Supplementation of progesterone via controlled internal drug release inserts during ovulation synchronization protocols in lactating dairy cows.

Our objective was to determine the effect of exogenous progesterone (P4) during a timed artificial insemination (TAI) protocol on pregnancies per AI (P/AI) in dairy cows not previously detected in estrus. Lactating cows (n=3,248) from 7 commercial dairy herds were submitted to a presynchronization protocol (2 injections of PGF(2alpha) 14 d apart; Presynch), and cows in estrus after the second PGF(2alpha) received AI (EDAI; n=1,583). Cows not inseminated by 12 to 14 d after the second PGF(2alpha) injection were submitted to a TAI protocol (GnRH on d 0, PGF(2alpha) on d 7, and GnRH+TAI 72h after PGF(2alpha)). At onset of the TAI protocol, cows were balanced by parity and days in milk and assigned randomly to receive no exogenous P4 (control, n=803) or a controlled internal drug release (CIDR) insert containing 1.38g of P4 from d 0 to 7 (CIDR, n=862). Blood samples were collected at the second PGF(2alpha) injection of the Presynch and on the day of the first GnRH injection of the TAI protocol for P4 determination. When P4 in both samples was <1 ng/mL, cows were classified as anovular, whereas cows having at least 1 sample >or=1 ng/mL were classified as cyclic. Concentration of P4 at 11 to 14 d after AI was determined in a subgroup of cows (n=453) from 2 herds. Pregnancy was diagnosed at 40+/-5 and 65+/-5 d after AI. Proportion of cows inseminated on estrus after the second PGF(2alpha) injection of the Presynch protocol differed among herds (range=26.7 to 59.8%). Overall P/AI for EDAI cows at 40+/-5 and 65+/-5 d were 36.2 and 33.7%, respectively, and pregnancy loss was 8.8%. Proportion of cyclic cows at the onset of the TAI protocol differed among herds (range from 66.5 to 86.3%), but did not differ between treatments (control=72.4%, CIDR=74.1%). Treatment affected P/AI at 40+/-5 (control=33.3%, CIDR=38.1%) and 65+/-5 (control=30.0%, CIDR=35.1%) d after AI but did not affect pregnancy loss (8.6%). Cyclic cows had greater P/AI at 40+/-5 (38.2 vs. 29.3%) and 65+/-5 d (35.1 vs. 26.1%) after AI, but cyclic status had no effect on pregnancy loss. Treatment affected P4 concentration after AI, with more CIDR cows having P4 >or=1 ng/mL (94.4 vs. 86.9%) and P4 >or=3.2 ng/mL (81.8 vs. 68.0%) at 11 to 14 d after AI compared with control cows. Treatment of cows not previously detected in estrus with a CIDR insert during a TAI protocol increased proportion of cows with functional CL after AI and P/AI.

Large variation in steroid concentrations and insulin-like growth factor binding proteins exists among individual small antral follicles collected from within cows at random stages of the estrous cycle.

Variation in the biochemical status of individual small (< or = 5 mm diameter) antral follicles within the ovaries of a cow at any given time likely influences the capacity for undergoing recruitment, selection, and establishing dominance. The objectives of this study were to provide insight into the magnitude of variation in follicular fluid concentrations of steroids and activities of IGFBP that exists among individual small antral follicles within and between cows, and to determine the relationships between follicular fluid IGFBP and steroid concentrations in these follicles. A total of 108 small antral follicles were collected from 6 cows at random stages of the estrous cycle, with 10 to 26 follicles/cow. Concentrations of steroids (ng/mL of follicular fluid) in the overall population of follicles ranged from 0.1 (lowest detectable limit) to 51 for estradiol (E2), 4 to 1,149 for progesterone (P4), and 5 to 504 for androstenedione (A4). Concentrations of E2 and A4 were associated positively (r = 0.2; P < 0.02), but E2 (r = -0.4) and A4 (r = -0.4) were associated negatively, with P4. The proportion of variation in steroid concentrations accounted for by differences among animals (P < 0.05) was small for E2 (12%), moderate for P4 (43%), and greatest for A4 (74%). Least differences between minimum and maximum concentrations of steroids observed in follicles from within a cow were 21-, 5.5-, and 3.5-fold for E2, P4, and A4, respectively, whereas the greatest differences between minimum and maximum concentrations were 505-, 108-, and 26-fold for E2, P4, and A4, respectively. Ranges of IGFBP concentrations (arbitrary densitometer units) detected in fluid from a sub-sample of 43 follicles were 1.18 to 4.50 for IGFBP-3, 0.54 to 4.68 for IGFBP-2, 0.07 to 2.56 for IGFBP-4, and 0.01 to 6.71 for IGFBP-5. Concentrations of E2 were correlated negatively with each IGFBP (r = -0.4 to -0.8; P < 0.05) except IGFBP-3. In contrast, concentrations of A4 were correlated positively with IGFBP-3 (r = 0.4; P < 0.05) but were not correlated with other IGFBP. Concentrations of P4 were correlated positively (r > 0.4; P < 0.05) with IGFBP-4 and -5. The results indicate that steroid concentrations and IGFBP activities vary substantially among small antral follicles collected from within and among individual animals and that increasing production of E2, the hallmark of a developing follicle, was associated with reduced activity of all IGFBP except IGFBP-3, thereby implicating these IGFBP in the regulation of follicular recruitment.

Effect of gonadotropin treatment on size, number, and cell proliferation of antral follicles in cows.

To determine the effects of gonadotropins on the size, number, and cell proliferation of antral ovarian follicles, cows received FSH-P or vehicle beginning on Day 2 after estrus, and ovaries were collected 6, 12, 24, or 48 hr after the initiation of FSH-P treatment or 24 or 48 hr after the initiation of vehicle treatment. Ovaries also were collected from untreated cows on Day 2 after estrus (pretreatment). Before fixation, all visible antral follicles were counted and their surface diameters were recorded. Proliferating cells were immunolocalized in fixed follicles by using a specific primary antibody against proliferating cell nuclear antigen (PCNA), and the labeling index (LI; percentage of cells staining positively for PCNA) was determined for granulosa and thecal cells. After 48 hr of treatment, FSH-P-treated cows had fewer (P < 0.01) small antral follicles and more medium and large antral follicles (P < 0.01 and P < 0.05, respectively) compared with vehicle-treated cows. Granulosa cell LI was negatively correlated (P < 0.05) with follicular diameter for vehicle-treated but not for FSH-P-treated cows. Analysis of covariance using follicular diameter as a covariate to adjust to a common diameter indicated that granulosa cell LI was greater (P < 0.05) at 24 and 48 hr in FSH-P-treated than in vehicle-treated cows; conversely, thecal cell LI was greater (P < 0.01) at 48 hr in FSH-P-treated compared with vehicle-treated cows but did not differ at 24 hr. Across all groups, the LI of cells located within the antral half of the granulosa cell layer was greater (P < 0.01) than that of cells located within the basal half. In conclusion, the stimulation of follicular development by exogenous gonadotropins increased or maintained the proliferation of granulosa and thecal cells concomitant with continued follicular growth. Therefore, enhanced follicular cell proliferation may be an important mechanism by which FSH-P superinduces the growth of antral follicles in cows.