Ovarian response, ova recovery and fertility in merino ewes superovulated either during the luteal phase of their oestrous cycle or after intravaginal progestagen treatment

Five groups of merino ewes were treated with 1000 i.u. of pregnant mare serum gonadotropin (PMSG) as a single injection per ewe. Three of these groups received treatment on days 7,9 and 11 of their oestrous cycle. Oestrus was synchronized with 125 mg of prostaglandin F2(alpha) (PG) given two days after PMSG. Oestrus in the other two groups was synchronized by intravaginal progesterone sponges inserted for 14 days. In one group, the sponges were inserted nine days after oestrus onset. In the other group the stage of the oestrous cycle was unknown. In both these groups, PMSG was given a day prior to sponge removal. No significant differences were recorded for either the mean numbers of corpora lutea, unovulated follicles or ova recovery between the five groups. However, progestagen synchronized ewes yielded significantly more fertilized ova (p < 0.05) than PG synchronized ewes.     

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.     

Effects of bromocriptine administration during the follicular phase of the oestrous cycle on prolactin and gonadotrophin secretion and follicular dynamics in merino monovular ewes

Two experiments using Spanish Merino ewes were conducted to investigate whether the secretion of prolactin during the follicular phase of the sheep oestrous cycle was involved in the patterns of growth and regression of follicle populations. In both experiments, oestrus was synchronized with two cloprostenol injections which were administered 10 days apart. Concurrent with the second injection (time 0), ewes (n = 6 per group) received one of the following treatments every 12 h from time 0 to 72 h: group 1: vehicle injection (control); group 2: 0.6 mg bromocriptine (0.03 mg per kg per day); and group 3: 1.2 mg bromocriptine (0.06 mg per kg per day). In Expt 1, blood samples were collected every 3 h from 0 to 72 h, and also every 20 min from 38 to 54 h to measure prolactin, LH and FSH concentrations. In Expt 2, transrectal ultrasonography was carried out every 12 h from time 0 until oestrus, and blood samples were collected every 4 h to measure prolactin, LH and FSH concentrations. Ovulation rates were determined by laparoscopy on day 4 after oestrus. Bromocriptine markedly decreased prolactin secretion, but did not affect FSH concentrations, the mean time of the LH preovulatory surge or LH concentrations in the preovulatory surge. Both doses of bromocriptine caused a similar decrease in LH pulse frequency before the preovulatory surge. The highest bromocriptine dose led to a reduction (P < 0.01) in the number of 2-3 mm follicles detected in the ovaries at each time point. However, bromocriptine did not modify the total number or the number of newly detected 4-5 mm follicles at each time point, the number of follicles > 5 mm or the ovulation rate. In conclusion, the effects of bromocriptine on gonadotrophin and prolactin secretion and on the follicular dynamics during the follicular phase of the sheep oestrous cycle indicate that prolactin may influence the viability of gonadotrophin-responsive follicles shortly after luteolysis.     

11-Ketotetranor PGF metabolites,a suitable indicator for measuring prostaglandin release during the normal oestrous cycle and early pregnancy in the goat

Peripheral plasma levels of 15-ketodihydro-PGF_2α, 11-ketotetranor PGF metabolites and progesterone were measured during normal oestrous cycle and early pregnancy in six goats. The does were synchronized before the start of the study by means of 10 mg of PGF_2α. Blood samples were collected twice daily until day 12 of the oestrous cycle and subsequently every 3 h until the onset of oestrus, at which time the does were mated. The blood sampling protocol was repeated until day 28 of pregnancy. High pulsatile peaks of 15-ketodihydro-PGF_2α and 11-ketotetranor PGF metabolites were observed during the last days of the oestrous cycle, indicating PGF_2α releases. This coincided with a fall in progesterone levels. During early pregnancy no such peaks of prostaglandin metabolites were recorded and high levels of progesterone were maintained. In the goat, analysis of the 11-ketotetranor PGF metabolites seems to be a better indicator of PGF_2α release than the analysis of 15-ketodihydro-PGF_2α. The former metabolites are more long-lived in the circulation and are thus easier to detect.     

Analyses of the variation in the interval from an injection of prostaglandin F2α to oestrus as a method of studying patterns of follicle development during dioestrus in dairy cows

Lactating dairy cows experiencing normal oestrous cycles were injected once with either 0.5 mg of an analogue of prostaglandin F_2α (PGF) (Cloprostenol, 435 cows) or 25 mg of a PGF-Tham salt (237 cows) when at days 7–16 of the cycle (oestrus = day 0). In these two trials, 91% and 93% of the cows were detected in oestrus from 3–10 days post-injection increasing from 81% to 98% with advancing dioestrus. Over 70% of detected cows injected on day 7 (early dioestrus) or day 16 (late dioestrus) were in oestrus from 48 to 72 h post-injection. Comparable response rates among cows injected on days 11 and 12 (mid-dioestrus) were less than 30% with most response intervals being at 4 and 5 days post-injection (73 h–120 h). The variability in response intervals generally decreased with advancing dioestrus. A regression model for ordinal data, with post-injection interval to oestrus as the ordinal response and stage of cycle at injection as the explanatory variable, showed that both the interval to oestrus and the variation in this interval varied with the stage of cycle at injection.These response intervals appear to reflect a wave-like pattern in ovarian follicle development during dioestrus. The probability of the presence of a follicle in a less advanced stage of development at the time of PGF injection is greatest among animals treated during mid-dioestrus.     

Oestrogenic effects of ICI 182,780, a putative anti-oestrogen,on the secretion of oxytocin and prostaglandin F2α during oestrous cycle in the intact ewe

The effect of ICI 182,780, oestrogen antagonist, on the concentrations of oxytocin and uterine PGF_2α was investigated in intact Border Leicester Merino cross ewes during the late oestrous cycle. Twelve cyclic ewes (n=6 per group) were randomly assigned to receive, at 6 h intervals, intra-muscular injection of either peanut oil or ICI 182,780 (1.5 mg kg⁻¹ day⁻¹) in oil for 2 days, starting at 1900 h on day 13 until 1300 h on day 15 post-oestrus. Hourly blood samples were collected via a jugular catheter from 0800 h on day 14 for 37 h and then daily over days 16, 17 and 18 post-oestrus. Peripheral plasma concentrations of oxytocin, the metabolite of prostaglandin F_2α, 15-keto-13,14-dihydro-prostaglandin F_2α, (PGFM) and progesterone were measured by radioimmunoassay. All ewes treated with ICI 182,780 exhibited functional luteal regression as indicated by a marked reduction in plasma progesterone concentrations to less than 1000 pg/ml over the period of 18–36 h during sampling period on days 14 and 15 of the oestrous cycle. In five of six vehicle-treated ewes, progesterone concentrations declined between day 16 and day 18 post-oestrus. In the remaining control ewe, progesterone concentrations reach less than 1000 pg/ml within 36 h of the commencement of the sampling period. During the frequent sampling period, the number of oxytocin pulses in the ICI 182,780 treated ewes was significantly higher compared to control ewes (2.7±0.3 vs. 0.8±0.3). The mean amplitude of oxytocin pulses observed was also greater (70.4±19.5 pg/ml) in ewes treated with ICI 182,780, but was not significantly different from control ewes (33.5±12.9 pg/ml). Oxytocin pulses may however have occurred following the initial two ICI 182,780 injections but before commencing blood sampling. The oxytocin pulses were detected at a mean of 3.2±0.2 h following each injection with ICI 182,780 during blood sampling. In the ICI 182,780-treated ewes, the pulsatile pattern of plasma PGFM in jugular blood samples over the 37 h sampling period on days 14 and 15 post-oestrus had a higher amplitude (512.9±158.9 vs. 121.7±78.7 pg/ml) and pulse area (618.1±183.3 vs. 151.5±102.9 (pg/ml)τ) compared to the vehicle-treated ewes (P<0.05) respectively. The average number of PGFM pulses observed per ewe was 3.0±0.7 in the ICI 182,780-treated group and was significantly (P<0.02) higher than the number of pulses (0.5±0.3) observed in ewes treated with vehicle alone. The PGFM pulses were detected at 4.2±0.6 h following each injection with ICI 182,780 during blood sampling. The percentage of PGFM pulses that occurred coincidently with a significant elevation of oxytocin concentrations was 44.4% in ICI 182,780-treated compared to 66.6% in control ewes. We conclude that administration of oestrogen antagonist ICI 182,780 accelerated development of the luteolytic mechanism by enhancing pulsatile secretion of oxytocin and PGFM which suggests that ICI 182,780 acts as an agonist for oxytocin and prostaglandin F_2α release in intact ewes when administered at 1.5 mg/kg/day over Day 13 to 15 post-oestrus.     

Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes

Continuous intravenous infusion of oxytocin (3 micrograms/h) between Days 13 and 21 after oestrus delayed return to oestrus by 7 days (length of cycle 23.3 +/- 0.6 days compared to 16.6 +/- 0.2 days in control ewes). At a lower infusion rate (0.3 micrograms/h) oxytocin delayed luteolysis in only 2 of 5 ewes. Treatment from Day 14, when luteolysis had already begun, was ineffective. Delay of luteal regression by oxytocin had no effect on the length of subsequent cycles. Measurement of circulating progesterone concentrations and luteal weight showed that prolongation of the oestrous cycle was due to prevention of luteal regression. Luteal regression and behavioural oestrus were induced during continuous oxytocin administration begun on Day 13 when cloprostenol was given on Day 15 (mean cycle length, 17.3 +/- 0.21 days). Continuous oxytocin infusion from Day 13 blocked the rise in uterine oxytocin receptor concentrations which normally precedes oestrus. Mean receptor concentrations in caruncular and intercaruncular endometrium and in myometrium were 76, 36 and 9 fmol/mg protein on Day 17 in ewes receiving continuous oxytocin (3 micrograms/h); in control ewes these values were 675, 638 and 130 fmol/mg protein respectively at oestrus. Receptor concentrations on the day of oestrus in ewes receiving oxytocin and cloprostenol were not significantly different from those in control ewes (649, 852, and 109 fmol/mg protein respectively). Since cloprostenol, a PGF-2 alpha analogue, overcame the antiluteolytic action of oxytocin, it is suggested that continuous oxytocin treatment may inhibit uterine production of PGF-2 alpha, possibly by down regulating the uterine oxytocin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle

Based on the premise that superovulation in cattle is optimal when superstimulation is initiated at the time of follicular wave emergence, the present study was done in beef heifers to determine if the superovulatory response following a single bolus of gonadotrophin treatment after follicle ablation (induced wave) at random stages of the oestrous cycle is comparable to the same gonadotrophin treatment at mid-dioestrus (spontaneous wave). In Experiment 1, heifers were assigned to nonablation (n = 18) and ablation (n = 20) groups. In nonablated heifers, superstimulatory treatment was given as a single subcutaneous injection (Folltropin-V, 400 mg) at mid-dioestrus to coincide with emergence of the spontaneous follicular wave 8 to 12 days after oestrus. In ablated heifers, the same superstimulatory treatment was given 1 day after ablation of all follicles ≥ 5 mm at random stages of the oestrous cycle to coincide with emergence of the ablation-induced wave. In both the nonablation and ablation groups, PGF_2α (Estrumate, 500 μg) was given 48 h after the superstimulatory treatment and artificial insemination was done 60 and 72 h later. Reproductive tracts were collected at the time of slaughter 6 or 7 days after insemination. Observations made in Experiment 1, indicated that some ablated heifers had only partial luteal regression at the time of insemination, while some others exhibited behavioral oestrus as early as 24 h after PGF_2α treatment. The design was amended in Experiment 2 to address these problems. Heifers were assigned to nonablation (n = 17), ablation-alone (n = 20) or ablation plus progestogen (n = 20) groups. Follicle ablation, superstimulatory treatment, artificial insemination and collection of reproductive tracts were done as in Experiment 1. However, all heifers were given two doses of PGF_2α (500 μg/dose) 48 and 60 h after superstimulatory treatment to ensure complete luteal regression, and heifers in the ablation plus progestogen group received a norgestomet ear implant at the time of follicle ablation to prevent early ovulations. The implant was removed at the time of the second PGF_2α treatment. In Experiments 1 and 2, the means for the ovarian and superovulatory responses were not significantly different between groups. Averaged over the nonablation and all ablation groups for Experiments 1 and 2, the mean number of corpora lutea, fertilized ova and transferable embryos were 22.9 vs 18.6, 7.3 vs 7.8 and 5.4 vs 5.6, respectively. In summary, follicle ablation at random stages of the oestrous cycle followed by a single bolus of gonadotrophin treatment 1 day later resulted in a superovulatory response that was comparable to the same superstimulatory treatment administered around the time of spontaneous wave emergence at mid-dioestrus. The ablation/superstimulation method described herein offers the advantage of initiating superstimulatory treatment forthwith and assuring that treatment is concomitant with wave emergence to achieve an optimal superovulatory response. Moreover, the full extent of the oestrous cycle is available for superstimulation and the need for detecting oestrus or ovulation and waiting 8 to 12 days to initiate treatment is eliminated.     

The effect of the stage of the oestrous cycle and exogenous sex steroids on the transport of starch grains through the genital tract of the ewe, a preliminary study

The starch grain test was studied in two ewes to determine the effect of the stage of the oestrous cycle on their transport through the Fallopian tubes. There was very little difference when the test was performed at oestrus or day 10 of the oestrous cycle. Bilateral ovariectomy had very little effect on the transport of starch grains. When progesterone in oil was administered by intramuscular injection to a bilaterally ovariectomised ewe and an intact ewe there was a marked increase in the number of grains which were recovered. Oestradiol benzoate in oil delayed the recovery of grains and reduced their numbers.     

Multiple ovulation resulting from low level administration of PMSG to cattle at different stages of the oestrous cycle

Two experiments were carried out to determine whether differences in sensitivity to exogenous gonadotrophin which exist during the oestrous cycle can be used effectively in the induction of multiple pregnancy in cattle. In Experiment I, Hereford heifers and cows were injected with 800 IU pregnant mare serum gonadotrophin (PMSG) on approximately day 10 of the oestrous cycle, followed 48 h later by 30 mg prostaglandin F_2α (PGF_2α). Controls were treated with PGF_2α alone. Mean ovulation rates based on rectal palpation were 1.33 ± 0.10 (range: 1–2) and 3.05 ± 0.68 (range: 1–13) for 21 control and 21 treated animals, respectively (P < 0.02). In Experiment II, Hereford cows were treated with 800 IU PMSG on either day 5 (14 cows) or day 10 (12 cows) of the oestrous cycle, followed 48 h later by PGF_2α. Mean numbers of ovulations for animals that became pregnant were 1.50 ± 0.26 (range 1–3) and 3.80 ± 0.74 (range 1–7), respectively (P < 0.02). A high incidence of embryonic wastage occurred by 120 days of gestation in animals treated on day 10. The results of this study indicate that taking advantage of an animal's reduced responsiveness to exogenous gonadotrophin during the early portion of the oestrous cycle may be useful in developing methods for inducing multiple births with exogenous gonadotrophins.     

Induction of male behaviour in ovariectomized ewes and ovariectomized-androgenized ewes chronically implanted with oestradiol-17β or testosterone

Normal mature ewes and ewes that had been androgenized with testosterone (T) between days 30–80 or 50–100 of fetal life were ovariectomized and given 100 mg implants of either oestradiol-17β (E) or T. The T implants caused a sustained elevation in plasma T levels but the E implants did not produce stable plasma levels of E. The implants were weighed on removal from the ewes and daily release rates for E and T were 14.4 ± 5.8 μg/kg/day and 24.2 ± 5.3 μg/kg/day respectively.The implants of E induced oestrous behaviour in both the non-androgenized and the androgenized ewes, some of these animals remaining in oestrus for up to 11 days. The ewes also began to mount each other after 1–9 days of treatments; the androgenized ewes also showed male-like aggressive behaviour whereas the non-androgenized ewes did not.The T implants induced oestrous behaviour in both androgenized and non-androgenized ewes. However, the non-androgenized ewes never mounted other ewes, nor did they show aggressive behaviour, whereas the androgenized ewes did.Prenatal androgenization clearly alters the ability of a ewe to respond to exogenous steroids by increasing its propensity to show masculine behaviour. Nevertheless, non-androgenized ewes may also show masculine behaviour during chronic steroid treatment.     

Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).     

Reproductive performance of Coopworth ewes following oral doses of zearalenone before and after mating

The reproductive performance of Coopworth ewes after administration of zearalenone was determined in two trials. In Trial I, zearalenone was administered to groups of 33 ewes at rates of 0, 1.5, 3.0, 6.0, 12.0 and 24.0 mg/ewe/day for 10 days, starting on Day 7 of the oestrous cycle before mating. There was a linear decline (P less than 0.001) in ovulation rate with dose of zearalenone; also cycle length decreased and duration of oestrous increased with increasing dose levels. Reductions in the incidence of ovulation and in fertilization were seen only at doses of 12 and 24 mg. In Trial 2, groups of 50 ewes were given the same range of doses of zearalenone for 10 days, starting 5 days after mating to entire rams. There was no effect of zearalenone treatment after mating on pregnancy rate or embryonic loss. These results indicate that the effects of zearalenone, administered orally, on ewe reproduction, at the dose levels examined, were restricted to ewes exposed before mating. Intakes of zearalenone of 3 mg/ewe/day or more during this period would be reflected as depressed ovulation rates and lower lambing percentages.     

Pulsatile release of oxytocin into the circulation of the ewe during oestrus, mating and the early luteal phase

Two experiments were designed to investigate release patterns of oxytocin into plasma during oestrus and the early luteal phase. In Exp. 1, blood samples were collected from 5 ewes every 30 min for 10 h during 6 days around oestrus and the early luteal phase. During oestrus concentrations of oxytocin were generally low (1.27 +/- 0.54 pg/ml; mean +/- s.d.) but with occasional pulses up to 6 pg/ml. By Day 5 mean basal concentrations had risen to 4.5 +/- 2.1 pg/ml with a fluctuating release pattern. In Exp. 2, a method was developed for continuous blood sampling from conscious, unrestrained ewes. On the predicted day of oestrus following an untreated oestrous cycle, 8-ml blood samples were collected every minute for two 35-min periods (8 ewes: 16 sampling periods). For 6 ewes a ram was introduced to the pen for part of this time, and resulting behaviour was recorded. Additional blood samples were assayed for LH and progesterone to determine the stage of the cycle. Overall mean oxytocin concentrations ranged from 1.5 +/- 0.53 to 6.8 +/- 5.25 pg/ml in different animals. Ewes which were both in oestrus and exposed to the ram showed a pulsatile oxytocin release pattern consisting of low baseline concentrations with short-duration pulses superimposed (duration 1-4 min; amplitude 2.5-31.7 pg/ml; frequency 3.18/h). Coitus was not temporally associated with pulsatile release. However, the importance of the presence of the ram was indicated by total separation of 2 oestrous ewes from the ram until after experimentation. In these animals only 1 pulse of oxytocin was detected in 2.7 h of sampling. It is concluded that, although mean oxytocin concentrations at oestrus were low, short duration pulses were released into the plasma at this time. This effect may be dependent on the presence of a ram.     

Comparison of oestrous synchronization regimens for lactating dairy cows.

The objective of this experiment was to evaluate various programmes for synchronization of oestrus. The focus of the study was to evaluate rates of detection of oestrus, synchrony of oestrus, pregnancy rate, and effect of ovarian status at initiation of the programmes on rates of detection of oestrus and pregnancy rate. Spring-calving, lactating dairy cows (n = 2009) were allocated at random to one of six treatments: (1) A (n = 335), progestogen (controlled intravaginal drug release; CIDR) inserted per vaginum 10 d before breeding season for 8 d, 10 microg of buserelin at CIDR insertion, PGF2alpha treatment on the day prior to CIDR removal, and AI of cows detected in oestrus within 6 d after CIDR withdrawal; (2) B (n = 330), as in A, plus 1 mg of oestradiol benzoate i.m. 10 h post CIDR withdrawal; (3) C (n = 347), as in A, except buserelin was replaced by 10 mg of oestradiol benzoate; (4) D (n = 335), as in A, plus PGF2alpha and oestradiol benzoate at CIDR insertion; (5) E (n = 332), CIDR containing a 10 mg oestradiol benzoate capsule inserted per vaginum for 12 d; or (6) F (n = 330), as in E, plus PGF2alpha on the day prior to CIDR withdrawal. The oestrous detection rate (number of cows detected in oestrus within 6 days of CIDR withdrawal as a proportion of the number of cows submitted for synchronization of oestrus) and oestrous synchrony (oestrous detection rate within 2 d of CIDR withdrawal), respectively, were greater (P<0.05) following B (95.7% of 330, 98.7% of 316) compared with any of the other programmes for synchronization of oestrus (A: 87.5 of 335, 79.4% of 293; C: 86.7% of 347, 80.0% of 301; D: 90.1% of 335, 89.8% of 302; E: 74.4% of 332, 70.4% of 247; F: 76.4% of 330, 78.5% of 252). The oestrous detection rate was reduced (P<0.05) among cows in metoestrus administered E (64.0% of 50) relative to similar cows administered F (82.8% of 64). Pregnancy rate was greater (P<0.05) following B (57.9% of 330) than A (48.9% of 335, P = 0.06), C (43.2% of 347), E (40.0% of 332), and F (35.1% of 330) but not D (59.3% of 302), when based on those cows presented for oestrous synchronization programmes. In conclusion, 1 mg of oestradiol benzoate administered 10 h post CIDR withdrawal (B) resulted in the best overall oestrous detection, oestrous synchrony, and pregnancy rates, which would be beneficial to a fixed-time AI program.     

Luteinizing hormone (LH) release after single injections of a synthetic LH-releasing hormone (LH-RH) in the ewe at three different reproductive stages and comparison with natural LH release at oestrus

The possibility was investigated of using single i.v. injections of a synthetic luteinizing hormone-releasing hormone (LH-RH) to manipulate the reproductive pattern of the ewe.Single i.v. injections of 150 μg synthetic LH-RH were given on Day 12 of the oestrous cycle, during seasonal anoestrus and on Day 16 $\text{post}$-$\text{partum}$ in ewes which lambed during the breeding season. Blood samples were obtained at 5-, 10- or 15-minute intervals for 1 hour before and for 3 hours after treatment. Plasma LH concentrations were measured using a specific double antibody radioimmunoassay, the development of which is described. Laparotomy was performed on each animal 2–3 days after treatment.The treatment induced LH peaks in all animals and ovulation in the majority. There was no significant difference between the groups in the LH response. The LH release was, however, much less than that found in untreated ewes sampled every 15 minutes for 18 hours during oestrus.     

Coincident increases in oxytocin receptor expression and EMG responsiveness to oxytocin in the ovine cervix at oestrus

This study has localised oxytocin receptor (OTR) mRNA expression within the cervix of non-pregnant ewes and related this to changes in the sensitivity of the cervical musculature to administered oxytocin (OT) during the oestrous cycle by recording electromyographic (EMG) activity. Cervices were collected from 34 ewes at specified time points throughout the cycle. OTR mRNA expression was localised by in situ hybridisation and results were expressed as optical density measurements from autoradiographs in each of four different cervical regions. EMG recordings were made for up to 8 h per day from four non-pregnant ewes undergoing seasonal oestrous cycles between Days −3 and +3 relative to oestrus (Day 0). The highest concentrations of OTR mRNA were detectable within the luminal epithelium (LE) of the cervix, with values increasing from Day 15 of the cycle, peaking during the follicular phase (P<0.001, compared to the mid-luteal phase) and returning to basal by Day 2. There was a small but significant increase in OTR mRNA hybridisation (above basal/luteal phase values) within the stromal cells (STR) adjacent to the lumen (P<0.05) during the same time period, but no differences from basal values were detectable in the dense collagenous annular ring or in tissue superficial to this. Analysis of pooled EMG activity recorded daily from the cervix indicated that endogenous contractile activity was higher on Day 0 than on the Days +1 (P<0.05), −2, +2 and +3 (P<0.001). The response to bolus intravenous (i.v.) injections of 25 mU OT (25 mU) varied with day of the cycle. This dose produced a measurable and significant response on Days 0 (P<0.001) and +1 (P<0.001), but not on any of the other days, indicating that the sensitivity of the cervical musculature to OT peaked on these days. These data show that the cervix is highly responsive to OT at oestrus. This coincides with an increase in OTR mRNA expression in the luminal epithelial cells, suggesting the likely production of an intermediary messenger between the epithelial and smooth muscle cells.     

Effects of chronic treatment with a GnRH agonist on oestrous behaviour and on the secretion of LH and progesterone in the ewe

A study was carried out to investigate a novel approach to oestrus synchronization in the ewe by treatment with a gonadotrophin releasing hormone (GnRH) agonist. Groups of ewes were initially treated on Day 2, 10 or 14 of the oestrous cycle with 10 mug GnRH analogue (D-Ser(Bu(t)) 6 des Gly GnRH ethylamide) per ewe per day for 14 days. Behavioural oestrus was inhibited during GnRH agonist treatment and recurred from 8 to 38 days after the treatment in an unsynchronized manner. Luteal activity during treatment was not impaired but reduced progesterone concentrations occurred in cycles after the treatment. The rhythm of ovarian function, generally characterized by prolonged follicular development, was impaired. During the treatment and subsequent recovery period, integrity of pituitary function was examined by measuring luteinizing hormone (LH) after GnRH agonist was injected, and after stimulation test doses of 150 ng natural GnRH were administered. During treatment there was, with time, a decline in pituitary response to the agonist which suggested that pituitary release of LH was exhausted. After the 14-day treatment the stimulation test with GnRH revealed a gradual return to normal responsiveness although this was not complete three weeks after the treatment when compared to control ewes. This lowered pituitary activity could cause the impaired ovarian function.     

Effects of synchronization and frequency in insemination on fertility.

Fifty-four normally cycling, non-lactating mares were given 2 injections (i.m.) of PGF-2 alpha (10 mg) 14 days apart without regard to stage of the oestrous cycle. At 19 days after the first PGF-2 alpha treatment, a single i.m. injection of either hCG (3300 i.u.) or a GnRH-analogue (500 micrograms) was administered. Each mare was inseminated with 100 X 10(6) motile spermatozoa at one of the following frequencies: once only on Day 20; every other day during oestrus or at least on Days 19 and 21; or daily during oestrus or at least on Days 19, 20, 21 and 22. Eighteen control mares received saline injections on Days 0 and 14, and were inseminated either on the 4th day of oestrus or every other day or daily beginning on the 2nd day of oestrus. More (P greater than 0.05) PGF-2 alpha treated mares displayed their 1st day of oestrus on Days 14 to 20 than control mares (80.6 versus 27.8%). During cycle 1, fewer (P greater than 0.05) treated mares became pregnant compared to controls; 38.9, 25.0 and 66.7% for PGF-2 alpha + hCG, PGF-2 alpha + GnRH-A and control mares, respectively. After three cycles, the pregnancy rates for mares inseminated every other day or daily were higher (P less than 0.05) than mares inseminated only once during oestrus (88.9 and 88.2 versus 64.7%).     

Oestrogen and progesterone receptor immunoreactivity and c-fos expression in the ovine cervix.

Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0-3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0-3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0-3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.