Variable progesterone response and estradiol secretion in prepubertal beef heifers following treatment with norgestomet implants

Two experiments were conducted to determine the effects of norgestomet ear implants on progesterone response and estradiol secretion in prepubertal beef heifers. In the first experiment, 47 beef heifers were treated with norgestomet. The implants were implanted subcutaneously for 9 d. After implant removal, blood samples were taken from heifers 2 to 4 d per week for 40 d. Following progesterone determination in jugular venous plasma, heifers were classified according to their progesterone response: 1) no response (Group 1); no rise in progesterone above 1 ng/ml throughout the sampling period; 2) one cycle (Group 2); one increase in progesterone above 1 ng/ml for at least 2 d followed by no further increase in progesterone during the sampling period; and 3) two cycles (Group 3); a rise in progesterone above 1 ng/ml for at least 2 d followed by another cycle of normal duration. Heifers treated with norgestomet were classified as 23 with no response, 9 with 1 cycle and 15 with 2 cycles. Concentrations of estradiol were measured in jugular venous samples on Day 2 after implant removal. Mean concentrations of estradiol were greater in Group 3 than in Group 1 (P < or = 0.01). In Experiment 2, 29 prepubertal beef heifers were assigned randomly to either a 9-d treatment with norgestomet (n = 14) or to serve as untreated controls (n = 15). Blood plasma samples were collected daily from Days 0 to 44 after implant removal. After progesterone determination, heifers were classified as 8 with no response, 4 with 1 cycle and 3 with 2 cycles in the control group, and 5 with no response, 3 with 1 cycle and 6 with 2 cycles in the norgestomet group (frequencies did not differ; P > 0.1). Jugular venous blood plasma was also collected at 4-h intervals from 0 h to 96 h after implant removal and concentrations of estradiol were measured. Patterns of estradiol secretion differed (P < or = 0.05) and overall mean concentrations of estradiol over the first 96 h following implant removal were greater (P < or = 0.01) in norgestomet-treated heifers versus the controls. We conclude that norgestomet can produce a variable progesterone response with heifers with 2 cycles secreting more estradiol. Implants of norgestomet also causes more acute secretion of estradiol in prepubertal beef heifers.     

Neonatal exposure to estradiol decreases hypothalamic allopregnanolone concentrations and alters agonistic and sexual but not affective behavior in adult female rats

Exposure of developing female rats to estradiol during the perinatal period induced long-lasting dysregulation of gonadal axis and decreased cerebrocortical and plasma concentrations of allopregnanolone. We have now examined the effects of neonatal estradiol administration in female rats on hypothalamic allopregnanolone concentrations and on exploratory, affective, agonistic and sexual behaviors as well as social learning. A single administration of β-estradiol 3-benzoate (EB, 10 μg) on the day of birth resulted in a delay of vaginal opening, acyclicity and ovarian failure. These alterations were associated with a significant decrease in the concentrations of allopregnanolone in the hypothalamus at 21 and 60 days, but not at 7 days, after birth. Neonatal administration of EB also increased agonistic behaviors in adult rats, such as dominant behaviors and following of an ovariectomized intruder, while living attacks unaffected. EB-treated rats showed also an increase in anogenital investigation, associated with a drastic reduction in spontaneous and induced female sexual behaviors (receptivity and proceptivity). In contrast, neonatal administration of EB did not affect locomotor activity, anxiety- and mood-related behaviors, the social transmission of flavor preferences, and seizures sensitivity. These effects of estradiol suggest that it plays a major role in regulation of both the abundance of allopregnanolone and the expression of agonistic and sexual behaviors, while failing to influence affective behaviors and social learning. Thus, the pronounced and persistent decrease in hypothalamic allopregnanolone concentration may be related to the manifestation of agonistic and sexual behaviors.     

Neonatal exposure to oestrogens alters the protein profiles and gene expression in the genital tract of adult male mice.

After neonatal administration of supraphysiological doses of oestradiol, the concentration of tissue proteins, in adult mice, was significantly reduced by 39, 45 and 56% in epididymis, vas deferens and seminal vesicle respectively. The protein profiles showed persistent alterations. In epididymis, 4 protein bands were differentially increased (14.4, 43 and 67 kDa) or reduced (24 kDa) in oestrogenized males. In vas deferens, 4 proteins were increased (14.4, 49,67 and 76 kDa) and one (34 kDa) virtually absent. In seminal vesicle, about 20 proteins of varying molecular weights (12-140 kDa) were differentially increased or decreased. Testosterone substitution, at adulthood, was unable to reverse these effects. Treatments with oestradiol during adult life induced persistent alterations in the protein profiles of the 3 organs but, in contrast to neonatal treatment, these alterations could be reversed by androgen therapy. A cDNA library has been constructed with RNA prepared from adult seminal vesicle and screened by differential hybridization. Neonatal oestrogenization strongly reduced the abundance of some mRNA species. Eleven recombinants containing putative oestrogen-sensitive sequences were isolated. Two of them, having an insert of about 500 base pairs, were used for dot-blot hybridization. Results showed that the two clones contained sequences which were differently regulated by androgens.     

Neonatal exposure to estradiol valerate programs ovarian sympathetic innervation and follicular development in the adult rat

A single injection of estradiol valerate (EV) to 14-day-old rats (when the ovarian follicle population has been already established) disrupts cyclicity, increases the activity of key enzymes of androgen biosynthesis, and develops polycystic ovary by a causally related increase in ovarian noradrenaline (NA). The current study examined an early window of ovarian development to look for a specific stage of development at which estradiol can induce such changes in sympathetic activity and follicular development. A single dose of EV applied to rats before the first 12 h of life rapidly increases (after 24 h) the ovarian expression of nerve growth factor (Ngfb) and p75 low-affinity neurotrophic receptor (Ngfr) mRNAs. When adults, rats presented early vaginal opening, disrupted cyclicity, appearance of follicular cyst, absence of corpus luteum, and infertility. Total follicles decreased, mainly due to a reduced number of primordial follicles, suggesting that estradiol acts in the first stages of folliculogenesis, when primordial follicles are organizing. These changes paralleled a 6-fold increase in NA concentration. No changes in NA content were found in the celiac ganglia, suggesting a local, non-centrally mediated effect of estradiol. Surgical section of the superior ovarian nerve (the main source of sympathetic nerves to the ovary) to rats neonatally treated with EV decreased intraovarian NA, delayed vaginal opening, and blocked the development of follicular cyst and that of preovulatory follicles. Therefore, we can conclude that early exposure to estradiol permanently modifies ovarian sympathetic activity and causes profound changes in follicular development, leading to the polycystic ovary condition.     

Pregnancy diagnosis based on the fecal progesterone concentration in beef and dairy heifers and beef cows

The present study was undertaken to examine whether pregnancy diagnosis was possible by measuring fecal progesterone concentrations in beef and dairy heifers and beef cows. Rectal fecal samples collected on days 18–24 after insemination or days 11–17 after embryo transfer were mixed with methanol and shaken for preparation of a fecal solution. After centrifugation, the supernatant was extracted with petroleum ether followed by an enzyme immunoassay for progesterone. All pregnant animals showed fecal progesterone concentrations greater than 50 ng/g of fecal material on days 18–24 after AI or estrus. In non-pregnant animals, however, the fecal progesterone concentrations ranged widely from 5 to 180 ng/g of fecal material. In non-pregnant cattle, the percentage of cattle with <50 ng progesterone/g of fecal material compared with the total number was 37–60% on days 18–20, whereas the percentages increased more than 70% to a maximum of 78.1% on day 23. When 50 ng/g was considered as the cut-off value, the sensitivity and specificity of positive pregnancy tests were less than 70% on days 21–24, and 100% for negative pregnancy tests on days 18–24. There were significant differences in the mean fecal progesterone concentrations between pregnant and non-pregnant cattle on days 19–24. These results suggest that feces can be utilized to substitute for plasma and milk to measure progesterone for the purpose of pregnancy diagnosis in heifers and cows.     

Relationship of pregnancy rate to peripheral concentrations of progesterone and estradiol in beef cows

The relationship between pregnancy rate and concentrations of progesterone (P(4)) and estradiol-17beta (E(2)) in serum was examined in inseminated beef cows. Jugular blood was collected twice daily on Days 4 through 7 and Days 14 through 17 after estrus to establish patterns of secretion of P(4) and E(2). Pregnancy rate was determined by palpation per rectum at 45 d. Mean concentrations of each hormone, ratio of E(2):P(4) and regressions of hormone on day were the variables measured for each of the 2 periods. Cows were classified into low (n=26), medium (n=50) and high (n=26) groups for each variable. The relationship of pregnancy rate to each variable was tested using Chi-square analyses. Pregnancy rates to the first service decreased linearly as relative mean concentrations of E(2) increased on Days 14 through 17 (P<0.05) but were not affected by any of the other hormonal variables studied during either period. Pregnancy rates to the second service were not related to concentrations of P(4) or E(2) during the luteal phase before mating (Days 14 through 17). The effects of pregnancy on concentrations of E(2) and P(4) also were tested. On Days 14 through 17, P(4) increased slightly in pregnant cows and declined slightly in nonpregnant cows (P<0.05), but pregnancy did not affect E(2) during either period or P(4) on Days 4 through 7. In summary, pregnancy rate to the first service decreased significantly as concentrations of E(2) increased on Days 14 through 17.     

Effects of circulating progesterone and insulin on early embryo development in beef heifers

The aims of this study were to determine the effect on early embryo development of feeding a diet formulated to enhance circulating insulin concentrations and secondly to investigate the association between early embryo development and maternal progesterone concentrations in beef heifers. The study was carried out in 32 Simmental x Holstein Friesian heifers 22-25 months of age weighing 506+/-7kg and in condition score 3.1+/-0.1. Animals were fed two diets that were isoenergetic and isonitrogenous, but that would encourage either propionate (diet A) or acetate (diet B) production in the rumen. The rationale was that propionate would induce a greater insulin release in response to feeding. Animals were fed a 50:50 mix of the two diets for 14 days at 0.8x maintenance, with straw provided ad libitum. Animals were then fed one of the experimental diets for 3 weeks prior to synchronisation of oestrus and insemination and for a further 16 days following mating. All heifers were blood sampled daily from oestrus synchronisation and eight animals on each diet underwent daily transrectal real-time ultrasonography to determine the day of ovulation. All heifers were slaughtered at Day 16 after mating. While feeding of diet A (propionic) caused a significant (P<0.05) increase in the plasma insulin to glucagons ratio differences in insulin were not significantly different. This is probably due to the fact that insulin concentrations were quite high as the heifers used in the present study were in good body condition making further increases in insulin difficult to achieve. Diet did not affect size of ovulatory follicle (DIET A: 15.1+/-0.7mm; diet B: 14.6+/-0.7mm), day of ovulation (diet A: 3.5+/-0.2 days; diet B: 3.4+/-0.2 days), mean plasma progesterone concentration (diet A: 4.7+/-0.4ng/ml; diet B: 5.2+/-0.3ng/ml), corpus luteum weight (diet A: 6.0+/-0.2g; diet B: 6.0+/-0.2g) or pregnancy rate (diet A: 81.3%; diet B: 81.3%). However, the proportion of well-elongated (>10cm) embryos on Day 16 was higher in animals fed diet A than in those fed diet B (84.6% versus 38.5%; P<0.05). While progesterone concentration did not differ between pregnant and non-pregnant heifers, progesterone did show an earlier post-ovulatory rise in heifers with well-elongated (>10cm) embryos with levels in these animals significantly higher on Days 4 and 5 than in heifers with small (<10cm) embryos at slaughter. This study demonstrated an enhancement in early embryo development in animals fed a diet generating an increased insulin:glucagon ratio that was not related to circulating maternal progesterone concentrations. However, across diets, enhanced embryo development was associated with elevated plasma progesterone on Days 4 and 5 following mating.     

Effect of low levels of exogenous progesterone on puberty in beef heifers

Twenty eight prepuberal Charolais-Angus cross heifers approximately 15 months old and averaging 334 kg were implanted intravaginally with either a placebo (control, 9 heifers) or progesterone containing implant (treated, 19 heifers). Implants were left in place 3 days. Seventeen heifers (8 controls and 9 treated) retained implants. Plasma progesterone concentrations were < .4 ng/ml in control heifers and 1.5 +/- .59 ng/ml in treated heifers while implants were in place. Seven heifers, all in the treated group, were observed in estrus within 4 days after implant removal. All but one heifer observed in estrus had elevated plasma progesterone concentrations 12 days after implant removal. None of the heifers not observed in estrus had elevated progesterone levels at this time. This study indicates that low levels of exogenous progesterone (1 to 2 ng/ml plasma for 3 days) will induce estrus in prepuberal heifers following its withdrawal.     

Luteal function and estrus in peripubertal beef heifers treated with an intravaginal progesterone releasing device with or without a subsequent injection of estradiol

The objectives of this experiment were to determine if treatment of beef heifers with progesterone (P4) using an intravaginal device alone or in combination with estradiol benzoate (EB) would induce estrus and cause development of corpora lutea (CL) with a typical life span. Peripubertal heifers (n = 311) were used when about 40% of the heifers had a functional CL. The heifers were assigned to receive one of the following treatments on Day 0: 1) a sham device for 7 d (C, n = 108); 2) an intravaginal device containing P4 for 7 d (P, n = 102); or 3) an intravaginal device containing P4 for 7 d plus an injection of 1 mg EB 24 to 30 h after device removal (PE, n = 101). Serum concentrations of P4 were determined on Days -7, 0, 8, 15 and 22. Weight and age of the heifers at the start of the trial averaged 292 +/- 45 kg and 365 +/- 38 d, respectively. A greater (P < 0.0001) proportion of the heifers from the PE than P group was in standing estrus (81 vs 37%) and formed normal CL (68 vs 44%) after device removal. Of the heifers exhibiting estrus, a greater (P < 0.05) proportion of PE (94%) than P (80%) heifers was active 1 to 3 d after implant removal. Short-term progesterone treatment increased the proportion of heifers in estrus and those forming normal CL, and adding EB to the progesterone treatment further enhanced these responses.     

Effects of estradiol and progesterone on rat uterine ribonuclease inhibitor activity

The effects of estradiol (E2) and progesterone on rat uterine RNase inhibitor activity were investigated. RNase inhibitor activity was found to be most prominent in nuclear and cytosolic fractions of the uterus. Uterine RNase inhibitor activity in the 45,000 x g cytosolic fraction was stimulated by E2 over a 6-24 hr treatment period. Progesterone did not produce a significant change in the uterine cytosolic RNase inhibitor activity during the same period.     

Binding of progesterone to the proteins of the uterine luminal fluid. Identification of uteroglobin as the binding protein.

1. The uterine luminal fluid of rabbits treated with estradiol and progesterone contains a protein factor with high affinity for [3-H] progesterone which is not present in the uterine secretion of control rabbits treated with estradiol. 2. This progesterone dependent factor is shown by gel filtration and polyacrylamide gel electrophoresis to be identical with the uterus specific protein uteroglobin, which seems to be required during the preimplantation phase. Uteroglobin specific antiserum, prepared in guinea pigs, completely inhibits the progesterone binding activity of the proteins of the uterine fluid. 3. Progesterone binding to uteroglobin is dependent upon millimolar concentrations of dithioerythritol. At saturation, one molecule of progesterone binds per uteroglobin molecule and the apparent association constant is 2 x 10-6 M-1 at 0 degrees C. 4. The progesterone binding species of uteroglobin exhibits a molecular weight of around 12 000 on polyacrylamide gels containing dodecylsulfate, and of 15 000 upon gel filtration, indicating a non-globular shape. This molecule is compased of two subunits of similar molecular size which are held together by a disulfide bridge among other forces.     

A critical period in the onset of parturition in rats and uterine sensitivity to estradiol and progesterone

The purpose of this paper was to impair normal parturition in rats in order to measure tissue levels of progestins and estrogens and compare these results with those of normal parturition in rats. Abnormal parturition was obtained by injection of isotonic saline into the uterine lumen of pregnant rats at the end of pregnancy or by handling the uterus. After each of these treatments on day 21 of pregnancy, parturition was impaired in 70 to 98% of the rats. When the treatments were carried out earlier or later in pregnancy, there was little or no impairment. Our results indicate transient discrepancies in plasma and tissue levels of steroids 6 h after treatment on day 21: 20 alpha-HP concentrations increased in treated rats compared to controls (uteri: 470%; p less than 0.01; ovaries: 89%; p less than 0.001); concomitantly, there was a sharp rise in P concentrations in uteri (+ 74% : p less than 0.05) and ovaries (+ 52%; p less than 0.05). Inversely, uterine concentrations of E2 decreased 6 h after treatment compared to controls (- 30%; p less than 0.05), although there was a transient rise of E2 in the ovaries (+ 30%; p less than 0.05). Twenty-four hours later, E2 concentrations were always lower in the uteri (- 30%; p less than 0.01). No change in E2 levels was noted in the uteri or ovaries of either the control or treated rats. The physiological significance of these changes and their consequences on uterine reactivity at term have been discussed. The data demonstrate that day 21 was a critical period in the parturient activity of the rat uterus which appears to be primarily affected by uterine levels of E2 between days 21 and 22 of pregnancy.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Neonatal opiate withdrawal alters the reactivity of adult rats to the hot-plate

Prenatal exposure of rats to 0.2 mg LAAM/kg/day but not to 0.05 mg LAAM/kg/day resulted in faster hot-plate escape latencies in 6 mo old offspring. No differences in tail-flick latencies were observed at 7 mo of age in offspring exposed to either dose of LAAM prenatally. Subsequent testing of littermates at 16 mo of age revealed that the greater sensitivity to the hot-plate observed in rats prenatally exposed to LAAM is apparently a result of neonatal withdrawal rather than a primary consequence of the drug. The data are discussed in relation to possible effects of drug or withdrawal on central nervous system development.     

Neonatal exposure to UVR alters skin immune system development, and suppresses immunity in adulthood

Neonates have a developing immune response, with a predisposition towards induction of tolerance. As the immune system develops, immunity rather than tolerance is induced, with this development of immunity occurring in response to external factors such as the environment. As ultraviolet radiation (UVR) suppresses immunity, it is likely that the effect of UVR on the neonatal immune system would be augmentation of the suppressive response. In support, childhood exposure to UVR has been linked with an increased incidence of melanoma; consistent with an increase in suppression. To address this, phenotypic and functional immune system studies were undertaken at 8 weeks after one single exposure of solar-simulated UVR to mice, when mice had reached adulthood. Subtle changes were observed in cell populations resident in the skin-draining lymph nodes (LNs) and there also appeared to be a subtle, but not statistically significant, increase in the production of interleukin-10 and interferon-γ. Importantly, these changes also corresponded with significant suppression of the contact hypersensitivity response in irradiated mice compared with their control counterparts. This suppression was apparent when antigen sensitisation occurred during the neonatal or adult period, and thus did not appear to be analogous to UVR-induced suppression in adults. Although the percentage of T regulatory cells was increased in the skin-draining LNs, they were induced in a different manner to those induced following adult UVR exposure, with no increase in function on a per-cell basis. It therefore appears that one single neonatal exposure to UVR alters development of the immune system, leading to long-term implications for induction of immunity.     

Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers

Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n = 197, ∼2-years old) and they were inseminated 12-18 h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n = 69; (2) progesterone supplementation using a Controlled Internal Drug Release Device (CIDR) from Day 3 to 6.5, n = 64; or (3) progesterone supplementation using a CIDR from Day 4.5 to 8, n = 64. Body condition (BCS) and locomotion scores (scale of 1-5) were recorded for all animals. Animals with a locomotion score ≥4 (very lame) were excluded. Embryo survival rate was determined at slaughter on Day 25. Conceptus length and weight were recorded and the corpus luteum (CL) of all pregnant animals was dissected and weighed. Supplementation with exogenous progesterone increased (P < 0.05) peripheral progesterone concentrations, but did not affect embryo survival rate compared with controls. Mean CL weight, conceptus length and conceptus weight were not different between treatments. There was a positive relationship (P < 0.04) between the increase in progesterone concentrations from Days 3 to 6.5 and embryo survival rate in treated heifers and a similar trend existed between the increase from Days 4.5 to 8 (P < 0.06). There was also a positive relationship (P < 0.05) between the progesterone concentration on Day 6.5 and the embryo survival rate in treated heifers. A direct correlation was seen between locomotion score and embryo survival rate, with higher (P < 0.05) early embryo survival rates in heifers with a lower locomotion score. In conclusion, supplementation with progesterone at different stages of early embryo development increased peripheral progesterone concentration and resulted in a positive association between changes in progesterone concentration during the early luteal phase and embryo survival rate. Supplementation with progesterone had no effect on either CL weight or conceptus size in pregnant animals. Lameness had a significant negative effect on early embryo survival.     

Effect of acute nutritional restriction on incidence of anovulation and periovulatory estradiol and gonadotropin concentrations in beef heifers

The effects of acute nutritional restriction on follicular dynamics, incidence of anovulation, and periovulatory estradiol and gonadotropin concentrations were studied in two replicates using beef heifers exhibiting regular estrous cycles. Heifers fed a diet supplying 1.2 maintenance (1.2 Mn) were synchronized using an intravaginal progesterone-releasing device for 8 days. One day before device removal, heifers were allocated randomly, within replicate, to a diet supplying 0.4 Mn (n = 20), or kept at 1.2 Mn (n = 21). On the sixth day after detected ovulation, heifers received 500 microg of synthetic prostaglandin F(2alpha) (PGF(2alpha)) to induce luteolysis, estrus, and ovulation of the first dominant follicle (DF). Animals were inseminated and returned to a diet of 1. 2 Mn. Pregnancy diagnosis was performed 30 days later. The maximum diameter subsequently attained by the DF present at progesterone withdrawal was smaller (P < 0.01) in heifers fed 0.4 Mn. Two heifers fed 0.4 Mn failed to ovulate this DF (P > 0.10). Growth rate (P < 0. 01) and maximum diameter (P < 0.001) of the DF in the first follicular wave of the next estrous cycle was also reduced in heifers fed 0.4 Mn. After prostaglandin administration, a further 10 heifers fed 0.4 Mn failed to ovulate the first DF of this cycle, and it regressed (P < 0.001), causing anovulation in 12 of 20 heifers within 13-15 days (P < 0.001). Anovulation of the DF present at progesterone withdrawal was preceded by a proestrous estradiol increase but absence of a gonadotropin surge (2 of 2 heifers), while neither endocrine event was detected before anovulation of the DF of the first new follicular wave (2 of 2 heifers). In cases in which ovulation of the first DF of the new cycle occurred, fertility was similar (P > 0.10) in heifers fed either 0.4 (n = 7) or 1.2 Mn (n = 20). In conclusion, acute nutritional restriction of cyclic heifers from 1.2 to 0.4 Mn decreased the growth rate and maximum diameter of DFs and induced failure of the DF to ovulate in 60% of heifers, but, within the confines of limited animal numbers, did not compromise fertility in heifers that ovulated.     

Estrus and ovulation in beef cows following use of progesterone-releasing devices, progesterone and estradiol valerate

Two hundred nonsuckling beef cows were treated with either 1) a progesterone-releasing intravaginal device (PRID) for 12 days; 2) PRID plus an IM injection of 200 mg progesterone (PRID-P); 3) PRID plus 5-mg IM injection of estradiol valerate (PRID-EV); or 4) PRID-EV-P. Cows were started on treatment on one of the first eight days of the estrous cycle. The number of cows which had P levels above 1 ng/ml one day after PRID removal was 12 to 50% lower in PRID-EV and PRID-EV-P groups than in PRID and PRID-P groups (P < 0.05). The proportion of cows showing estrus by 96 hours after PRID removal was 38, 36, 77, and 88% (P < 0.05) for the PRID, PRID-P, PRID-EV and PRID-EV-P groups, respectively. Thirty-one percent fewer cows treated with PRID on days 5 through 8 of the estrous cycle showed estrus by four days after PRID removal than those treated on days 1 through 4. In addition, 18 to 22% more cows had P levels above 1 ng/ml among cows treated with PRID or PRID-P on days 5 through 8 than among cows treated similarly on days 1 through 4. It was concluded that effective synchronization of estrus is achieved only when estrogen is used in conjunction with PRID in cows treated for twelve days during the first eight days of an estrous cycle.