Injection Of PGF2 Alpha Research Articles

Gonadotropin-releasing hormone-induced ovulation and luteinizing hormone release in beef heifers: Effect of day of the cycle

The COSynch protocol has been used to synchronize ovulation and facilitate fixed-time AI in beef cattle. Establishment and maintenance of pregnancy was negatively affected, in previous studies, by GnRH-induced ovulation of small dominant follicles (</=11 mm). The reason for the presence of small follicles at the second GnRH (GnRH 2) is not clear. The objectives of this study were 1) to determine the effect of ovulatory response at the first GnRH (GnRH 1) on diameter and variation in diameter of the largest follicle at GnRH 2, and 2) to determine the effect of day of the cycle (stage of a follicular wave) on GnRH-induced luteinizing hormone (LH) release, and the resulting ovulatory response after GnRH 1 and 2. Two experiments used pubertal beef heifers synchronized to be on different days of the estrous cycle (d 2, 5, 10, 15, and 18 after estrus) in which a dominant follicle would or would not respond to GnRH 1. Ovulatory response to GnRH 1 did not affect size or variation in diameter of the largest follicle at GnRH 2 in Exp. 1 or 2. In Exp. 1, ovulatory response after GnRH 1 (0/14(a), 12/13(b), 4/13(ac), 9/13(bc), and 2/10(a) in the d 2, 5, 10, 15, and 18 groups; (a-c)P < 0.05) and GnRH 2 (13/14(a), 12/13(a), 12/13(a), 2/13(b), and 2/10(b) in the d 2, 5, 10, 15, and 18 groups, respectively; (a,b)P < 0.05) was affected by day of the cycle. In Exp. 2, day of the cycle also affected the proportion of heifers ovulating after GnRH 1 (0/7(a), 8/8(b), 0/6(a) 5/8(ab), and 5/8(ab) of the d 2, 5, 10, 15, and 18 heifers, respectively; (a-c)P < 0.05) and GnRH 2 (3/7(ab), 8/8(b), 5/6(b), 1/8(a), and 2/8(a) of the d 2, 5, 10, 15, and 18 heifers, respectively; (a,b)P < 0.05). In both experiments, heifers receiving GnRH 1 on d 15 and 18 had a greater (P < 0.05) occurrence of luteolysis before PGF(2alpha) injection and expression of estrus than heifers treated on d 2, 5, and 10. The GnRH-induced LH surge was of greatest magnitude in heifers receiving GnRH 1 on d 18 of the cycle followed by d 5, 15, 10, and 2 (9,054(b), 5,774(bc), 4,672(c), 2,548(c), and 915(d) arbitrary units; respectively; (a-d)P < 0.05). In summary, ovulatory response to GnRH 1 did not affect size of the dominant follicle at GnRH 2. Day of the cycle when GnRH 1 was delivered affected dominant follicle size at GnRH 2. Treatment with GnRH 1 in the earlier part of the estrous cycle (on or before d 10) increased the proportion of dominant follicles that were large enough to respond to GnRH 2 (>/=10 mm) and increased ovulatory response after GnRH 2.

Utilization of dried distillers grains for developing beef heifers1,2,3

A 2-yr study was conducted at 2 locations to determine if supplementing beef heifers with dried distillers grains (DDG) as an energy source affected growth or reproduction. Spring-born crossbred heifers (n = 316) were blocked by age or sire and age and assigned randomly to DDG or control (dried corn gluten feed, whole corn germ, urea) supplement. Heifers received prairie hay in amounts sufficient for ad libitum intake and 0.59% of BW DDG or 0.78% of BW control supplement (DM basis). Supplements were formulated to be isocaloric, but protein degradability differed. Supplemental undegradable intake protein intake from DDG averaged 267 g/animal daily and reached 318 g/animal daily; control supplemental undegradable intake protein intake averaged 90 g/animal daily and peaked at 107 g/animal daily. Initial pubertal status was determined by 2 blood samples collected 10 d apart, and monthly BW were collected from November through January; then biweekly BW and blood samples were collected from February until May yearly. Heifers were synchronized with 2 injections of PGF2alpha 14 d apart; estrus was detected and heifers were artificially inseminated for 5 d and placed with bulls 10 d later. Conception and pregnancy rates were determined via transrectal ultrasonography. Initial age, BW, and BCS did not differ (P > 0.92) for control and DDG heifers. Final BW, ADG, and final BCS also were not affected (P > 0.31) by supplementation. Estimated age and BW at puberty did not differ (P > 0.23) between treatments, and the proportions of pubertal heifers did not differ at the initiation of the experiment (P > 0.82), at the beginning of the 14-d sampling intervals, or before synchronization. Estrus synchronization rate (75.9%), time of estrus, and overall pregnancy rate (89.5%) were not affected (P > 0.14) by treatment. However, a greater proportion (P = 0.008) of DDG than control heifers conceived to AI (75.0 vs. 52.9%), resulting in greater (P = 0.07) AI pregnancy rates for DDG heifers (57.0 vs. 40.1%). Body weight or BCS at pregnancy diagnosis did not differ (P > 0.52) between DDG and control heifers. Supplementing beef heifers with DDG during development did not affect age at puberty but improved AI conception and pregnancy rates compared with an isocaloric control supplement.

Developmental Programming: Follicular Persistence in Prenatal Testosterone-Treated Sheep Is Not Programmed by Androgenic Actions of Testosterone

Testosterone (T) treatment during early-midgestation (30-90 d; term is 147 d) leads to reproductive cycle defects. Daily ultrasonography in prenatal T-treated female sheep during the first two breeding seasons revealed an increase in the number of large follicles and follicular persistence. The objective of this study was to determine whether follicular persistence in prenatal T-treated females was programmed by the androgenic actions of T. Pregnant Suffolk ewes were injected with 100 mg (im; twice weekly) of T propionate or dihydrotestosterone (DHT, a nonaromatizable androgen) in cottonseed oil from d 30 to d 90 of gestation. Prior to daily transrectal ovarian ultrasonography, estrus was synchronized with two injections of 20 mg of prostaglandin F2alpha (PGF2alpha) given 11 d apart in two consecutive years. In yr 1 ultrasonography began 14 d after PGF2alpha, during the presumptive luteal phase, and continued until subsequent ovulation and corpora lutea were detected (10-13 d). In yr 2, ultrasonography began 2 d before the last PGF2alpha injection and concluded 25 d after the last PGF2alpha injection. Daily changes in appearance and disappearance of ovarian follicles and follicular sizes were assessed. Prenatal DHT, but not prenatal T, treatment increased the total number of follicles by increasing the number of small follicles. Prenatal T, but not DHT, treatment increased (P<0.05) the number of large follicles with the majority of prenatal T-treated females manifesting follicular persistence. The data indicate that occurrence of large-sized follicles and follicular persistence in prenatal T-treated females are not programmed by androgenic actions but likely are programmed by estrogenic actions stemming from aromatization of T to estradiol.

Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers1,2

The objectives of the current study were to evaluate the correlation between reproductive status and steady-state levels of Myxovirus resistance gene (MX2) mRNA in peripheral blood leukocytes (PBL) of dairy heifers and the reliability of using change in MX2 messenger RNA (mRNA) for identification of nonpregnant heifers 18 to 19 d after AI. Holstein heifers (n = 266), 13 +/- 1 mo of age, were assigned randomly to be inseminated (BRED; n = 214) or not (NONBRED; n = 52). Estrous cycles of all heifers were synchronized with an intravaginal insert containing progesterone for 7 d. At insert removal, heifers received an injection of PGF2alpha. Heifers in the BRED group were inseminated on detection of estrus or at a fixed time, 72 h after insert removal concomitant with a GnRH treatment. Heifers in the NONBRED group received an injection of GnRH 48 h after insert removal. Blood samples collected on d 0 (d of AI or estrus) and 18 were used to determine steady-state levels of MX2 mRNA. Samples collected on d 0, 7, 14, and 21 were analyzed for progesterone concentration. Pregnancy was determined retrospectively by progesterone concentration on d 21 and was diagnosed at 30 +/- 1 and 60 +/- 3 d after AI. The fold change in levels of MX2 mRNA from d 0 to 18 was greater for heifers classified and diagnosed as pregnant on d 21 (P < 0.05) and 30 +/- 1 (P < 0.05) and 60 +/- 3 (P < 0.05) d after AI compared with nonpregnant (bred but not pregnant) and NONBRED heifers. Heifers that experienced pregnancy loss from 21 to 30 +/- 1 (P = 0.11) or 21 to 60 +/- 3 (P = 0.08) d of gestation tended to have smaller fold increases in MX2 mRNA expression than those that maintained pregnancy. The sensitivity (range 57.1 to 65.6%) and negative predictive values (range 47.9 to 57.1%) of determining reproductive status on d 18 according to the change in the level of MX2 mRNA expression in PBL were low, and the correlation between diagnosis of pregnancy by fold change in MX2 mRNA expression and other methods was small (r = 0.20 to 0.36). The current study indicates that increased expression of MX2 mRNA in PBL is related to pregnancy approximately 21, 30, and 60 d after AI in dairy heifers and that losses that occurred later in pregnancy were associated with lower fold increases in MX2 mRNA. However, using the change in MX2 mRNA expression was not a reliable method for diagnosis of pregnancy at 18 d after AI because of the low sensitivity and negative predictive value.

Comparison of Two Types of CIDR-based Timed Artificial Insemination Protocols for Repeat Breeder Dairy Cows

This study compared two types of controlled internal drug release (CIDR)-based timed artificial insemination (TAI) protocol for treatment of repeat breeder dairy cows. In the first trial of the experiment, 55 repeat breeder cows were randomly assigned to the following two treatments. (1) In the EB group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 1 mg estradiol benzoate (EB) plus 50 mg progesterone (P4; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 1 mg EB on Day 8 and were subjected to TAI 30 h later (n=27). (2) In the gonadotrophin releasing hormone (GnRH) group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 250 microg gonadorelin (GnRH; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 250 microg GnRH on Day 9 and were subjected to TAI 17 h later (n=28). In the second trial, 41 repeat breeder cows that were confirmed as not pregnant in the first trial were randomly assigned to the same two treatments used in the first trial (an EB group of 20 cows and a GnRH group of 21 cows). The ovaries of 15 cows from each group were examined by transrectal ultrasonography in order to observe the changes in ovarian structures, and blood samples were collected for analysis of serum P4 concentrations. The pregnancy rates following TAI in the first (18.5 vs. 32.1%) and second (40.0 vs. 38.1%) trials and the combined rates (27.7 vs. 34.7%) did not differ between the EB and GnRH groups. The proportions of cows with follicular wave emergence within 7 days did not differ between the EB (12/15) and GnRH groups (13/15). The interval to wave emergence was shorter (P<0.01) in the GnRH group than in the EB group, but there was no difference in the mean diameters of dominant follicles on Day 7 between the groups. Moreover, the proportions of cows with synchronized ovulation following a second EB or GnRH treatment did not differ between the groups. In conclusion, treatment with either EB or GnRH in a CIDR-based TAI protocol results in synchronous follicular wave emergence, follicular development, synchronous ovulation, and similar pregnancy rates for TAI in repeat breeder cows.

Ovarian, hormonal, and reproductive events associated with synchronization of ovulation and timed appointment breeding of Bos indicus-influenced cattle using intravaginal progesterone, gonadotropin-releasing hormone, and prostaglandin F2α1

The objectives of this study were to 1) compare cumulative pregnancy rates in a traditional management (TM) scheme with those using a synchronization of ovulation protocol (CO-Synch + CIDR) for timed AI (TAI) in Bos indicus-influenced cattle; 2) evaluate ovarian and hormonal events associated with CO-Synch + CIDR and CO-Synch without CIDR; and 3) determine estrual and ovulatory distributions in cattle synchronized with Select-Synch + CIDR. The CO-Synch + CIDR regimen included insertion of a controlled internal drug-releasing device (CIDR) and an injection of GnRH (GnRH-1) on d 0, removal of the CIDR and injection of PGF2alpha (PGF) on d 7, and injection of GnRH (GnRH-2) and TAI 48 h later. For Exp. 1, predominantly Brahman x Hereford (F1) and Brangus females (n = 335) were stratified by BCS, parity, and day postpartum (parous females) before random assignment to CO-Synch + CIDR or TM. To maximize the number of observations related to TAI conception rate (n = 266), an additional 96 females in which TM controls were not available for comparison also received CO-Synch + CIDR. Conception rates to TAI averaged 39 +/- 3% and were not affected by location, year, parity, AI sire, or AI technician. Cumulative pregnancy rates were greater (P < 0.05) at 30 and 60 d of the breeding season in CO-Synch + CIDR (74.1 and 95.9%) compared with TM (61.8 and 89.7%). In Exp. 2, postpartum Brahman x Hereford (F1) cows (n = 100) were stratified as in Exp. 1 and divided into 4 replicates of 25. Within each replicate, approximately one-half (12 to 13) received CO-Synch + CIDR, and the other half received CO-Synch only (no CIDR). No differences were observed between treatments, and the data were pooled. Percentages of cows ovulating to GnRH-1, developing a synchronized follicular wave, exhibiting luteal regression to PGF, and ovulating to GnRH-2 were 40 +/- 5, 60 +/- 5, 93 +/- 2, and 72 +/- 4%, respectively. In Exp. 3, primiparous Brahman x Hereford, (F1) heifers (n = 32) and pluriparous cows (n = 18) received the Select Synch + CIDR synchronization regimen (no GnRH-2 or TAI). Mean intervals from CIDR removal to estrus and ovulation, and from estrus to ovulation were 70 +/- 2.9, 99 +/- 2.8, and 29 +/- 2.2 h, respectively. These results indicate that the relatively low TAI conception rate observed with CO-Synch + CIDR in these studies was attributable primarily to failure of 40% of the cattle to develop a synchronized follicular wave after GnRH-1 and also to inappropriate timing of TAI/GnRH-2.

GnRH‐antagonist mediated down‐regulation of the estrous cycle in marmosets

Because of its small size and unproblematic captivity behavior the marmoset monkey is an attractive New World primate model for early developmental questions. However, superovulation protocols used in Old World monkeys and women are not successful in the female marmoset. A novel protocol is needed to utilize these New World monkeys as an efficient animal model for in vitro fertilization experiments or embryo stem cell research. To create such a protocol we first examined the effects of long-term estrous cycle control, secondly, in a dose-finding study, we determined the length of a down-regulation protocol with a gonadotropic releasing hormone (GnRH)-antagonist. Twenty-nine female marmosets were grouped according to the number of estrous cycles, which had been controlled for a period of 12 months in which 88 cycles were monitored. Application of PGF2alpha in the mid-luteal phase led to immediate onset of the follicular phase. The blood progesterone concentration rapidly declined and increased again on day 9-11. The results show that the controlled ovarian cycle length and progesterone response are not altered by the number of PGF2alpha injections. The rapid decline was similar in all groups, indicating that all animals, independent of the number of controlled cycles, react equally to multiple PGF2alpha injections. To determine the proper dosage for a GnRH-antagonist (Cetrorelix), 12 animals in three groups of four female marmosets were treated with two different dosages and a sham dosage. Cetrorelix was applied in the mid-luteal phase, three times over 2 days. In both Cetrorelix-treated animal groups the early progesterone levels matched those in the controls. In the low-dose treatment group [0.01 mg/100 g body weight (BW)] the expected progesterone rise on day 10 was delayed between 9 and 15 days whereas in the high-dose treatment group (0.1 mg/100 g BW) the progesterone rise was delayed between 21 and 41 days. In the low-dose group the steepness of the slope from day 20 onwards was almost identical to that of the control group. This was reflected in the bioCG levels measured. Based on the GnRH-antagonist studies, complete ovarian down-regulation in female marmosets can be achieved by applying a low-dose regimen, and intrinsic gonadotropins would not interfere with an ovarian superstimulation protocol.

Induction of Estrus by Prostaglandin F2.ALPHA. Administration in the Vaginal Vestibules of Miniature Pigs

Estrus induction is an important step in embryo production. It has been difficult to induce estrus in miniature pigs by intramascular (i.m.) injection of prostaglandin F2alpha (PGF2(2alpha)) in the early luteal stage of the estrous cycle. In the present study, we injected two different doses of PGF2(2alpha) i.m. and into the submucosa of the vaginal vestibule (i.ves.) of miniature pigs, and examined the effect of these treatments on estrus induction. Fifteen miniature pigs were divided into five experimental groups (control, saline injected i.m.; PGF2alpha treated, 1.0 or 1.5 mg of PGF2alpha injected twice i.m. or i.ves.), and the estrus length and concentrations of 17beta-estradiol (E2) and progesterone (P) in the blood were examined. Estrus length was significantly shortened by a large amount of PGF2alpha injected i.ves. In addition, the concentration of P in the blood significantly decreased after two injections of PGF2alpha (i.m. or i.ves.). These results suggest that in miniature pigs, administration of at least 3.0 mg of PGF2alpha is required for the induction of luteolysis and injection of PGF2alpha into the vaginal vestibule is a useful method of estrus induction.

Immunization of Goats against Inhibin Increased Follicular Development and Ovulation Rate

In the present study, two experiments were conducted to induce superovulation in goats using passive and active immunization against inhibin. In the first experiment, two groups of goats were given an intravenous injection of either 10 ml normal goat serum (control; n=6) or inhibin antiserum developed against [Tyro30]-inhibin alpha (1-30) (passively immunized; n=6) 48 h before treatment with PGF2alpha. In the second experiment, two groups of goats were immunized with inhibin vaccine (actively immunized; n=5) or Freund's adjuvant (control; n=5) followed by three booster immunizations at 4 week intervals. Blood samples were collected for determination of FSH, LH, estradiol-17beta, and progesterone. Ultrasonography was used to determine ovarian activity at PGF2alpha injection and ovulation rate one week after estrus. In both experiments, there was a significant increase in plasma FSH concentration compared with the controls. However, the pattern of the FSH levels was different between the passively and actively immunized goats. The numbers of follicles in passively and actively immunized goats (22.4 +/- 2.3 and 18.6 +/- 2.1, respectively) were significantly greater than those in the controls (2.6 +/- 0.4 and 2.3 +/- 0.4, respectively). In addition, the ovulation rate was greater in the immunized animals compared with the controls. Therefore, either passive or active immunization against inhibin could be used to induce superovulation in goats.

POSSIBLE MECHANISM FOR TESTICULAR FOCAL NECROSIS INDUCED BY hCG IN RATS

Possible mechanisms for testicular focal necrosis induced by human chorionic gonadotropin (hCG) were examined in Fischer 344 rats. A single s.c. injection of 2000 IU/kg hCG produced focal necrosis 2 days later in testicular tissues such as the seminiferous tubules in the frontal lower part of the testis (FLPT) of 11-week-old F344/Jcl rats. This hCG-induced necrosis was suppressed by an oral treatment (concomitant or delayed by 3 hr) with cyclooxygenase inhibitors (indomethacin, rofecoxib) or prostaglandin (PG) receptor blocker (AA-2414). Focal necrosis was also induced by intratesticular injection of PGF(2alpha) or PGE(2) with this necrosis suppressed by previous oral treatment with AA-2414, and the PGF(2) level in the testis increased 4 hr after hCG treatment. These findings suggested that de novo synthesis of PGs beginning at 3-4 hr was responsible for induction of necrosis. No necrosis was induced by hCG in the Leydig cell-devoid testis produced by ethane dimethanesulfonate treatment. Necrosis of spontaneously-induced Leydig cell tumor mass was also induced by hCG, suggesting that Leydig cells are responsible for induction of necrosis. An injection of dye into the testicular artery and laser Doppler flowmetry revealed a continuous reduction of blood flow at the FLPT at 6-48 hr after hCG treatment; contrary to this, the upper part showed an early recovery from the reduced flow. From these results, the mechanism of the hCG-induced necrosis was concluded to be: 1) hCG stimulates Leydig cells to synthesize PGs de novo; 2) PGs induce the intratesticular arteries to contract in the FLPT; and 3) obstruction of blood flow (ischemia) for more than 12 hr induced focal necrosis in the testis.

Effect of the Dominant Follicle Aspiration before or after Luteinizing Hormone Surge on the Corpus Luteum Formation in the Cow

Luteinizing hormone (LH) surge and follicle rupture act as trigger to start corpus luteum (CL) formation. Thus, we aimed to investigate whether a dominant follicle that has not been exposed to an LH surge can become a functional CL. For this purpose, follicular fluid from the dominant follicles (DF) of cows was aspirated before or after a GnRH-induced LH surge, and subsequent CL formation was observed. Holstein cows were divided into four groups as follows: Luteal phase, a DF was aspirated 7 days after GnRH injection; Pre-LH surge, a DF was aspirated 42 h after PGF(2alpha) injection during the mid luteal phase; Post-LH surge, a DF was aspirated 24 h after GnRH injection following PGF(2alpha); and Intact follicle, ovulation was induced by GnRH injection after PGF(2alpha). Observation of morphological changes in the aspirated follicle using color Doppler ultrasonography and blood sampling was performed on Days 0, 3, 6, and 9 (Day 0 = follicle aspiration). CL formation following DF aspiration was observed only in the Post-LH surge group. In both the Luteal phase and Pre-LH surge groups, however, none of the cows showed local blood flow at the aspirated site or CL formation. Luteal blood flow area, CL volume, and plasma progesterone concentration in the Post-LH surge group were no different from those in the Intact follicle group. The present results clearly demonstrate that rather than follicle rupture, it is the LH surge that is essential for CL formation in cows.

Fish meal supplementation alters uterine prostaglandin F2α synthesis in beef heifers with low luteal-phase progesterone1,2

The objective of the current study was to evaluate the effect of omega-3 fatty acids in fish meal on mitigating uterine PGF2alpha synthesis in heifers with low luteal-phase concentrations of progesterone. Animals were individually fed a corn silage-based diet supplemented with fish meal (5% of DMI; n = 12) or corn gluten meal (6% of DMI; n = 13). Estrous cycles were synchronized using PGF2alpha beginning on d 25 of supplementation. Random heifers from each supplement group (n = 6 fish meal, and n = 7 corn gluten meal) were given three additional i.m. injections of PGF2alpha (25 mg) at 12-h intervals beginning at 0600 on d 3 after estrus to induce formation of corpora lutea that secrete lower concentrations of progesterone. Jugular blood samples were collected daily commencing on d 1 and continuing through d 16 of the estrous cycle to determine serum progesterone concentrations. Oxytocin was administered i.v. (100 IU) to heifers on d 16 after estrus to stimulate uterine PGF2alpha synthesis. Before statistical analyses, heifers were sorted to either normal or low luteal-phase progesterone as determined from serum progesterone on d 9 of the estrous cycle. After sorting, treatment groups consisted of 1) normal luteal progesterone + fish meal (n = 6); 2) low luteal progesterone + fish meal (n = 6); 3) normal luteal progesterone + corn gluten meal (n = 6); and 4) low luteal progesterone + corn gluten meal (n = 7). Serum concentrations of the PGF2alpha metabolite following oxytocin stimulation tended (P = 0.09) to be greater in heifers with low luteal-phase progesterone compared with heifers with normal luteal-phase progesterone. Fish meal supplementation mitigated this response in heifers with low luteal-phase progesterone (P < 0.05), but had no effect on heifers with normal luteal-phase progesterone. In conclusion, the omega-3 fatty acids in fish meal seem to decrease uterine PGF2alpha synthesis in heifers with low luteal-phase serum concentrations of progesterone.

Effect of administration of human chorionic gonadotropin after artificial insemination on concentrations of progesterone and conception rates in beef heifers1,2

The objective of this study was to determine whether administration of hCG approximately 5 d after AI would increase plasma progesterone concentrations and conception rates in beef heifers. Heifers from two locations (Location 1: n = 347, BW = 367 +/- 1.72 kg; Location 2: n = 246, BW = 408 +/- 2.35 kg) received melengestrol acetate (0.5 mg.heifer(-1).d(-1)) for 14 d and an injection of PGF2alpha (25 mg i.m.) 19 d later. Heifers were observed for estrus continuously during daylight from d 0 to 4.5 after PGF2alpha and artificially inseminated approximately 12 h after the onset of estrus. Half of the heifers inseminated at Location 1 were assigned randomly to receive an injection of hCG (3,333 IU i.m.) 8 d after PGF2alpha, and a blood sample was collected from all heifers 14 d after PGF2alpha for progesterone analysis. Half of the heifers inseminated at Location 2 were administered hCG on d 9 after PGF2alpha, and a blood sample was collected from all heifers 17 d after PGF2alpha. Heifers at Location 1 had a 94% synchronization rate, exhibited estrus 2.45 +/- 0.03 d after PGF2alpha, and received hCG 5.55 +/- 0.03 d after AI. Heifers at Location 2 had an 85% synchronization rate, exhibited estrus 2.69 +/- 0.03 d after PGF2alpha, and received hCG 6.31 +/- 0.03 d after AI. Progesterone concentrations were greater (P < 0.01) for hCG-treated heifers than for controls at both locations (8.6 vs. 4.6 ng/mL for treatment vs. control at Location 1, and 11.2 vs. 5.6 ng/mL for treatment vs. control at Location 2). Pregnancy status was determined by ultrasound approximately 50 d after AI. Conception rates (65 vs. 70% for treatment vs. control, respectively) did not differ at Location 1. Conception rates tended (P = 0.10) to be increased with hCG treatment at Location 2 (61 vs. 50% for treatment vs. control, respectively). A second experiment was conducted with 180 heifers at a third location to determine the effects of hCG administration 6 d after timed insemination at approximately 60 h after PGF2alpha in heifers synchronized as in Exp. 1. Pregnancy rate to timed AI did not differ between hCG-treated (62%) and control heifers (59%). Final pregnancy rate after timed AI and bull exposure (92%) was not affected by treatment. In summary, administration of hCG 5 to 6 d after AI did not improve conception or pregnancy rates at two out of three locations evaluated, suggesting insufficient progesterone is not a major factor contributing to early pregnancy failure in beef heifers.

Prostaglandin F2α-mediated Activation of Apoptotic Signaling Cascades in the Corpus Luteum during Apoptosis: INVOLVEMENT OF CASPASE-ACTIVATED DNase

Prostaglandin F(2alpha) (PGF(2alpha)) acting via a G protein-coupled receptor has been shown to induce apoptosis in the corpus luteum of many species. Studies were carried out to characterize changes in the apoptotic signaling cascade(s) culminating in luteal tissue apoptosis during PGF(2alpha)-induced luteolysis in the bovine species in which initiation of apoptosis was demonstrable at 18 h after exogenous PGF(2alpha) treatment. An analysis of intrinsic arm of apoptotic signaling cascade elements revealed that PGF(2alpha) injection triggered increased ratio of Bax to Bcl-2 in the luteal tissue as early as 4 h posttreatment that remained elevated until 18 h. This increase was associated with the elevation in the active caspase-9 and -3 protein levels and activity (p < 0.05) at 4-12 h, but a spurt in the activity was seen only at 18 h posttreatment that could not be accounted for by the changes in the Bax/Bcl-2 ratio or changes in translocation of Bax to mitochondria. Examination of luteal tissue for FasL/Fas death receptor cascade revealed increased expression of FasL and Fas at 18 h accompanied by a significant (p < 0.05) induction in the caspase-8 activity and truncated Bid levels. Furthermore, intrabursal administration of specific caspase inhibitors, downstream to the extrinsic and intrinsic apoptotic signaling cascades, in a pseudopregnant rat model revealed a greater importance of extrinsic apoptotic signaling cascade in mediating luteal tissue apoptosis during PGF(2alpha) treatment. The DNase responsible for PGF(2alpha)-induced apoptotic DNA fragmentation was found to be Ca(2+)/Mg(2+)-dependent, temperature-sensitive DNase, and optimally active at neutral pH conditions. This putative DNase was inhibited by the recombinant inhibitor of caspase-activated DNase, and immunodepletion of caspase-activated DNase from luteal lysates abolished the observed DNA fragmentation activity. Together, these data demonstrate for the first time temporal and spatial changes in the apoptotic signaling cascades during PGF(2alpha)-in-duced apoptosis in the corpus luteum.

Effect of addition of a progesterone intravaginal insert to a timed insemination protocol using estradiol cypionate on ovulation rate, pregnancy rate, and late embryonic loss in lactating dairy cows1

The objectives of this study were to determine the effects of incorporating a progesterone intravaginal insert (CIDR) between the day of GnRH and PGF2alpha treatments of a timed AI protocol using estradiol cypionate (ECP) to synchronize ovulation on display of estrus, ovulation rate, pregnancy rate, and late embryonic loss in lactating cows. Holstein cows, 227 from Site 1 and 458 from Site 2, were presynchronized with two injections of PGF2alpha on study d 0 and 14, and subjected to a timed AI protocol (100 mixrog of GnRH on study d 28, 25 mg of PGF2alpha on study d 35, 1 mg of ECP on study d 36, and timed AI on study d 38) with or without a CIDR insert. Blood was collected on study d 14 and 28 for progesterone measurements to determine cyclicity. Ovaries were scanned on d 35, 37, and 42, and pregnancy diagnosed on d 65 and 79, which corresponded to 27 and 41 d after AI. Cows receiving a CIDR had similar rates of detected estrus (77.2 vs. 73.8%), ovulation (85.6 vs. 86.6%), and pregnancy at 27 (35.8 vs. 38.8%) and 41 d (29.3 vs. 32.3%) after AI, and late embryonic loss between 27 and 41 d after AI (18.3 vs. 16.8%) compared with control cows. The CIDR eliminated cows in estrus before the last PGF2alpha injection and decreased (P < 0.001) the proportion of cows bearing a corpus luteum (CL) at the last PGF2alpha injection because of less ovulation in response to the GnRH and greater spontaneous CL regression. Cyclic cows had greater (P = 0.03) pregnancy rates than anovulatory cows at 41 d after AI (33.8 vs. 20.4%) because of decreased (P = 0.06) late embryonic loss (16.0 vs. 30.3%). The ovulatory follicle was larger (P < 0.001) in cows in estrus, and a greater proportion of cows with follicles > or = 15 mm displayed estrus (P < 0.001) and ovulated (P = 0.05) compared with cows with follicles <15 mm. Pregnancy rates were greater (P < 0.001) for cows displaying estrus, which were related to the greater (P < 0.001) ovulation rate and decreased (P = 0.08) late embryonic loss for cows in estrus at AI. Cows that were cyclic and responded to the presynchronization protocol (high progesterone at GnRH and CL at PGF2alpha) had the highest pregnancy rates. Incorporation of a CIDR insert into a presynchronized timed AI protocol using ECP to induce estrus and ovulation did not improve pregnancy rates in lactating dairy cows. Improvements in pregnancy rates in cows treated with ECP to induce ovulation in a timed AI protocol are expected when more cows display estrus, thereby increasing ovulation rate.

Induction of ovulation with GnRH and PGF2a in lactating Bos taurus × Bos indicus cows

Induction of ovulation for timed artificial insemination (TAI) with the Ovsynch protocol was evaluated in 49 anoestrous and lactating Bos taurus x Bos indicus cows. Palpation per rectum and transrectal ultrasonography were used on Days -30, -20, -10 and 0 (start of treatment) to confirm anoestrus but with the presence of follicles > or = 10 mm, and every other day during treatment to determine ovarian activity. Cows were randomly assigned to: (1) Ovsynch (n = 24; Day 0, 200 microg GnRH; Day 7, 150 microg PGF2alpha; Day 9, 200 microg GnRH + TAI 16 to 20 h later) and (2) control (n = 25; no treatment). Rates of ovulation for the first GnRH injection, detection of a corpus luteum (CL) at PGF2alpha injection, pregnancy and induction of cyclicity were greater (P < 0.05) with Ovsynch. There was no effect of body condition score (P > 0.05). In conclusion, the Ovsynch protocol was not effective in obtaining acceptable pregnancy rate for TAI, but it was effective for induction of cyclicity in anoestrous and lactating Bos taurus x Bos indicus cows under tropical conditions.

Semen characteristics and libido in boars treated repeatedly with PGF2alpha.

The objective was to determine the effects of repeated injections of PGF2alpha (Lutalyse; Pfizer) on semen and libido characteristics in terminal-line boars. Semen was collected once weekly from wk 0 to 15 and on four consecutive days during wk 16. Boars received an i.m. injection of 10 mg of PGF2alpha (n = 11) or 2 mL of vehicle (n = 11) immediately before entering the collection room. For the weekly collections, semen volume (220.3 +/- 3.2 mL; mean +/- SE), gel weight (38.7 +/- 0.7 g), total sperm cells (65.4 +/- 1.2 billion), motile sperm cells (67.4 +/- 0.6 %), and sperm velocity (125.9 +/- 1.2 microm/s), were affected by time (P < 0.01) but not by treatment (P > 0.10). Sperm concentration (0.31 +/- 0.01 billion/mL) was not affected (P > 0.10) by time or treatment. The percentage of morphologically normal sperm cells, assessed at wk 16, did not differ (P = 0.39) between groups (80.8 +/- 1.0). Libido was evaluated from wk 0 to 16. There were no effects of treatment or time (P > 0.10) on the period from injection to the start of ejaculation (225.6 +/- 9.1 s). Duration of ejaculation was affected by treatment (P < 0.01; 472.0 +/- 43.1 s and 280.4 +/- 43.1 s, for PGF2alpha-treated and control boars, respectively) and time (P < 0.01). During the intensive collection period (wk 16), semen volume (200.1 +/- 7.1 mL), gel weight (39.2 +/- 1.5 g), sperm concentration (0.19 +/- 0.01 billion/ mL), total sperm cells (39.5 +/- 3.6 billion), motile sperm cells (65.6 +/- 2.2%), and sperm velocity (117.8 +/- 3.7 microm/ s) were affected by time (P < 0.10) but not by treatment (P > 0.10). The period from injection to the start of ejaculation tended to decrease (by 44%) during the intensive collection period in PGF2alpha-treated boars, but not in controls (treatment x time, P = 0.07). Regardless, the period from injection to the start of ejaculation did not differ (P = 0.63) between groups on d 4 of the intensive collection period. Duration of ejaculation was affected by treatment (P < 0.01; 459.1 +/- 24.1 s for PGF22alpha-treated boars vs. 303.1 +/- 24.1 s for controls) but not by time (P > 0.10). Overall, there were no exceptional positive or negative effects of long-term treatment with PGF2alpha on semen characteristics and libido in boars.

Ovarian follicular development in cattle selected for twin ovulations and births.

Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2alpha on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 24, 48, and 72 h after PGF2alpha. Jugular vein blood was collected from each animal at PGF2alpha injection and at 24-h intervals until slaughter. Ovaries of twinner cows contained more small (< or = 5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large (> or = 10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, androstenedione, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 24, and 48 h and of estradiol, androstenedione, and testosterone in follicular fluid of large follicles at 0 h after PGF2alpha for twinner vs. control cows (follicular status x time x population, P < 0.01). Binding activities of IGFBP-5 and -4 were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estro-gen-active (estradiol:progesterone ratio > 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I.

Relative Changes in mRNA Expression of Angiopoietins and Receptors Tie in Bovine Corpus Luteum during Estrous Cycle and Prostaglandin F2.ALPHA.-induced Luteolysis: A Possible Mechanism for the Initiation of Luteal Regression

Local angiogenesis and angiolysis in the corpus luteum (CL) relate to the luteal function. Recent studies indicate that angiopoietins (ANPT) and their receptors Tie regulate remodeling of microvasculature. We therefore examined 1) the relative changes in the expression of mRNA for ANPT-1, ANPT-2, Tie1 and Tie2 in bovine CL by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) during the estrous cycle and prostaglandin F2alpha (PGF2alpha)-induced luteolysis, and 2) the effect of ANPT-2 on progesterone (P4) release from CL at the late stage of the estrous cycle by an in vitro microdialysis system (MDS). The CLs were classified into 4 stages (early: Day 2-5, n=7, mid: Day 8-12, n=15, late: Day 15-17, n=9, regressing: Day >18, n=19). The levels of ANPT-1 mRNA in early and regressing CL were lower than those in mid and late CL, whereas ANPT-2 mRNA expression did not change during the estrous cycle. The Tie2 mRNA expression decreased as the CL aged. During PGF2alpha-induced luteolysis, ANPT-2 mRNA expression was acutely and temporally increased at 2 h after PGF2alpha injection. The expression of ANPT-1 mRNA was decreased from 4 h after PGF2alpha injection and kept low levels. In the experiment with the in vitro MDS, an infusion of ANPT-2 (100 ng/ml) acutely inhibited P4 release from late CL. Overall, results suggest that decrease of ANPT-1 mRNA is a basic mechanism of vascular remodeling in CL. In addition, ANPT-2 might play a role in regulation of P4 secretion in CL during luteolysis.

Passive Immunoneutralization of Endogenous Inhibin Increases Ovulation Rate in Miniature Shiba Goats

We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2alpha injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin alpha (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 microg PGF2alpha 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17beta increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 +/- 2.2) than in the control group (2.2 +/- 0.6), and the mean number of follicles > or = 4 mm in diameter was 10.0 +/- 0.8 in the inhibin-AS group compared with 2.4 +/- 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2alpha injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.