 ± 24 cm³). In ISO rams, the number of white pixels was higher (P < 0.01) in May (348 ± 74) than in March (94 ± 21) and differed significantly (P < 0.01) from that of the STI rams in May (160 ± 33). In ISO rams, the number of grey pixels was higher (P < 0.05) in May (107 ± 3) than it was in March (99 ± 1). Stimulated and ISO rams did not differ significantly in mean LH plasma concentrations (0.8 ± 0.5 v. 0.9 ± 0.4 ng/ml), LH pulses (2.1 ± 0.5 v. 2.2 ± 0.2) and amplitude (2.0 ± 0.4 v. 3.2 ± 0.7 ng/ml, respectively). Stimulated rams had significantly higher testosterone concentrations than ISO rams from April to the end of the experiment. Stimulated rams performed more (P < 0.05) mountings with intromission (3.0 ± 0.4) than did ISO rams (1.5 ± 0.5). In conclusion, after 3 months in the continuous presence of ewes in estrus in spring, rams had higher TV and some testicular echogenic parameters were modified than isolated rams. Although exposed rams also had higher levels of testosterone after 2 months in the presence of estrous ewes, their LH pulsatility at the end of the study was not modified.     

Reproductive performance of heifers induced to oestrous asynchrony by suprabasal plasma progesterone levels

Suprabasal progesterone concentrations around oestrus have induced disturbances in oestrous behaviour and ovulation. To determine whether fertility in such an altered oestrus can be maintained at normal levels with additional inseminations (AI) until ovulation, fertility was compared in heifers (n = 11) inseminated in normal oestrous cycles and thereafter in cycles in which the animals were treated with progesterone in order to create suprabasal concentrations after luteolysis. The treatment consisted of silicone implants containing 10.6 mg kg⁻¹ of progesterone inserted subcutaneously on Day 8 of the oestrous cycle (day of ovulation designated Day 0) and removed on Day 25. Both in control oestrous cycles and oestrous cycles under progesterone treatment, growth of the ovulatory follicle and ovulation were determined by frequent ultrasound scanning. Blood was collected frequently for further analysis of progesterone, oestradiol-17β and luteinising hormone (LH). Insemination was performed 12 h after onset of standing oestrus. if ovulation did not occur 24 h after AI, heifers were inseminated again until ovulation. Pregnancy was diagnosed by ultrasound 25 days after ovulation.In control oestrous cycles, plasma progesterone decreased to 0.3 ± 0.3 nmol 1⁻¹. Duration of oestrus was 22.9 ± 2.0 h, the interval from onset of oestrus to ovulation was 32.4 ± 2.3 h and the interval from LH peak to ovulation was 28.6 ± 1.4 h. The interovulatory interval was 20.7 ± 0.6 days. In oestrous cycles in treated heifers, progesterone decreased to 1.0 ± 0.3 nmol l⁻¹ (P > 0.10) and the interovulatory interval was prolonged to 23.5 ± 1.0 days (P < 0.05). Standing oestrus lasted 47.2 ± 12.0 h (P = 0.09, n = 7). The interval from the onset of oestrus to ovulation was 59.4 ± 13.0 h (P = 0.08) and the interval from LH peak to ovulation 25.8 ± 1.3 h (P > 0.10). The prolonged oestrus was associated with increased (P < 0.05) growth of the ovulatory follicle and higher (P < 0.05) release of oestradiol-17β. Conception rates were 90% and 46% (P < 0.05), and the numbers of AI per heifer were 1.1 ± 0.1 and 3.4 ± 0.6 (P < 0.01) for control oestrous cycles and after treatment, respectively.The induction of suprabasal concentrations of progesterone caused asynchronies similar to those observed in cases of repeat breeding. The repeated AI did not maintain fertility at normal levels. It is suggested that the extended growth of the ovulatory follicle may cause impaired oocyte maturation or it may alter the maternal milieu owing to the prolonged release of oestradiol.     

Interactions between dietary protein,ovulation rate and follicle stimulating hormone level in the ewe

Ovulation rate (OR) was studied in two experiments using mature Border Leicester × Merino ewes in which oestrous cycles were synchronized using a prostaglandin analogue. In both experiments a basal ration of 500 g of lucerne/barley pellets was provided. In the first experiment, ewes were fed individually iso-energy supplements of 500 g of either peas, lupins, soybean pellets or lucerne/barley pellets. The ovulation studied at laparoscopy occurred approximately 34 days after starting the supplementary feeding. Ewes fed lupins or soybean pellets had higher (P < 0.05) OR's than the ewes fed the other diets.In the second experiment, ewes were fed either iso-protein supplements of peas or lupins or casein supplement (170 or 100 g of protein) either formalin treated or untreated. The ovulations studied at laparoscopy occurred approximately either 17 or 34 days after the first feeding of the supplement. Follicle stimulating hormone (FSH) levels in plasma were measured over the 8 days prior to the second ovulation. There were no differences (P < 0.05) in OR's at the first ovulation. However, by the second, ewes fed peas had the highest (P < 0.05) OR while those fed lupins or protected casein had similar OR's. These tended to be higher than in the ewes fed untreated casein. FSH levels were generally higher from 8 days to 3 days prior to ovulation in ewes which were to have twin ovulations compared to those having single ovulations.The results confirm that feeding high energy or high protein will increase OR. There are independent effects of energy and protein. The results suggested that the ovulation rate may be related to FSH levels.     

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone.Treatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h⁻¹, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml⁻¹ h⁻¹, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles.It is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.     

Plasma inhibin A in heifers: relationship with follicle dynamics, gonadotropins, and steroids during the estrous cycle and after treatment with bovine follicular fluid

The relationship between follicle growth and plasma inhibin A, FSH, LH, estradiol (E), and progesterone was investigated during the normal bovine estrous cycle and after treatment with steroid-free bovine follicular fluid (bFF) to arrest follicle development. In the first study, four heifers were monitored over three prostaglandin (PG)-synchronized cycles. Blood was collected every 2-8 h, and ovaries were examined daily by ultrasonography. Inhibin A was measured using a modified enzyme-linked immunosorbent assay that employed a new monoclonal antibody against the alpha subunit of bovine inhibin. Plasma inhibin A ( approximately 50 pg/ml before luteolysis) rose steadily during the induced follicular phase (P < 0.05) to a peak ( approximately 125 pg/ml) coincident with the preovulatory E/LH/FSH surge. After ovulation, inhibin A fell sharply (P < 0.05) to a nadir ( approximately 55 pg/ml) coincident with the secondary FSH rise. During the next 3 days, inhibin A increased to approximately 90 pg/ml in association with growth of the new dominant follicle (DF). Plasma E also rose twofold during this period, whereas FSH fell by approximately 50%. Inhibin A was negatively correlated with FSH (r = -0.37, P < 0.001) and positively correlated with E (r = 0.49, P < 0.0001). Observations on eight cycles (two cycles/heifer), in which growth of the ovulatory DF was monitored from emergence to ovulation, showed that the first-wave DF (DF1) ovulated in three cycles and the second-wave DF (DF2) in five cycles. After PG, plasma inhibin A and E increased similarly in both groups, with concomitant falls in FSH. In the former group, the restricted ability of DF1 to secrete both inhibin A and E was restored after luteolysis. Results indicate that dynamic changes in the secretion of both E and inhibin A from the DF contribute to the fall in FSH during the follicular phase and to the generation and termination of the secondary FSH surge, both of which play a key role in follicle selection. In the second study, bFF (two dose levels) was administered to heifers (n = 3-4) for 60 h starting from the time of DF1 emergence. Both doses suppressed FSH (P < 0.05) and blocked DF1 growth to the same extent (P < 0.01), although inhibin A levels were only marginally raised by the lower dose (not significant compared to controls). The high bFF dose raised (P < 0.001) inhibin A to supraphysiological levels ( approximately 1 ng/ml). A large "rebound" rise in FSH occurred within 1 day of stopping both treatments, even though the inhibin A level in the high-dose bFF group was still approximately threefold higher than that in controls. This indicates that desensitization of gonadotropes to inhibin negative feedback is a contributory factor, together with reduced ovarian output of E, in generation of the post-bFF rebound in FSH.     

The influence of azaperone treatment at weaning on reproductive function in sows: ovarian activity and endocrine profiles during the weaning-to-ovulation interval

Azaperone can reduce stress caused by weaning and relocation of breeding sows, but its effects on reproductive processes are still poorly understood. The primary aim of this study was to describe and compare the endocrine and ovarian activity in ultrasonographically monitored second parity sows, with or without azaperone treatment at weaning (2 mg/kg BW i.m.). The intervals from weaning to the onset of estrus and ovulation were both greater (P<0.05) in azaperone-treated (n=12) than in control sows (n=12) by ~12 h. Mean daily growth rates of identified antral follicles were less (P<0.05) in azaperone-treated than in control sows (1.08±0.17 v.1.23±0.18 mm/day; mean±SD) and treated animals exceeded (P<0.05) controls in the mean ovulation rate (13.7±1.3 v. 12.6±1.2). A transient suppression of cortisol release was observed in the treatment group (at 10 and 30 min after azaperone injections) but circulating cortisol concentrations were variable in both groups of sows for the remainder of the study. The preovulatory rise in LH and estradiol secretion was delayed (P<0.05), and the duration of the LH surge was greater (P<0.001) in azaperone-treated sows compared with their control counterparts. The amplitude of episodic fluctuations in serum cortisol concentrations was correlated with the number of stillborn piglets in control sows (r=0.63, P=0.04). The amplitude and concentration of the preovulatory rise in estradiol secretion were negatively correlated with ovulatory response and litter size (r=−0.63 to −0.82, P<0.05), whereas the time at which the LH surge ended was directly related to the number of live-born piglets (r=0.82, P=0.002) in azaperone-treated animals. The present results indicate that administration of azaperone at weaning had a profound effect on preovulatory LH secretion as well as growth kinetics and estrogenicity of ovarian antral follicles. However, the causative associations among various characteristics of the preovulatory LH discharge, ovarian and adrenal steroid secretion post-weaning, and reproductive variables in sows remain equivocal.     

Gonadotrophin secretion during the periovulatory period in Galway and Finnish Landrace ewes and Finnish Landrace ewes selected for high ovulation rate

Rates of ovulation differed significantly (P less than 0.01) among ewes of the different genetic lines. However, of the reproductive characteristics studied, only progesterone concentration at the height of luteal function, duration of oestrus, and interval from onset of oestrus to peak of the preovulatory gonadotrophin surge showed significant positive association with rate of ovulation. The pattern of secretion of LH during the periovulatory period did not differ in the Galway and Finnish Landrace breeds. The total amount of LH secreted during the preovulatory surge did not differ amongst lines. Similarly, no difference in the plasma concentration of LH at the height of the preovulatory surge was noted among Galway and reference Finnish Landrace lines. However, the concentration of LH at the height of the surge was significantly (P less than 0.05) reduced in the selected Finnish Landrace line. Plasma concentrations of FSH during the preovulatory period were significantly (P less than 0.05) elevated in the breed (Galway) with the lowest prolifcacy. When contrasted with either of the Finnish Landrace lines, the magnitudes of the preovulatory surge of FSH and the secondary surge of FSH were significantly greater (P less than 0.05) in Galway ewes. These results suggest that genetic difference in rate of ovulation among sheep breeds is not tightly coupled to quantitative differences in plasma concentration of gonadotrophic hormones during the periovulatory period.     

Effects of oestradiol-17 beta, progesterone or bovine follicular fluid on the plasma concentrations of FSH and LH in ovariectomized Booroola ewes which were homozygous carriers or non-carriers of a fecundity gene

The plasma concentrations of FSH and LH were measured in ovariectomized Booroola FF and ++ ewes before and after treatment with subcutaneous implants of oestradiol-17 beta (0, 2 or 8 cm Silastic capsules; 5 ewes/genotype per dose) or progesterone (0, 1 or 3 Silastic envelopes; 5 ewes/genotype per dose) or subcutaneous injections of steroid-free bovine follicular fluid (bFF; 0, 0.5, 1.0, 2.5 or 5 ml; 4 ewes/genotype per dose). During the first 50 h after implantation of oestradiol or progesterone, or the first 24 h after bFF treatment, the FSH and LH concentrations in plasma were not different between the genotypes although there were significant effects of the steriods and bFF with respect to dose (P less than 0.05). At 6 days after steroid implantation, no gene-specific effects were noted for the plasma concentrations of FSH although significant effects of dose of oestradiol (P less than 0.01) but not progesterone were noted. Also at 6 days after steroid implantation, no gene-specific differences in the pulsatile patterns (i.e. peak frequency or amplitude) of plasma LH concentrations were noted although there were significant effects of steriod dose (P less than 0.05) on frequency and/or amplitude. It is concluded that the higher ovulation-rate in FF than ++ Booroola ewes is unlikely to be due to gene-specific differences in the sensitivity of the hypothalamic-pituitary axis to ovarian hormones.     

Suppression of the secondary FSH surge with bovine follicular fluid is associated with delayed ovarian follicular development in heifers

Holstein heifers were given 5 injections (twice/day) of 10 ml charcoal-extracted bovine follicular fluid (bFF; N = 6) or 10 ml saline (N = 5) beginning 12 h after the onset of oestrus. Blood samples were collected for determination of plasma concentrations of FSH, LH, progesterone and oestradiol-17 beta. Treatment with bFF suppressed the secondary FSH surge (P less than 0.01). Cessation of bFF injections was followed by a rebound period during which FSH was elevated compared with controls (P less than 0.01). Daily ultrasonographic examinations revealed that follicular growth occurred in waves, with 4 of 5 control heifers exhibiting 3 waves and the other 2 waves. In contrast, 5 of 6 bFF-treated animals exhibited 2 waves and the other 3 waves. Appearance of follicles in the first wave was delayed in bFF-treated heifers (Day 3.3 +/- 0.3 compared with Day 1.4 +/- 0.2; P less than 0.0001) and appearance of the dominant follicle of the first wave was delayed (Day 4.5 +/- 0.3 compared with Day 1.8 +/- 0.2; P less than 0.0001). Follicles in the second wave appeared later in animals treated with bFF (Day 12.7 +/- 0.4 compared with Day 10.4 +/- 0.6; P less than 0.01), and the dominant follicle of this wave also appeared later (Day 13.0 +/- 0.5 compared with Day 10.6 +/- 0.5; P less than 0.01). Oestradiol-17 beta increased during the early luteal phase, but this increase occurred later in heifers treated with bFF (peak concentrations on Day 6.3 +/- 0.6 compared with Day 4.2 +/- 0.2; P less than 0.05). LH, progesterone and cycle length were not affected by bFF. Delayed follicular growth associated with suppression of FSH suggests that the secondary FSH surge is important in the initiation of follicular development early in the bovine oestrous cycle, and thus may play a role in the regulation of ovarian follicular dynamics.     

Use of bovine follicular fluid to increase ovulation rate or prevent ovulation in sheep

Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0.1, 0.5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1.0 ml bFF twice daily (1.9 +/- 0.2 ovulations/ewe, mean +/- s.e.m. for N = 34) or 5.0 ml once daily (2.0 +/- 0.2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1.4 +/- 0.1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (greater than or equal to 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17 beta in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17 beta and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1-2.5 mm diam.) or medium (3-4.5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.     

Calm Merino ewes have a higher ovulation rate and more multiple pregnancies than nervous ewes

In 1990, two selection lines of Merino sheep were established for low and high behavioural reactivity (calm and nervous temperament) at the University of Western Australia. Breeding records consistently showed that calm ewes weaned 10% to 19% more lambs than the nervous ewes. We hypothesise that calm ewes could have a higher ovulation rate than nervous ewes and/or calm ewes could have a lower rate of embryo mortality than nervous ewes. We tested these hypotheses by comparing the ovulation rate and the rate of embryo mortality between the calm and nervous lines before and after synchronisation and artificial insemination. Merino ewes from the temperament selection lines (calm, n=100; nervous, n=100) were synchronised (early breeding season) for artificial insemination (day 0) (intravaginal sponges containing fluogestone acetate and eCG immediately after sponge withdrawal). On day-17 and 11 ovarian cyclicity and corpora lutea, and on days 30 and 74 pregnancies and embryos/foetuses were determined by ultrasound. Progesterone, insulin and leptin concentrations were determined in blood plasma samples from days 5, 12 and 17. Ovarian cyclicity before and after oestrus synchronisation did not differ between the lines, but ovulation rate did (day-17: calm 1.63; nervous 1.26; P<0.01; day 11: calm 1.83; nervous 1.57; P<0.05). Ovulation rate on day 11 in nervous ewes was higher than on day-17. Loss of embryos by day 30 was high (calm: 71/150; nervous: 68/130); but nervous ewes had a lower proportion (15/47) of multiple pregnancies compared with calm ewes (30/46; P<0.01). Reproductive loss between days 30 and 74 represented 7.3% of the overall loss. Temperament did not affect concentrations of progesterone, but nervous ewes had higher insulin (32.0 pmol/l±1.17 SEM; P=0.013) and lower leptin (1.18 μg/l±0.04 SEM; P=0.002) concentrations than calm ewes (insulin: 27.8 pmol/l±1.17 SEM; leptin: 1.35 μg/l±0.04 SEM). The differences in reproductive outcomes between the calm and nervous ewes were mainly due to a higher ovulation rate in calm ewes. We suggest that reproduction in nervous ewes is compromised by factors leading up to ovulation and conception, or the uterine environment during early pregnancy, that reflect differences in energy utilisation.