Factors affecting conception and expression of oestrus in anoestrous cows treated with progesterone and oestradiol benzoate

Nutrient balance affects the resumption of ovarian cyclic activity following calving in dairy cattle. However, few data are available about the relationships between nutrient balance and expression of oestrus or conception. It was hypothesised that previously anoestrous cows that conceived to first insemination and cows that expressed oestrus at the subsequent expected return to oestrus would be less likely to be in negative energy balance (i.e. would have higher body condition score, higher glucose, insulin-like growth factor (IGF), leptin or insulin concentrations, and lower non-esterified fatty acids (NEFA), blood urea nitrogen (BUN), beta hydroxy butyrate (BOH) concentrations 12-15 days after insemination) than herd mates not conceiving or expressing oestrus. Anoestrous cows were treated with progesterone and oestradiol benzoate (Day 0 = end of treatment period) and retrospectively categorised as conceiving (n = 108) or not conceiving (n = 108) to insemination. A subset of cows not conceiving to insemination were categorised as expressing (n = 44) or not expressing (n = 44) oestrus between Days 14 and 28 after initial insemination. Cows conceiving had a lower NEFA concentration (P = 0.014) than non-conceiving cows. Cows subsequently detected in oestrus had higher body condition scores (P = 0.016), IGF concentrations (P = 0.008) and milk protein percentages (P = 0.038), and lower BOH concentrations (P = 0.018) than cows not expressing oestrus. No difference in concentrations of leptin, insulin, glucose, blood urea nitrogen or milk yield were found between cows conceiving or not conceiving and those detected in oestrus or not detected in oestrus (P > 0.1). It is concluded that some measures of metabolic status at the time of pregnancy recognition affects the probability of pregnancy and of subsequent expression of oestrus in those treated, anoestrous cows not conceiving.     

Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes.

The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 x 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day-1) for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.     

The effect of progesterone, oestradiol and HCG on cell-mediated immunity in pregnant mice.

During pregnancy in mice, cell-mediated immunity as measured by a contact allergic reaction to picryl chloride was diminished (P less than 0.001). Mice in which delay of implantation was maintained by progesterone, and mice with progesterone- and oestradiol-maintained pregnancies, also showed a reduction in the inflammatory response. The response of pseudopregnant mice did not differ from that of the non-pregnant controls. Young mice sensitized before complete immunological competence gave a 50% response. The response doubled in animals given a second sensitization. The extent of the response in females with delay of implantation varied inversely with the dose of progesterone. A range of oestrogen doses gave the same depression in the response when given to pseudopregnant animals. Administration of HCG to pseudopregnant mice also reduced the inflammatory response.     

Plasma progesterone concentrations, ovarian and endocrinological response and sperm transport in ewes with synchronized oestrus

Two experiments involving 24 and 54 Australian Merino ewes were conducted in which the establishment of a cervical population of spermatozoa and several endocrinological events were studied after several regimens for the synchronization of oestrus. Intravaginal sponges impregnated with 500 mg (Exp. 1) or 200, 400 or 600 mg (Exp. 2) progesterone resulted in the maintenance of plasma progesterone concentrations of 1.5-4.9 ng/ml over a 12-day insertion period compared with 1.9-6.9 ng/ml during dioestrus in control ewes. In Exp. 1 basal concentrations of less than or equal to 0.25 ng/ml plasma were attained by 4 h after sponge withdrawal and this decline was much more rapid than in normal luteolysis. This was associated with fewer spermatozoa recovered from the cervix 2 h after insemination, and PMSG had no significant effect. In Exp. 2 injection of a supplementary dose of progesterone at sponge withdrawal resulted in a rapid increase in plasma progesterone concentrations followed by an equally rapid decrease and an attenuation of the rise in plasma oestradiol-17 beta, the LH surge, and the onset of oestrus. The numbers of spermatozoa recovered 4 h after insemination were not increased, and PMSG had no significant effect. Two factors were significant, namely the dose of progesterone in the sponge (600 mg greater than 400 or 200 mg, P less than 0.05) and stage of oestrus when inseminated (mid- or late oestrus greater than early). The data demonstrated that an adequate dose of progesterone/progestagen incorporated into intravaginal sponges and accurate timing of insemination relative to the LH surge are the most important factors involved in penetration of the cervix by spermatozoa.     

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.     

Egg proteins in cod serum. Natural occurrence and induction by injections of oestradiol 3-benzoate

1. Before the uptake of water that precedes spawning, eggs of cod (Gadus morhua L.) contained 30% dry matter, of which 80% was protein. Some 75% of this protein was soluble in 0.5m-sodium chloride. The major components in the extract were two similar lipoproteins, of molecular weight about 400000, containing 21% lipid, some two-thirds of which was phospholipid, and about 0.5% protein phosphorus. 2. These lipoproteins were identified by immunochemical methods in the serum of female cod with developing ovaries, but not in the serum of male or of immature female fish. 3. The concentrations of egg proteins in the serum of female cod were determined by a serial-dilution double-diffusion immunological method, and were shown to increase with development of the ovaries, reaching a value of about 32mg/ml when the weight of the ovaries was 10% of the weight of the fish. 4. Immature male and female cod were injected intramuscularly with a solution of oestradiol-17beta 3-benzoate in oil and the concentration of egg proteins in their serum was measured by the immunodiffusion method. The serum contained no detectable egg proteins before injection of the fish, but 30mug of oestradiol benzoate/kg gave rise to detectable amounts of egg proteins in 10 days, and with 300mug or 1mg of oestradiol benzoate/kg the concentration of egg proteins rose to 32mg/ml. The values for male and female cod were similar and represented about one-half of the total serum protein. 5. With a dose of 1mg of oestradiol benzoate/kg, egg proteins were first detected in the serum 2 days after injection and the concentration increased up to 10 days. 6. Serum samples taken before and 10 days after an injection of 1mg of oestradiol benzoate/kg were fractionated by gel-filtration on Sephadex G-200. The difference curves obtained from fractionation curves after and before injection confirmed the values of the concentrations of egg proteins obtained from the immunodiffusion test and showed that the concentrations of the normal serum components fell by 20-50% of the initial value, the high-molecular-weight globulins showing the most marked fall. 7. Egg proteins were detected in the liver and testes of the injected fish, but not in the ovaries.     

Oestrogen and progesterone concentrations in plasma and oviductal tissue of ewes exhibiting a natural or induced oestrus

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.     

The induction of oestrus in ewes during the non-breeding season using pre-used CIDRs and oestradiol-17β treatment

The fertility obtained in sheep after the use of intravaginal progesterone devices is related to the content of progesterone of the device. The hypothesis of this study was that the reproductive response of anoestrous ewes to the ram-effect could be improved by the administration of oestradiol-17β in conjunction with CIDRs treatment—using previously used CIDRs in a 5-day progestagen priming. Therefore, the objective was to determine if oestradiol-17β treatment increases fertility of anoestrous ewes primed with used CIDRs and stimulated by the ram-effect. The hypothesis was tested with CIDRs that had been previously used for 12 or 18 days. The trial was performed during the non-breeding season using 158 Corriedale ewes. Ewes had been isolated from rams since Day −35 (Day 0 = introduction of the rams). A CIDR (0.3 g progesterone, InterAg, Hamilton, New Zealand) was inserted on Day −5 in all ewes with CIDR that had been previously used for 12 days (n = 62) or 18 days (n = 96). Also on Day −5, 29 and 53 ewes that had received CIDRs of 12 or 18 days, respectively, received an intra-muscular treatment of 50 μg of oestradiol-17β (E groups). The ewes that did not receive the oestradiol-17β treatment remained as the control group (C group). Overall the treatment groups were thus: C12 (n = 33), C18 (n = 43), E12 (n = 29), and E18 (n = 53). On Day 0 all CIDRs were withdrawn, and ewes were placed with 18 rams and 20 ewes hormonally induced to exhibit oestrus. Sexual receptivity of ewes treated with CIDRs was estimated from marks on the rumps of the ewes daily from Day 0 to Day 5, and the pregnancy status diagnosed with transrectal ultrasonography on Day 40. The percentage of ewes exhibiting oestrus and pregnancy rates were lower in ewes synchronized with previously used CIDRs for 18 days, compared to those used for 12 days. The responses of ewes in oestrus were 39.4, 14.0, 65.5, and 32.1% for the C12, C18, E12, and E18 groups respectively, with pregnancy rates of 30.3, 14.0, 34.5, and 17.0%. Administration of oestradiol-17β increased the frequency of oestrous response in ewes that were treated with CIDRs previously used for 12 days (P < 0.05), but not in those treated with CIDRs used for 18 days. It could be concluded that the administration of oestradiol-17β only improved the percentage of ewes responding to oestrus when CIDRs previously used for 12 days were used for 5 days before the introduction of rams. No positive effect on fertility was observed irrespective of the period during which CIDR had been previously used.     

Plasma gonadotrophins and oestradiol during oestrus in the cow

LH values between -2 and +4 h (0 h = LH peak) were higher than baseline. FSH values were also raised at this time and between +16 and +30 h. Oestradiol values between -20 and 0 h were higher than during +4 to +20h.     

Early identification of non-pregnant and pregnant ewes in the field using circulating progesterone concentration

A method for early indentification of non-pregnant and pregnant ewes is described. It is applicable to field research situations where mating data for individual ewes cannot be collected and requires three plasma progesterone measurements from each ewe over a 12-days period.Ewes were diagnosed non-pregnant according to whether their lowest progesterone concentration (p) was below or above a “discriminatory value”. This value was chosen after examining the overall frequency distribution of values of log₁₀p.In experiment 1, $\text{25}\text{27}$ non-pregnant ewes and $\text{64}\text{65}$ pregnant ewes (20 to 31 days post-mating) were diagnosed correctly (96.7% accuracy). In experiment 2, $\text{40}\text{41}$ non-pregnant ews and $\text{45}\text{48}$ pregnant ewes (21 to 34 days post-mating) were diagnosed correctly (95.5% accuracy).     

Ram-induced ovulation in seasonally anovular merino ewes: Effect of oestradiol on the frequency of ovulation,oestrus and short cycles

Seasonally anovular Merino ewes can be induced to ovulate by introducing rams. This ovulation is rarely accompanied by oestrus, and the resulting corpus luteum may regress prematurely. Oestradiol (100 μg i.m.) delayed and depressed the ovulatory response (33/45 vs 33/34 for controls), but had no effect on the expression of oestrus (10/33 vs 7/33 in controls) or the frequency of short cycles (16/33 vs 15/33 for controls). The ovulation following premature regression of the CL was not accompanied by oestrus. It seems unlikely that the lack of oestrus and the formation of a CL with short life span are due to a deficiency of oestradiol.     

Serum hormone levels and occurrence of oestrus following use of an intravaginal device containing progesterone and oestradiol 17 β in heifers

A simple and inexpensive vaginal device made of silastic tubing containing progesterone and oestradiol 17 β was developed to release a sufficient amount of progesterone and oestrogen to control oestrus in cycling heifers. More than 90% of the heifers treated retained the device and oestrus was synchronised after removal. Based on a limited number of observations the conception rate following synchronised oestrus was satisfactory. The results also show that oestrus, ovulation and cyclic activity can be induced in prepuberal heifers using the above device.     

Effects of maturity of the potential ovulatory follicle on induction of oestrus and ovulation in cattle with oestradiol benzoate

The effect of maturity of the dominant follicle (DF) on the capacity of oestradiol benzoate (ODB) to induce oestrus and ovulation was examined in cattle. In experiment 1, 31 prepubertal heifers each received an intravaginal progesterone insert (IPI) and 1mg ODB i.m./500kg BW (ODB1). Daily ovarian ultrasonography detected emergence of a new follicular wave 3.1+/-0.1 days after ODB1. The IPI was removed when newly emerged DF were "young" (1.3+/-0.1 days after emergence; YDF; n=15) or "mature" (4.2+/-0.1 days; MDF; n=16), and 24h later, heifers received 0.75mg ODB/500kg BW (ODB2; n=16) or no further treatment (NoODB2; n=15). Most of the heifers receiving ODB2 were observed in oestrus (15/16) and ovulated (12/16), as compared to 0/15 and 1/15 in the NoODB2 group, respectively (P<0.01). In experiment 2, 32 heifers received ODB1 on day 6 of the oestrous cycle, and new follicular wave emergence was detected 3.2+/-0.1 days later. Heifers received an injection of prostaglandin-F2alpha (PGF) when the DF was young (1.1+/-0.1 days after emergence; YDF; n=16) or mature (4 days; MDF; n=16), and then ODB2 24h later or no further treatment (NoODB2). The interval from PGF to oestrus was greater (P<0.01) in the YDF-NoODB2 (70+/-3.9h) as compared to MDF-NoODB2 group (57+/-1.8h). Inclusion of ODB2 reduced (P<0.01) this interval to 47.0+/-0.7h without regard to the maturity of the DF (maturityxODB2, P<0.05) and also reduced (P<0.05) the interval to ovulation. In experiment 3, 21 suckling anoestrous cows received an IPI and ODB1 at 29.3+/-1.7 days postpartum. The IPI were removed either 1 day (YDF; n=9) or 3.9+/-0.1 days (MDF; n=9) after emergence of a new follicular wave and every cow received ODB2. Oestrus was subsequently detected in all but one animal. Ovulation of the newly emerged DF was detected within 48h of ODB2 in nine of nine cows of the MDF group, and in four of nine of the YDF group (P<0.05). During the subsequent ovulatory cycle, luteal size and plasma concentrations of progesterone were greater (P<0.01) in the MDF group compared to the YDF group. We conclude that behavioural oestrus is readily induced by 0.75mg ODB i.m./500kg BW. Maturity of the DF appeared to have little influence on the ability of the DF to ovulate in heifers. In contrast, young DF in lactating anoestrous cows were less likely to respond to the ovulatory cue provided, and luteal development was compromised in those that did ovulate.     

The effects of oestradiol benzoate, progesterone, relaxin and ovariectomy on cervical extensibility in the late pregnant rat

Cervical extensibility increased from Day 16 to term in the pregnant rat. Following ovariectomy on Day 16 of pregnancy the cervix became as inextensible by Day 20 as that of non-pregnant animals. Fetal growth was maintained in rats ovariectomized on Day 16 if given oestradiol benzoate plus progesterone but cervical extensibility only increased to a small extent. Relaxin given to these animals further increased cervical extensibility, suggesting a role for this hormone.     

Effects of continuous elevated cortisol concentrations during oestrus on concentrations and patterns of progesterone, oestradiol and LH in the sow

This study investigated the effect of continuous elevated cortisol concentrations during standing oestrus on time of ovulation and patterns of progesterone, oestradiol and luteinising hormone (LH) in sows. The elevation of cortisol concentrations was achieved through repeated intravenous injections of synthetic adrenocorticotropic hormone (ACTH) every 2 h for approximately 48 h, from the onset of the second standing oestrus after weaning. Treatment was terminated when ovulation was detected (monitored by transrectal ultrasonography every 4h) or when the sow had received a maximum of 24 injections. The dose of ACTH (2.5 microg/kg) was chosen to mimic the cortisol concentrations seen during mixing of unfamiliar sows. The sows (n=14) were surgically fitted with jugular vein catheters and randomly divided into a control (C group where only NaCl solution were injected) or an ACTH group. Blood samples were collected every 2 h. In parallel with the blood sampling, saliva samples for cortisol analyses were taken from eight sows before onset of treatment and from four of the sows during treatment. There was no difference in time from onset of standing oestrus to ovulation between the two groups. The interval between the peaks of oestradiol and LH to ovulation was prolonged in the ACTH group compared to the C group (p<0.05), with a tendency towards an earlier decline of oestradiol in the ACTH group. Cortisol and progesterone concentrations were significantly elevated during treatment in the ACTH group (p<0.001), with cortisol peak concentrations occurring between 40 and 80 min after each ACTH injection. Cortisol concentrations in saliva and plasma were highly correlated (p<0.001). In conclusion, elevated cortisol concentrations from the onset of standing oestrus increase progesterone concentrations and prolong the interval between oestradiol and LH peaks to ovulation, the latter possible due to an early decline in oestradiol concentrations and a change of the LH peak outline. The effect these hormonal changes have on reproductive performance need to be further investigated. Saliva samples might be a useful and non-invasive method to assess cortisol concentrations in sows.