J.P. Kastelic

Composition and Characteristics of Follicular Waves during the Bovine Estrous Cycle

The characteristics of anovulatory and ovulatory follicular waves for 18 interovulatory intervals with two waves were studied in Holstein heifers. Daily ultrasonic monitoring of individually identified follicles was used. Waves were detectable retrospectively as a cohort of 4 to 6 mm follicles on a mean of day 0 (day of ovulation) for the anovulatory wave and day 10 for the ovulatory wave. For each wave, the follicles which became dominant versus subordinate did not differ in diameter on the first day of the wave, but the dominant follicle was significantly larger than the subordinates on the following day. On the average, the subordinates ceased growing 4.4 days after the origin of a wave. The dominant follicle of the anovulatory wave grew linearly (1.8 ± 0.1 mm/day) to an average of 15.8 ± 0.5 mm, remained static for a mean of 6 days and then regressed linearly (−1.0 ± 0.1 mm/day). The dominant ovulatory follicle grew slower (P<0.0001) (linear slope, 1.2 ± 0.1 mm/day) than the dominant anovulatory follicle. The diameter of the ovulatory follicle on the day before ovulation (16.2 ± 0.4 mm) was not different from the diameter of the dominant anovulatory follicle during the static phase. The numbers of growing, static and regressing 4 to 6 mm identified follicles did not differ between anovulatory and ovulatory waves. Ninety-five percent of the growing identified follicles were assignable to a wave (follicles emerging within two days of each other) and each wave emerged during a consistent and narrow time period (anovulatory wave, days −1, 0, or 1; ovulatory wave, days 8, 9, 10, or 11). It was concluded, therefore, that the formation of waves was a well-controlled phenomenon. There was a consistent temporal relationship between emergence of the ovulatory wave and onset of regression of the dominant follicle of the anovulatory wave (length of interval from beginning of ovulatory wave to beginning of regression of anovulatory follicle, approximately 3 days). Perhaps, therefore, the mechanism that caused regression of the subordinate follicles of the ovulatory wave also caused regression of the large, static, dominant follicle of the anovulatory wave.

Relationship between ultrasonic assessment of the corpus luteum and plasma progesterone concentration in heifers.

Abstract In nulliparous Holstein heifers, ultrasonography was used to measure cross-sectional areas of corpora lutea, central luteal cavities and luteal tissue on Days 2, 5, 8, and 11 and daily on Days 14 to 21 (pregnant heifers, n = 7) or Day 14 to the day of the subsequent ovulation (nonbred and bred nonpregnant heifers, n = 7 and n = 8, respectively). A blood sample for progesterone assay was collected prior to each ultrasound examination. Combined for the three reproductive statuses, luteal tissue area and plasma progesterone concentration increased (P

Effect of day of prostaglandin F2α treatment on selection and development of the ovulatory follicle in heifers

In previous studies of heifers with two follicular waves during an estrous cycle, the dominant follicle of Wave 1 was first detected ultrasonically on approximately the day of ovulation (Day 0) when its diameter was 4–5 mm. On average, it grew linearly for 6 days (growing phase), remained the same size for 6 days (static phase), and then regressed (regressing phase). The dominant follicle of Wave 2 was first detected on approximately Day 9 and became the ovulatory follicle. In the present experiment, nonbred and bred heifers were treated with a luteolytic dose of prostaglandin F2α (25 mg) on Days 5, 8, or 12, when the dominant follicle of Wave 1 was expected to be in the growing, static, and regressing phase, respectively. There were no significant effects of breeding status on any end point. The hypothesis that growth of the dominant follicle during Wave 1 and response to prostaglandin F2α treatment is different between bred and nonbred heifers was not supported. Ovulation occurred from the dominant follicle of Wave 1 in 5 of 5, 6 of 6 and 0 of 4 heifers treated on Days 5, 8, and 12, respectively (P<0.005). Wave 2 was not detected in the Day-8 heifers, but was the origin of the ovulatory follicle in the Day-12 heifers. The results supported the hypothesis that the dominant follicle of Wave 1 is viable (capable of ovulation) before detection of Wave 2. For heifers treated on Days 5, 8, and 12, the ovulatory follicle had a mean diameter of 13.8, 17.3, and 11.8 mm, respectively, on the day of treatment and a mean diameter of 16.0, 19.5, and 16.4 mm, respectively, on the day prior to ovulation (significant increase between treatment and day prior to ovulation for each group). The results supported the hypothesis that the static-phase dominant follicle of Wave 1 is capable of further growth after luteolysis, even though it has apparently reached maximum diameter. The interval from treatment to ovulation was significantly shorter in Day-5 heifers (mean, 3.0 days) than in Day-12 heifers (mean, 4.5 days). In summary, the viable dominant follicle present at the time of luteolysis increased in diameter and became the ovulatory follicle.

Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers.

Ultrasonography was used once daily to quantify corpora lutea, central luteal cavities, and luteinized tissue during interovulatory intervals (n=66) and during Days 0 to 60 of pregnancy (n=14) in nulliparous Holstein heifers (ovulation=Day 0). The corpus luteum of the estrous cycle was detectable by ultrasonography in most heifers from the day of ovulation (mean, Day 0.5) and extending into the regressive phase beyond the next ovulation (mean, Day 1.4+/-0.2 after the next ovulation). During pregnancy, the corpus luteum was detected until Day 60 (end of study). Maximal central luteal cavity area detected on Days 0 to 20 was used retrospectively to group luteal glands into four cavity categories: no, small, medium, and large. These categories corresponded to approximate cavity diameters of <2 mm, 2 to 5 mm, 6 to 10 mm, and >10 mm, respectively. The incidence of each cavity category was similar between interovulatory intervals and pregnancies (combined incidence, 17/80, 8/80, 33/80, and 22/80 for no, small, medium, and large cavities, respectively; total with cavities, 63/80, 79%). Mean day of first detection of a central cavity was earliest for large cavities during interovulatory intervals (means, Days 4.7, 4.4, and 3.0 for small, medium, and large cavities, respectively; P<0.04) and during pregnancies (means, Days 5.5, 4.2, and 3.3, respectively; NS). However, the day that the cavities reached maximum size (range of means, Days 5.5 to 7.0) did not differ among categories. Mean day of last detection of the central cavity was significantly different among cavity categories during interovulatory intervals (means, Days 9.3, 11.1, and 17.4 for small, medium, and large cavities, respectively) and pregnancies (means, Days 7.0, 8.8, and 20.2, respectively). Time of loss of central cavities was similar between nonbred and pregnant heifers, and there was no significant difference among cavity categories in the length of the interovulatory interval (mean, 20.1 d). Luteal tissue area was not significantly different among cavity categories during interovulatory intervals. There were no indications that cavities were functionally important. Luteal tissue area increased linearly in pregnant heifers on Days 21 to 60 (mean slope, 2.6 mm2/day).

Factors affecting the origin of the ovulatory follicle in heifers with induced luteolysis

Seventeen nulliparous Holstein heifers were treated with a luteolytic dose of prostaglandin F2α (PGF) on Day 8 (Day 0 = day of pretreatment ovulation) when the dominant follicle of the first follicular wave (Wave 1) was expected to be in the static phase. Wave 2 was first detected on Days 8 or 9 in 2 of the 14 and 3 of the 3 heifers that ovulated from Waves 1 and 2, respectively. That is, most heifers treated with PGF on Day 8 ovulated from the dominant follicle of Wave 1, except when Wave 2 emerged early, in heifers that ovulated from Wave 1, the dominant follicle increased (P < 0.0001) in diameter from Day 8 to the day prior to post-treatment ovulation (mean increase, 2.3 mm), even though it had apparently reached its maximum diameter before the induction of luteolysis. Heifers ovulating from the dominant follicle of Wave 1 differed from heifers ovulating from the dominant follicle of Wave 2 in the following ways: change in diameter of the ovulatory follicle from day of treatment to day of post-treatment ovulation (means, 2.3 versus 9.0 mm, P < 0.0001), diameter of Wave-1 dominant follicle on the day prior to post-treatment ovulation (means, 17.6 versus 13.7 mm, P < 0.0005); and length of the interval from day of treatment to day of post-treatment ovulation (means, 4.2 versus 6.3 days, P < 0.03). Following ovulation after the PGF-induced luteolysis, 11 heifers had two-wave estrous cycles and 5 had three-wave cycles. Three-wave cycles were associated with an earlier emergence of Wave 2 and a longer luteal phase (onset of luteal regression; Day 20.0 versus Day 17.5; P < 0.03).

Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers

Previous studies demonstrated that waves of follicular activity develop approximately every 9 d in cattle during the estrous cycle and early pregnancy. A dominant follicle develops from each wave and the remaining follicles (subordinates) begin to regress after a few days. In this study, intraovarian luteal and follicular interrelationships were examined during the follicular waves of the estrous cycle and pregnancy using data obtained by ultrasonography. During the estrous cycle, no intraovarian relationships were found between the ovary containing the corpus luteum and the ovary containing the dominant follicle (n = 165), or between the location of the corpus luteum and the characteristics of the dominant follicle. During pregnancy, however, the frequency distribution for the number of follicular waves with the dominant follicle and corpus luteum on the same or opposite ovaries differed (P<0.05) among Waves 1 to 10. The two structures (dominant follicle and corpus luteum) were more often in opposite ovaries during Waves 3 to 10 (combined frequency, 75%) than during Waves 1 and 2. During pregnancy, dominant follicles of consecutive waves differed (P<0.05) among Waves 1 to 8 in the frequency with which they appeared in the same versus the opposite ovary. The difference seemed primarily due to an increased frequency of consecutive follicles on the same ovary for Waves 4 to 8 (combined frequency, 80%). During both the estrous cycle and pregnancy, there was no significant intraovarian effect of the dominant follicle on the day of detection of the next dominant follicle, on the growth rate of the largest subordinate follicle, or on the length of the interval from wave origin to cessation of growth of the largest subordinate; these results indicate that previously postulated suppressive effects between follicles are exerted through systemic channels.

Continued periodic emergence of follicular waves in non-bred progesterone-treated heifers

The hypothesis was tested that continued periodic emergence of anovulatory follicular waves in pregnant heifers is associated with continued progesterone production. Daily transrectal ultrasonic examinations were used to monitor the diameter profiles of dominant follicles in 19 bred heifers and six non-bred progesterone-treated heifers; 15 of the 19 bred heifers became pregnant and were assigned to a pregnant-control group. Beginning on Day 10 (ovulation = Day 0), non-bred heifers were given a daily injection of 150 mg of progesterone in corn oil vehicle and bred heifers were given corn oil vehicle only. Treatment continued until approximately Day 100, except for non-pregnant heifers, in which treatment was discontinued at reovulation. In the non-bred progesterone-treated group, development of the dominant follicle for Waves 1–10 in relation to side of ovulation and development of successive dominant follicles in relation to side of the previous dominant follicle occurred with equal frequency (not significantly different) in the same and opposite ovaries. In both the pregnant-control and non-bred progesterone-treated groups, the maximum diameter of the dominant follicle of Wave 2 (means ± SEM, 12.7 ± 0.4 mm and 10.1 ± 0.4 mm, respectively) was smaller (P < 0.05) than for Wave 1 (15.1 ± 0.3 mm and 14.8 ± 0.5 mm). The maximum diameter of the dominant follicle of Wave 2 in the non-bred progesterone-treated group (10.1 ± 0.4 mm) was smaller (P < 0.05) than in the pregnant-control group (12.7 ± 0.4 mm). Similarly, the day-to-day profile of the dominant follicle of Wave 2 was less pronounced in the progesterone-treated group (P < 0.003). The dominant follicle of Waves 3–10 (pregnant-control group) and Waves 4, 5, 7 and 9 (non-bred progester-one-treated group) also was smaller (P < 0.05) than for Wave 1. The interval between successive waves was not different between pregnant-control (mean 8.9 ± 0.2 days) and non-bred progesterone-treated (9.6 ± 0.3 days) groups. The hypothesis that the continued periodic emergence of anovulatory follicular waves in pregnant heifers was associated with continued production of progesterone was supported by the continued periodic emergence of waves in the non-bred progesterone-treated heifers.

Basic principles and techniques for transrectal ultrasonography in cattle and horses

Abstract Diagnostic ultrasonography is a powerful tool for evaluating the reproductive tracts in horses and cattle. The technology should be considered for use in the embryo transfer industry. Ultrasound imaging technology provides rapid, non-invasive access to the internal reproductive organs. Dynamic structures may be visualized in the living animal — structures that were previously detectable only in the static state at necropsy or surgical removal. The potential for assessing reproductive structures in the same animals over time has afforded an unprecedented depth to investigations of the dynamic changes in biological structures (e.g., changes in the ovarian follicular population during the estrous cycle and early pregnancy, the process of ovulation, luteal dynamics, and the interactions between the conceptus and uterus). Ultrasound technology is expensive and requires a thorough working knowledge of anatomy and acoustic principles for maximal utilization. The potential, however, is great for future discoveries in both basic and clinical research. We believe that the best is yet to come as basic research utilizing ultrasonography evolves and ultrasonic imaging continues to be incorporated into clinical and research programs.

Accuracy of ultrasonography for pregnancy diagnosis on days 10 to 22 in heifers

Two operators independently conducted ultrasonic pregnancy examinations on nulliparous Holstein heifers on Days 10, 12, 14, 16, 18, 20 and 22, and assigned a diagnosis (pregnant or nonpregnant) and a score for degree of certainty in the diagnosis (1, 2 or 3 for low, intermediate or high, respectively). Pregnancy was retrospectively confirmed by the ultrasonographic detection of an embryo proper and embryonic heartbeat on Day 24 in 20 25 bred heifers; the five nonpregnant heifers were excluded from the analyses. Eleven nonbred heifers were included as an unequivocal source of nonpregnant heifers. Accuracy was not significantly greater than a guess (50%) before Day 18, but reached 100% on Days 20 and 22. Mean accuracy was higher (P<0.005) for nonpregnant (65 77 , 84%) than pregnant heifers (91.5 140 , 65%). For certainty score, there were main effects of day (P<0.0001), reproductive status (pregnant or nonpregnant, P<0.003), and an interaction of day and reproductive status (P<0.0001). The certainty score increased in all heifers among days and was higher (P<0.05) in pregnant than nonpregnant heifers on Days 16 to 20. For luteal area (area of corpus luteum, excluding area of fluid filled center, if present), there were significant main effects of day, reproductive status and a day by status interaction (P<0.0001 for each). Luteal area was approximately constant in pregnant heifers, but in nonpregnant heifers it was lower (P<0.05) on Days 16 to 22 than on Days 10 to 14. Uterine echotexture was scored on a scale of 1 to 3, characteristic of a diestrus, intermediate and estrus uterus, respectively. There were main effects of day and reproductive status (P<0.0001 for each) and an interaction of day and reproductive status (P<0.025). Uterine echotexture was approximately constant in pregnant heifers, but in nonpregnant heifers it was higher (P<0.05) on Days 16 to 22 than on Days 10 to 14. Pregnancy diagnosis on Days 10 to 14 was based on detection of the conceptus; however, detection of the conceptus was not accurate prior to visualization of the embryo proper (mean Day 22, range Days 20 to 24). In nonpregnant heifers, a correct diagnosis with high certainty was made when a small corpus luteum and uterine echotexture characteristic of estrus were detected. In the absence of these changes on Days 18 to 22, a diagnosis of pregnancy was made with high accuracy and intermediate or high certainty.

Suppression of dominant and subordinate ovarian follicles by a proteinaceous fraction of follicular fluid in heifers

Nulliparous Holstein heifers were examined ultrasonically once daily during an interovulatory interval (ovulation=Day 0). Follicles with a diameter>or=4 mm were sequentially identified. Heifers were randomized into four groups (n=4 heifers per group): untreated control heifers and those treated on Days 0 to 3, Days 3 to 6, or Days 6 to 11. Heifers designated for treatment were given an intravenous injection, twice daily, of a proteinaceous fraction of follicular fluid (PFFF; 16 ml) prepared by extracting bovine follicular fluid with activated charcoal. Mean cessation of growth of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 5.5) than in heifers treated on Days 0 to 3 (Day 1.5) or Days 3 to 6 (Day 3.5). Mean onset of regression of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 12.0) than in heifers treated on Days 0 to 3 (Day 5.0) or Days 3 to 6 (Day 7.5). Mean cessation of growth of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 3.0) than in heifers treated on Days 0 to 3 (Day 1.2). Mean onset of regression of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 7.0) than in heifers treated on Days 0 to 3 (Day 4.8). In heifers treated on Days 6 to 11, cessation of growth and onset of regression of the dominant follicle (means, Days 5.2 and 12.0, respectively) were not significantly different from those of the controls. The hypothesis that PFFF treatment on Days 0 to 3 would cause suppression of all follicles of Wave 1 was supported. The hypothesis that PFFF treatment on Days 3 to 6 would not alter growth of the dominant follicle of Wave 1 was not supported. The mean day of detection of the dominant follicle of Wave 2 was different (P<0.005) in control heifers (Day 8.5) than in heifers treated on Day 0 to 3 (Day 5.5) or Days 6 to 11 (Day 14.2). The mean length of the interovulatory interval was shorter (P<0.05) in control heifers (20.5 d) than in heifers treated on Days 6 to 11 (23.2 d). The hypothesis that PFFF treatment on Days 6 to 11 would delay the emergence of Wave 2 was supported. The proportion of heifers with 2-wave interovulatory intervals was 3 4 for control heifers and 0/4, 1/4, and 4/4 for heifers treated on Days 0 to 3, Days 3 to 6, and Days 6 to 11, respectively (3/4 vs 0/4, P<0.05); the remaining heifers had 3-wave interovulatory intervals. On average, in PFFF-treated heifers, follicles stopped growing 1 d after treatment was started, and Wave 2 was detected 3 d after treatment was stopped.