E. K. Inskeep

Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes

The effects of progesterone (P4) on follicular growth and fertility in ewes were examined. In Experiment 1, 22 ewes received either one or three packets of P4 (5 g/packet) or an empty packet subcutaneously (sc) from Days 5 to 15 of the estrous cycle (estrus = Day 0). On Day 6, P4-treated ewes received 12.5 mg of prostaglandin F2 alpha. Follicles > or = 3 mm in diameter were observed via transrectal ultrasonography daily from Day 4 through estrus, corpora lutea (CL) were observed 5 to 7 d after estrus. Ewes with low (LOW; < or = 1 ng/ml; n = 5), intermediate (MED; >1 and <2 ng/ml; n = 10), or normal (NOR; > or =2 ng/ml; n = 7) P4 in jugular plasma on Days 7 through 15 differed in follicular development. The largest follicle at estrus was larger in ewes with LOW vs. MED and NOR P4 (7.8 +/- 0.3 vs. 6.9 +/- 0.2 mm; P < 0.05). Treatments differed in proportions of multiple-ovulating ewes, in which the oldest ovulatory follicle was first observed before Day 10 (LOW: 3 of 3, MED: 6 of 10, NOR: 0 of 5, respectively; P < 0.05). Estradiol was higher early in the treatment period in LOW ewes than in MED and NOR ewes (day x treatment; P < 0.05). In Experiment 2, ewes received 5 mg of P4 in corn oil (low progesterone [LP]; n = 51) or 2 ml of corn oil (CON; n = 49) sc every 12 hr on Days 6 through 14 of the estrous cycle before mating. LP ewes received 15 mg of prostaglandin F2 alpha on Day 6. Mean serum P4 on Days 7 through 15 was 0.6 +/- 0.1 ng/ml in LP and 1.9 +/- 0.1 ng/ml in CON ewes. Eleven LP and 12 CON ewes were scanned daily from Day 4 through mating, and in all ewes (n = 93), CL were counted 10 d after mating and embryos were counted at 25, 40, and 60 d of gestation. In multiple-ovulating ewes, day of cycle of appearance was earlier for the oldest (Day 6.1 +/- 0.8 vs. 10.4 +/- 0.8) but not second oldest (Day 11.7 +/- 1.0 vs. 12.2 +/- 0.9) ovulatory follicles in LP compared with CON ewes. The conception rate was lower in LP (72%) than in CON ewes (98%; P < 0.01). However, numbers of CL 10 d after mating, and in pregnant ewes, numbers of embryos 25 d after mating and lambs born, did not differ with treatment. In summary, low P4 increased the size of the largest follicles and the age of the oldest ovulatory follicles. Embryos resulting from the ovulation of older and younger follicles in the same ewe did not differ in their ability to survive.

Determination of early pregnancy in ewes utilizing transrectal ultrasonography

Transrectal ultrasonography was used in ewes to determine the earliest day at which pregnancy could be detected, the number of embryos present, and the pattern of growth of the embryos. Twenty-one ewes were placed with 2 fertile rams and 20 ewes with 2 vasectomized rams. All ewes were treated to synchronize estrus and were observed for estrus twice daily. The 36 ewes that showed synchronized estrus were separated from the rams following mating. Transrectal ultrasonography was performed daily from estrus (Day 0) to Day 25 for all ewes and on Days 30, 35 and 40 post breeding for the 20 ewes mated to fertile rams. A 7.5 MHz transducer (human prostate, linear array) was utilized, with the ewes in dorsal recumbency in a tilting squeeze chute. Extraembryonic fluid and membranes were observed in the uterine horns ipsilateral to corpora lutea by Day 15 post breeding in all 17 ewes subsequently diagnosed as pregnant. Rhythmic pulsations (heartbeat) within the embryonic vesicles were first detected on Day 18 or 19. At least 1 embryo was detected by Day 20 in all the pregnant ewes, but not all the embryos were counted accurately until Day 25 (main effect of day; P < 0.05). Two ewes each had an embryo which died (absence of previously observed heartbeat) by Day 25 or Day 40, respectively, but each maintained the remaining embryos to term. The pattern of embryonic growth, as determined by crown-rump lengths on Days 20, 25, 30, 35 and 40, did not differ with the number of embryos carried (n = 1 to 4). In conclusion, transrectal ultrasonography was found to provide a rapid, accurate means for the early detection of pregnancy in ewes.

Relationships of hormonal patterns and fertility to occurrence of two or three waves of ovarian follicles, before and after breeding, in beef cows and heifers

Ovarian follicular waves were characterized before and after breeding in 52 lactating beef cows and 16 heifers. Effects of two (2 W) or three (3 W) waves of follicular development and associated patterns of concentrations of steroids in jugular serum on pregnancy rate were examined. Animals were observed for oestrus (= day 0) twice daily and inseminated artificially at second oestrus. Follicular development was monitored by ultrasonography and jugular blood samples were collected simultaneously on alternate days from day 6 after first oestrus until ovulation after the second oestrus and from day 6 after second oestrus (insemination) until next ovulation or day 24 of pregnancy. Pregnancy was determined by ultrasonography at 25 days after insemination. From individual patterns of growth or regression of the largest follicles, more of the 59 animals with oestrous cycles of 17 to 25 days had 2 W (51, 86%) than 3 W (8, 14%) during the oestrous cycle before breeding (P < 0.01). Cycles averaged 1.1 days longer (P < 0.10) and corpora lutea regressed later (P < 0.01) in animals with 3 W vs. those with 2 W, but mean oestradiol between 7 and 2 days before second oestrus (3.7 +/- 0.3 pg ml-1) did not differ between cycles with 2 W or 3 W. Ovulatory follicles in animals with 2 W differed from those with 3 W (P < 0.05) in day of detection (12.3 +/- 0.3 vs 16.5 +/- 0.5), growth rate (1.0 +/- 0.1 vs 1.5 +/- 0.1 mm day-1), interval from detection to ovulation (9.3 +/- 0.3 vs 6.3 +/- 0.7 days) and duration of dominance (4.0 +/- 0.2 vs 2.1 +/- 0.6 days). Pregnancy rates, 82% in cows with 2 W and 100% in cows with 3 W, did not differ (P > 0.05). During the period equivalent to an oestrous cycle after breeding, 29 (49%) of the 59 animals had 2 W and 30 (51%) had 3 W. Fewer animals with 2 W than 3 W after breeding became pregnant (16/23, 70% vs 26/27, 96%; P < 0.05), but patterns of concentrations of progesterone on days 6 through 14 or mean oestradiol on day 14 (2.6 +/- 0.2 pg ml-1) did not differ. In conclusion, fewer animals had 3 W than 2 W before breeding and fertility did not differ. During the equivalent of one oestrous cycle after breeding, approximately equal numbers of animals had 2 W or 3 W and fertility was greater for animals with 3 W.

Patterns of follicular development during the estrous cycle in monovular Merino del Pais ewes

Growth and regression of ovarian follicles with antral diameters > or = 2 mm were characterized during 15 estrous cycles by daily transrectal ultrasonography (7.5 MHz probe) in 9 ewes of Merino del Pais, a consistently monovular Spanish breed. Mean interovulatory interval was 17.5 +/- 0.5 days and ovulation rate was 1 in all ewes; of 60 to 116 follicles, > or = 2 mm observed during the entire estrous cycle, 13.0 +/- 1.2 reached a maximum diameter > or = 4 mm and 7.9 +/- 0.6 different follicles became the largest follicle in the animal at some point during the cycle. An average of 4.5 new follicles per ewe were detected each day, with no significant effect of day of cycle. Appearance of new follicles that grew to > or = 4 mm tended to differ during the first 8 days of the cycle, being highest on day 3 and lowest on day 6 (P < 0.10), but did not vary significantly during the last 6 days. Growth of new follicles from the day of detection to the next day differed between, but not within, periods, averaging 1.4 +/- 0.3 mm of the first 8 days of the cycle and 1.8 +/- 0.5 mm from day -6 through -1 (P < 0.05). Total number of follicles > or = 2 mm per ovary on days 1 through 8 varied with the interaction of ovary by day, being more variable in the non-CL ovary. During the last 7 days, a linear decline in total follicles was coupled with a linear increase in number of large follicles (P < 0.05). Differences in the size between the largest and second largest follicles were greater on days 5 through 8 than on days 1 through 4, did not differ with day of cycle on days -6 through -1, then increased on the last day from 1.5 mm to 2.9 mm (P < 0.001). In conclusion, the monovular Merino del Pais ewe showed a more rapid growth and turnover of ovarian follicles than other breeds studied, but identified 3-mm follicles did not emerge in other than a random distribution. There was little evidence of dominance until the ovulatory follicle had been identified.

Prostaglandins F in uterine and ovarian venous plasma from nonpregnant and pregnant ewes collected by cannulation

Periodic collections of uterine venous blood were obtained from three nonmated, three pregnant and two mated but nonpregnant ewes in which uterine veins were cannulated with polyvinyl tubing on day 11 postestrus. Frequent sampling was achieved in three of these ewes with additional cannulae in the ovarian veins. Blood samples were collected at 3-hr intervals from 0600 on day 12 to 1800 on day 13 and then 6-hr intervals through day 15. On day 13, three additional samples at 30-min intervals were collected between 1400 and 1530. Prostaglandins F (PGF) in plasma were quantified by radioimmunoassay. On day 12, one ewe in each group had at least one measurement which suggested an increased rate of release of PGF into the uterine vein. Seven of eight ewes on day 13 appeared to have increased rates of release of PGF from the uterus between 0900 and 1500. The highest level measured in each ewe during this period ranged from 2.7 to 11 ng per milliliter. Concentrations of PGF in ovarian venous plasma in two of three ewes were positively correlated (P less than .05) with concentrations of PGF in uterine venous plasma (r equals .64 in each ewe). No evidence was obtained that pregnant and nonpregnant ewes differ in rate or pattern of release of PGF from the uterus into the uterine vein on days 12 and 13. Comparisons could not be made with confidence concerning PGF either in uterine veins on days 14 and 15 or in ovarian veins on all days due to limited number of observations.

Aspects of regulation of uterine secretion of prostaglandins during the oestrous cycle and early pregnancy

Abstract Evidence that the pattern of secretion of prostaglandin E 2 (PGE 2 ) by the uterus: conceptus unit is consistent with that compound playing a role in the local antiluteolytic effect of the embryo is reviewed briefly. Utero-ovarian venous concentrations of PGE 2 increased on day 13 in pregnant compared to non-pregnant ewes, both absolutely and in relation to prostaglandin F 2α (PGF 2α ). The roles of oestrogen and progesterone in regulation of utero-ovarian venous concentrations of PGF 2α were examined during two intervals of the oestrous cycle and on day 13 of the cycle or pregnancy. The first interval (Experiment l, days 10 to 14) was chosen to study the initial rises of PGF 2α that precede luteolysis. At laparotomy on day 9, a catheter was inserted into a utero-ovarian vein. Eight ewes received no further treatment. At surgery, 12 ewes were given 10 mg of progesterone (i.m.) along with an intravaginal pessary containing 30 mg flurogestone acetate and were either ovariectomized (n=5) or lutectomized (n=7). Five ewes were ovariectomized only. The second interval (Experiment 2, days 14 to 17) was chosen to study the maximal rises of PGF 2α associated with the completion of luteal regression and the preovulatory increase of oestrogen. Ewes were laparotomized and catheterized on day 14 and either received no further treatment (n=5), or were lutectomized (n=6), ovariectomized (n=5) or ovariectomized and implanted with oestradiol-17β (n=5). Five utero- ovarian venous samples were collected, every 30 min for 2 hours beginning at 0600 and 1800 on each day in each experiment, and assayed for PGF 2α . In a third experiment, 15 bred and 12 nonbred ewes were laparotomized and catheterized on day 12. One third of each type remained intact, while two thirds were ovariectomized and half of those received 10 mg of progesterone (s.c.) and an intravaginal sponge containing 30 mg of progesterone on day 12. Utero-ovarian samples were collected at 15-minute intervals for 3 hours on day 13. Neither the average day of occurrence of the first peak of PGF 2α (control, 12.1; lutectomy plus progesterone, 11.6; ovariectomy plus progesterone, 12.0; ovariectomy, 12.2) nor the mean number of peaks detected in each ewe during the sampling periods on days 10 to 14 (2.8, 3.7, 3.2 and 3.4, respectively) differed among groups in Experiment 1. The mean number of peaks of PGF 2α detected in each ewe during days 14 to 17 did not differ among groups in Experiment 2. Neither of the patterns of concentrations of PGF 2α studied (mean within sampling period over time and natural logarithm of the variance within sampling period over time) differed among groups in either experiment. Concentrations of PGF 2α increased after ovariectomy in Experiment 3 and were restored to values similar to intact ewes by replacement therapy with progesterone. These data do not support the idea that increases in oestrogen are involved in regulating patterns of uterine secretion of PGF 2α during the ovine oestrous cycle or the initiation of maternal recognition of pregnancy. In addition, progesterone may not be necessary after day 9 for initial rises of PGF 2α to occur on days 12 and 13, but does appear to be necessary to sustain normal patterns of secretion of PGF 2α beyond day 14.

Short-term treatment with a controlled internal drug releasing (CIDR) device and FSH to induce fertile estrus and increase prolificacy in anestrous ewes

The objectives were to evaluate, in anestrous ewes, the effectiveness of a CIDR-G device (0.3 g progesterone) administered for 5 d to induce estrus; and FSH (Folltropin; 55 mg NIH-FSH-P1 equivalent) in saline:propylene glycol (1:4) 24 h before insert removal (Day 0), to increase ovulation rate and prolificacy. Ewes of mixed breeding were assigned at random to 3 treatments: control (C; n = 125), 5 d progesterone (P5; n = 257) and 5 d progesterone plus FSH (P5F; n = 271). Intact rams were joined at insert removal and ewes were observed every 24 h for 3 d. On Day 14, the ovulation rates of all ewes detected in estrus in the treated groups were determined using transrectal ultrasonography. Rams were removed on Day 26 to 31. Ewes were examined for pregnancy then, and again 20 to 25 d later to detect ewes that conceived to the second service period. Percentage of ewes marked by rams was higher in progesterone-treated (77%) than in C (20%; P < 0.01), but did not differ between P5 and P5F. The ovulation rate (1.95+/-0.04) did not differ due to FSH. Conception (68%) and pregnancy (52%) rates were higher in progesterone-treated (P < 0.01) than in C (0%) ewes. Estrous response varied quadratically with time after ram introduction, and the conception rate varied quadratically with the time of observation of onset of estrus. Over two service periods more progesterone-treated than C ewes lambed (65 vs 45%; P < 0.01). Lambs born per ewe exposed (0.7+/-0.1, 1.0+/-0.1, and 1.1+/-0.1 for C, P5 and P5F, respectively) was increased by progesterone (P < 0.05). Litter size to the first service period (1.59+/-0.04) and overall (1.54+/-0.03) did not differ among treatment groups. FSH-treated ewes tended to have more lambs (1.67+/-0.1) than did ewes receiving progesterone alone (1.5+/-0.1; P = 0.06) and than did ewes lambing to the second service period (1.5+/-0.1; P = 0.06). In summary, a 5-d progesterone pre-treatment of anestrous ewes induced estrous cycles and increased the pregnancy rates. A single injection of FSH only tended to increase litter size.

Role of progesterone in regulating uteroovarian venous concentrations of PGF2 α and PGE2 during the estrous cycle and early pregnancy in ewes

The role of progesterone in regulation of uteroovarian venous concentrations of prostaglandins F2 alpha(PGF2 alpha) and E2 (PGE2) during days 13 to 16 of the ovine estrous cycle or early pregnancy was examined. At estrus, ewes were either mated to a fertile ram or unmated. On day 12 postestrus, ewes were laparotomized and a catheter was inserted into a uteroovarian vein. Six mated and 7 unmated ewes received no further treatment. Fifteen mated and 13 unmated ewes were ovariectomized on day 12 and of these, 7 mated and 5 unmated ewes were given 10 mg progesterone sc and an intravaginal pessary containing 30 mg of progesterone. Uteroovarian venous samples were collected every 15 min for 3 h on days 13 to 16 postestrus. Mating resulted in higher mean daily concentrations of PGE2 in the uteroovarian vein than in unmated ewes. Ovariectomy prevented the rise in PGE2 with day in mated ewes but had no effect in unmated ewes. Progesterone treatment restored PGE2 in ovariectomized, mated ewes with intact embryos. Mating had no effect on mean daily concentrations of PGE2 alpha or the patterns of the natural logarithm (1n) of the variance of PGF2 alpha. Ovariectomy resulted in higher mean concentrations and 1n variances of PGF2 alpha on day 13 and lower mean concentrations and 1n variances of PGF2 alpha on days 15 and 16. Replacement with progesterone prevented these changes in patterns of mean concentrations and 1n variances of PGF2 alpha following ovariectomy. It is concluded that progesterone regulates the release of PGF2 alpha from the uterus, maintaining high concentrations while also preventing the occurrence of the final peaks of PGF2 alpha which are seen with falling concentrations of progesterone. This occurs in both pregnant and non-pregnant ewes. Progesterone is also needed to maintain increasing concentrations of PGE2 in mated ewes.

Prostaglandin F2α in the uterus of ewes during early pregnancy

Abstract PGF 2α has been postulated to be the uterine luteolytic factor. If this is true, then one might expect the levels of PGF 2α to be lower during early pregnancy. However, in the present study levels of PGF 2α were found to be significantly higher in both content (ng) and concentration (ng/gm) in the endometrium of day-13 pregnant ewes (2375, 152 respectively) compared to day-13 nonpregnant animals (1478, 89 respectively). These results imply that the luteotropic effects of the embryo must occur by some mechanism other than inhibition of production or release of the uterine luteolytic factor.

Increased Expression of Insulin-Like Growth Factor Binding Protein-1 During Induced Regression of Bovine Corpora Lutea

Abstract Three experiments were conducted to examine gene expression during induced luteal regression in the cow; the initial purpose was the identification of potential embryotoxins. In experiment 1, changes in gene expression in the corpus luteum (CL) were identified by differential display reverse transcription–polymerase chain reaction (DD-PCR) during the first 72 h of luteal regression in cows treated with prostaglandin F2α (PGF2α) on Days 4–7 after estrus. Expression of insulin-like growth factor–binding protein-1 (IGFBP-1) was up-regulated, with greatest expression at 24 h (P < 0.05) after treatment with PGF2α began. In experiment. 2, IGFBP-1 and its mRNA were quantified in CL collected 24 or 48 h after treatment with PGF2α on Day 4 or 10 after estrus. Because local mechanisms for exchange of hormones between the ovary and uterus are known in ruminants, uterine flushings were assayed for IGFBP-1 to seek evidence of local transfer of luteal IGFBP-1 to the uterus. IGFBP-1 mRNA was increased (P < 0.05) in CL 24 h after treatment when PGF2α that began on Day 10, and by 48 h after treatment that began on Day 4. Concentrations of IGFBP-1 increased (P < 0.05) in a pattern similar to mRNA, by 24 h on Day 10, and by 48 h on Day 4. Concentrations of IGFBP-1 in uterine flushings did not change on either day. Concentrations of progesterone decreased (P < 0.05) by 8 h after treatment with PGF2α that began on Day 10, but not until 24 h after treatment that began on Day 4. In experiment 3, cows received either saline or PGF2α and CL were collected 2 or 10 h after a single treatment, or 2 h after a second treatment that was given 8 h after the first. Expression of IGFBP-1 was increased by 2 h after treatment with PGF2α on both Days 4 and 10 after estrus. In conclusion, secretion of IGFBP-1 is increased during luteolysis, and may inhibit the steroidogenic effects of insulin-like growth factor-I (IGF-I), but no evidence was found to implicate IGFBP-1 in the embryotoxic effect of regressing CL.