P.M. Fricke

Follicular deviation and acquisition of ovulatory capacity in bovine follicles.

Abstract Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was ≥12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was ≤10 mm (0 of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 10 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P < 0.05) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 40 mg of LH when the largest follicle first achieved 7.0, 8.5, or 10.0 mm (experiment 3). No cows with a follicle of 7 mm (0 of 9) or 8.5 mm (0 of 9) ovulated, compared with 80% (8 of 10) of cows with 10-mm follicles. Thus, follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle.

Efficacy of decreasing the dose of GnRH used in a protocol for synchronization of ovulation and timed AI in lactating dairy cows

To determine the efficacy of reducing the dosage of GnRH used in a protocol for synchronization of ovulation and timed AI, primiparous and multiparous lactating Holstein cows (n = 237) were randomly assigned to 1 of 2 treatment groups. Ovulation was synchronized for cows in the first group using intramuscular injections of GnRH and PGF2 alpha as follows: Day 0, 100 micrograms GnRH; Day 7, 25 mg PGF2 alpha; Day 9, 100 micrograms GnRH. Ovulation was synchronized in the second group of cows using the same injection schedule and dosage of PGF2 alpha but only 50 micrograms GnRH per injection. All cows underwent a timed AI at 12 to 18 h after the second GnRH injection. The proportion of cows ovulating in response to the second GnRH injection (synchronization rate) and pregnancy status at 28 and 56 d post AI were determined using transrectal ultrasonography. The synchronization rate, double-ovulation rate, conception rate at 28 and 56 d post AI, and pregnancy loss from 28 to 56 d post AI did not differ statistically between treatment groups. For all cows, synchronization rate was 84.0%, and double-ovulation rate was 14.1%. Conception rates calculated using all cows receiving synchronization of ovulation were 41.1% at 28 d and 34.4% at 56 d post AI. Conception rates calculated for only synchronized cows were 47.6% at 28 d and 40.1% at 56 d post AI. For all cows, pregnancy loss from 28 to 56 d post AI was 13.5%, with an attrition rate of 0.5% per day. Estimated savings in hormone costs using 50 rather than 100 micrograms GnRH per injection for synchronizing ovulation were

Effect of milk production on the incidence of double ovulation in dairy cows

6.40 per cow and

Detection of anovulation by heatmount detectors and transrectal ultrasonography before treatment with progesterone in a timed insemination protocol

20.27 per pregnancy. Thus, decreasing the dosage of GnRH used for synchronization of ovulation and timed AI in lactating dairy cows reduces synchronization costs per cow and per pregnancy without compromising the efficacy of the synchronization protocol.

Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination

To determine the effect of parity and milk production on the incidence of double ovulation, the synchronization of ovulation, using GnRH and prostaglandin F2 alpha followed by timed AI (Ovsynch), was initiated at a random stage of the estrous cycle in lactating Holstein cows (n = 237). Ovulatory response at 48 h after the second GnRH injection and conception rate at 28 d post AI were determined by transrectal ultrasonography. Ovulation was synchronized in 84% of cows receiving the Ovsynch protocol. Of the synchronized cows, 14.1% exhibited a double ovulation and 47.6% conceived. Conception rate tended to be greater (P = 0.08) for cows exhibiting double (64.0%) rather than single ovulation (45.2%). To determine the effect of milk production on the incidence of double ovulation, cows were classified into low (< or = 40 kg/d) or high (> 40 kg/d) milk production groups based on the average milk production of 40.5 +/- 0.8 kg/d collected 2 d before AI. Although the incidence of double ovulation tended to increase linearly (P = 0.09) with increasing parity, the incidence of double ovulation was nearly 3-fold greater (P < 0.05) for cows in the high (20.2%) than the low (6.9%) milk production group. Furthermore, the increase in the incidence of double ovulation with parity apparently occurred because, within a parity group, the proportion of cows with high milk production was greater for the older cows. Twinning rate of cows that calved (n = 58) was 5.2%. In a secondary objective, cows were retrospectively classified as cystic or normal based on ultrasonographic ovarian morphology at the time of the second GnRH injection. Incidence of ovarian cysts was 11%, and the synchronization and conception rate of cows classified as cystic was 73.1 and 36.8%, respectively, which did not differ from that of normal cows. We conclude that milk production is the primary factor affecting the incidence of double ovulation in lactating dairy cows and may explain the effect of parity on twinning rate. In addition, Ovsynch appears to be an effective method for establishing pregnancy in lactating dairy cows with ovarian cysts.

Physiological and practical effects of progesterone on reproduction in dairy cattle.

Our objective was to determine the accuracy of identifying noncycling lactating dairy cows before the application of a timed artificial insemination (AI) protocol [with or without progesterone supplementation via a controlled internal drug-release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 locations in the Midwest, 1,072 cows were enrolled in a Presynch protocol (2 injections of PGF(2alpha) 14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF(2alpha) injection, with timed AI at 0 or 24 h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone > or =1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24 h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.

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

The objective was to determine if using a Double-Ovsynch protocol [DO; Pre-Resynch: GnRH-7 d-PGF(2α)-3 d-GnRH, 7 d later Breeding-Resynch: GnRH-7 d-PGF(2α)-56 h-GnRH-16 h-timed artificial insemination (TAI)] to resynchronize ovulation after a previous TAI would increase synchrony and pregnancies per AI (P/AI) compared with an Ovsynch protocol initiated 32 d after TAI (D32; GnRH-7 d-PGF(2α)-56 h-GnRH-16 h-TAI). Lactating Holstein cows at various days in milk and prior AI services were blocked by parity and randomly assigned to resynchronization treatments. All DO cows received the first GnRH injection of Pre-Resynch 22 d after TAI, and cows (n=981) diagnosed not pregnant using transrectal ultrasonography 29 d after TAI continued the protocol. Pregnancy status for all D32 cows was evaluated 29 d after TAI so fertility and pregnancy loss could be compared with that of DO cows. All D32 cows received the first GnRH injection of Ovsynch 32 d after TAI, and cows (n=956) diagnosed not pregnant using transrectal palpation 39 d after TAI continued the protocol. In a subgroup of cows from each treatment, ultrasonography (n=751) and serum progesterone (P4) concentrations (n=743) were used to determine the presence of a functional corpus luteum (CL) and ovulation to the first GnRH injection of D32 and Breeding-Resynch of DO (GnRH1), luteal regression after PGF before TAI, and ovulation to the GnRH injection before TAI (GnRH2). Overall, P/AI 29 d after TAI was not affected by parity and was greater for DO compared with D32 cows (39 vs. 30%). Pregnancy loss from 29 to 74 d after TAI was not affected by parity or treatment. The percentage of cows with a functional CL (P4 ≥1.0 ng/mL) at GnRH1 was greater for DO than D32 cows (81 vs. 58%), with most DO cows having medium P4 (60%; 1.0 to 3.49 ng/ml), whereas most D32 cows had either low (42%; <1.0 ng/mL) or high (36%; ≥3.5 ng/mL) P4 at GnRH1. Ovulation to GnRH1 was similar between treatments but was affected by serum P4 at GnRH. Cows with low P4 (<1.0 ng/mL) had the greatest ovulatory response (59%), followed by cows with medium (≥1.0 to 3.49 ng/mL; 38%) and then high (≥3.50 ng/mL; 16%) P4 at GnRH1. A greater percentage of DO cows were synchronized compared with D32 cows (72 vs. 51%) primarily due to a greater percentage of D32 than DO cows without a functional CL at the PGF injection before TAI (35 vs. 17%) or without complete CL regression before GnRH2 (17 vs. 7%). We conclude that DO increased fertility of lactating dairy cows during a resynchronization program primarily by increasing synchronization of cows during the Ovsynch protocol before TAI.

Effect of progesterone on magnitude of the luteinizing hormone surge induced by two different doses of gonadotropin-releasing hormone in lactating dairy cows

The discovery of progesterone (P4) and elucidation of the mechanisms of P4 action have an important place in the history of endocrinology and reproduction. Circulating P4 concentration is determined by a balance between P4 production, primarily by the corpus luteum (CL), and P4 metabolism, primarily by the liver. The volume of luteal tissue and number and function of large luteal cells are primary factors determining P4 production. Rate of P4 metabolism is generally determined by liver blood flow and can be of critical importance in determining circulating P4 concentrations, particularly in dairy cattle. During timed artificial insemination (AI) protocols, elevations in P4 are achieved by increasing number of CL by creating accessory CL or by supplementation with exogenous P4. Dietary manipulations can also alter circulating P4, although practical methods to apply these techniques have not yet been reported. Elevating P4 before the timed AI generally decreases double ovulation and increases fertility to the timed AI. Near the time of AI, slight elevations in circulating P4, possibly due to inadequate luteal regression, can dramatically reduce fertility. After AI, circulating P4 is critical for embryo growth and establishment and maintenance of pregnancy. Many studies have attempted to improve fertility by elevating P4 after timed AI. Our recent meta-analysis and manipulative study indicated small fertility benefits (3% to 3.5%) mostly in primiparous cows. Thus, previous research has provided substantial insight into mechanisms regulating circulating P4 concentrations and actions. Understanding this prior research can focus future research on P4 manipulation to improve reproductive success.

Effect of dry period length on reproduction during the subsequent lactation

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.

Effect of increasing GnRH and PGF2α dose during Double-Ovsynch on ovulatory response, luteal regression, and fertility of lactating dairy cows

Ovulation to the first GnRH injection of Ovsynch-type protocols is lower in cows with high progesterone (P4) concentrations compared with cows with low P4 concentrations, suggesting that P4 may suppress the release of LH from the anterior pituitary after GnRH treatment. The objectives of this study were to determine the effect of 1) circulating P4 concentrations at the time of GnRH treatment on GnRH-induced LH secretion in lactating dairy cows and 2) increasing the dose of GnRH from 100 to 200 μg on LH secretion in a high- and low-P4 environment. A Double-Ovsynch (Pre-Ovsynch: GnRH, PGF(2α) 7d later, GnRH 3d later, and Breeding-Ovsynch 7d later: GnRH, PGF(2α) 7d later, and GnRH 48 h later) synchronization protocol was used to create the high- and low-P4 environments. At the first GnRH injection of Breeding-Ovsynch (high P4), all cows with a corpus luteum ≥ 20 mm were randomly assigned to receive 100 or 200 μg of GnRH. At the second GnRH injection of Breeding-Ovsynch (low P4) cows were again randomized to receive 100 or 200 μg of GnRH. Blood samples were collected every 15 min from -15 to 180 min after GnRH treatment, and then hourly until 6h after GnRH treatment. As expected, mean P4 concentrations were greater for cows in the high- than the low-P4 environment. For cows receiving 100 μg of GnRH, the LH peak and area under the curve (AUC) were greater in the low- than in the high-P4 environment. Similarly, for cows receiving 200 μg of GnRH, the LH peak and AUC were greater in the low- than the high-P4 environment. Cows receiving 100 or 200 μg of GnRH had greater mean LH concentration in the low- than the high-P4 environment from 1 to 6h after GnRH treatment. On the other hand, when comparing the effect of the 2 GnRH doses in the high- and low-P4 environments, cows receiving 200 μg of GnRH had a greater LH peak and AUC than cows treated with 100 μg of GnRH both in the high- and low-P4 environments. For the high-P4 environment, mean LH was greater from 1.5 to 5h after GnRH treatment for cows receiving 200 μg of GnRH than for those receiving 100 μg of GnRH. In the low-P4 environment, mean LH was greater for cows receiving 200 μg of GnRH than for those receiving 100 μg of GnRH from 1 to 2.5h after GnRH treatment. We conclude that the P4 environment at GnRH treatment dramatically affects GnRH-induced LH secretion, and that a 200-μg dose of GnRH can increase LH secretion in either a high- or a low-P4 environment.