R.C. Chebel

Period of dominance of the ovulatory follicle influences embryo quality in lactating dairy cows.

Length of dominance of the ovulatory follicle and exposure to oestradiol (OE(2)) during proestrus can affect fertility. Lactating cows had their oestrous cycle pre-synchronized and were subjected to one of the four synchronization treatments. Cows in the oestrus detection (OD) treatment received GnRH on day 6 of the oestrous cycle, PGF(2alpha) 7 days later, and were inseminated at detected oestrus. The remaining cows were subjected to the Ovsynch (OVS) protocol (day 0 GnRH, day 7 PGF(2alpha), day 9 GnRH, and timed artificial insemination (AI) 12 h later) starting on day 3 (OVS3) or day 6 (OVS6 and OVS6E) of the oestrous cycle. Cows in the OVS6E treatment received an injection of 0.5 mg oestradiol cypionate 36 h before AI. Ovaries were examined by ultrasonography and blood was sampled for progesterone and OE(2) concentrations. Uteri were flushed 6 days after AI and recovered embryos-oocytes evaluated. Diameter of the ovulatory follicle at AI differed (P<0.01) among treatments, and it was the largest for OVS3 cows, which also had extended (P<0.01) length of follicular dominance. During proestrus, OD and OVS6E cows had increased (P<0.01) OE(2) concentrations. Fertilization was not altered by treatments, and maximum fertilization was achieved when the number of accessory spermatozoa was >7. Proportions of viable embryos in relation to embryos and embryos-oocytes recovered were smaller for OVS3 cows (P<0.01) than the other treatments, and embryos from OVS3 cows also had fewer (P<0.01) blastomeres and tended (P=0.09) to have a lower proportion of live blastomeres. Extending the period of follicle dominance did not alter fertilization but reduced (P<0.001) embryo quality. Embryo quality was compromised even when the dominance of the ovulatory follicle was extended by only 1.5 days.

Effect of fat source differing in fatty acid profile on metabolic parameters, fertilization, and embryo quality in high-producing dairy cows

The objectives were to evaluate the effects of source of fatty acids (FA) on embryo quality of dairy cows. A total of 154 Holstein cows were assigned randomly to 1 of 2 sources of FA supplemented at 2% of the dietary dry matter as calcium salts of either palm oil (PO) or linoleic and trans-octadecenoic acids (LTFA) from 25 d prepartum to 80 d in milk (DIM). Cows were presynchronized beginning at 30 +/- 3 DIM and then subjected to the Ovsynch protocol beginning on d 39 +/- 3 postpartum. Timed artificial insemination was performed 12 h after the final GnRH of the Ovsynch protocol with semen from a single sire of proven fertility. The uteri of cows were nonsurgically flushed at 5 d after artificial insemination for collection of embryos-oocytes. Ovaries were examined by ultrasonography throughout the synchronization protocol. Blood was sampled and plasma was analyzed for concentrations of metabolites and hormones. The body condition score and yields of milk and milk components were measured throughout the first 90 DIM. Treatment did not affect concentrations of nonesterified FA, beta-hydroxybutyrate, glucose, and progesterone in plasma. Body condition was similar between treatments. Milk production was similar between treatments, but concentrations of fat in milk and yields of fat and 3.5% fat-corrected milk decreased in cows fed LTFA, whereas concentration of true protein increased. Source of dietary FA did not influence ovulatory responses, diameter of the ovulatory follicle, and diameter of the corpus luteum during synchronization. Embryo-oocyte recovery relative to the number of corpora lutea did not differ between treatments. Fertilization tended to increase in cows fed LTFA compared with cows fed PO. Feeding LTFA improved the proportion of excellent-, good-, and fair-quality embryos, and embryos from cows fed LTFA had a greater number of blastomeres than embryos from cows fed PO. Feeding a more unsaturated source of FA improved fertilization and embryo development in lactating dairy cows, despite similar indicators of metabolic status.

Effect of reducing the period of follicle dominance in a timed artificial insemination protocol on reproduction of dairy cows

Objectives were to determine the effect of reducing the period of follicle dominance in a timed artificial insemination (AI) protocol on pregnancy per AI (P/AI) in Holstein cows. In experiment 1, 165 cows received 2 injections of PGF(2alpha) at 36 and 50 d in milk (DIM). At 61 DIM, cows were assigned randomly to Cosynch 72 h (CoS72: d 61 GnRH, d 68 PGF(2alpha), d 71 GnRH) or to a 5-d Cosynch 72 h with 1 (5dCoS1: d 61 GnRH, d 66 PGF(2alpha), d 69 GnRH) or 2 injections of PGF(2alpha) (5dCoS2: d 61 GnRH, d 66 and 67 PGF(2alpha), d 69 GnRH). Blood was sampled at the first GnRH, first PGF(2alpha), and at the second GnRH of the protocols and assayed for progesterone. Ovulatory responses to GnRH were evaluated by ultrasonography. Cows were considered synchronized if they had concentrations of progesterone >or=1 ng/mL and <1 ng/mL on the days of the PGF(2alpha), and the second GnRH of the protocols, respectively, and if they ovulated within 48 h of the second GnRH injection. In experiment 2, 933 cows were assigned randomly to CoS72 or 5dCoS2. Blood was assayed for progesterone and ovaries were scanned as in experiment 1. Plasma on the days of the first PGF(2alpha) and final GnRH of the timed AI protocols was assayed for estradiol in 75 cows. Pregnancy was diagnosed on d 38 and 66 after AI. In experiment 1, the proportions of cows with corpora lutea (CL) regression on the day of AI differed and were 79.0, 59.1, and 95.7% for CoS72, 5dCoS1, and 5dCoS2, respectively. Cows that ovulated to the first GnRH of the Cosynch tended to have lesser CL regression than cows that did not ovulate (73.0 vs. 86.4%). Protocol synchronization differed between treatments and they were greater for CoS72 (69.4%) and 5dCoS2 (78.4%) than for 5dCoS1 (42.3%). In experiment 2, CL regression was lesser (91.5 vs. 96.3%) but detection of estrus at timed AI (30.9 vs. 23.6%) was greater for CoS72 than 5dCoS2, and cows in estrus had increased P/AI (46.2 vs. 31.9%). Cows in CoS72 ovulated a larger follicle and had greater concentrations of estradiol on the day of AI than cows in 5dCoS2, but protocol synchronization tended to increase in cows receiving the 5dCoS2. When all 933 cows were evaluated, P/AI was greater for 5dCoS2 than for CoS72 (37.9 vs. 30.9%). Similarly, when only cows with progesterone <1 ng/mL on the day of AI were evaluated, P/AI was greater for 5dCoS2 than for CoS72 (39.3 vs. 33.9%). Treatment with PGF(2alpha) on d 5 and 6 after GnRH resulted in increased luteolysis and allowed for reducing the interval from GnRH to timed AI, which increased P/AI. Reducing time of follicle dominance in timed AI protocols improves fertility of lactating dairy cows.

Reduced progesterone concentration during growth of the first follicular wave affects embryo quality but has no effect on embryo survival post transfer in lactating dairy cows

Fertility of lactating dairy cows is associated with reduced progesterone (P(4)) concentration compared with nonlactating animals. The objective of the current study was to determine whether P(4) during growth of the first follicular wave (FFW) affects embryo quality. Lactating Holstein cows at 33±3 days post partum were allocated to one of three treatments. Cows in the FFW and FFW with P(4) (FFWP) treatments started the superstimulation protocol on day 1 of the estrous cycle and second follicular wave (SFW) cows started the superstimulation protocol on estrous cycle day 7. Cows were superstimulated with 400  mg of NIH-FSH-P1 (FSH) given twice daily for 5 days, two prostaglandin F(2α) (PGF(2α)) injections given with the ninth and tenth injections of FSH, GNRH given 48  h after the first PGF(2α) injection, and timed insemination 12 and 24  h after the GNRH injection. Cows in the FFWP treatment received two intravaginal P(4) inserts during the superstimulation. Embryos were recovered 6.5 days after artificial insemination and excellent/good and fair embryos were frozen and transferred. Blood was sampled daily from estrous cycle day 0 until insemination from donor cows. During the superstimulation protocol, P(4) was (P<0.01) greatest for SFW cows followed by FFWP and FFW cows respectively. The percentage of embryos-oocytes from SFW and FFWP cows classified as excellent/good and fair embryos was (P=0.02) greater than those of FFW cows. Pregnancy per embryo transfer was not (P≥0.73) affected by embryo donor treatment. Reduced embryo quality of cows induced to ovulate the follicles from the first follicular wave is a consequence of reduced P(4) during follicle growth.

Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses

Two experiments evaluated the influence of altering the concentrations of progesterone during the development of the ovulatory follicle on the composition of the follicular fluid, circulating LH and PGF(2α) metabolite (PGFM), and expression of endometrial progesterone receptor and estrogen receptor-α. In both experiments, the estrous cycles were presynchronized (GnRH and progesterone insert followed by insert removal and PGF(2α) 7 d later, and GnRH after 48 h) and cows were then enrolled in 1 of 2 treatments 7 d later (study d -16): high progesterone (HP) or low progesterone (LP). In experiment 1 (n=19), cows had their estrous cycle synchronized starting on study d -9 (GnRH and progesterone insert on d -9, and insert removal and PGF(2α) on d -2). In experiment 2 (n=25), cows were submitted to the same synchronization protocol as in experiment 1, but had ovulation induced with GnRH on study d 0. In experiment 1, plasma was sampled on d -4 and analyzed for concentrations of LH; the dominant follicle was aspirated on d 0 and the fluid analyzed for concentrations of progesterone, estradiol, and free and total IGF-1. In experiment 2, follicular development and concentrations of progesterone and estradiol in plasma were evaluated until study d 16. Uterine biopsies were collected on d 12 and 16 for progesterone receptor and estrogen receptor-α protein abundance. An estradiol/oxytocin challenge for PGFM measurements in plasma was performed on d 16. In experiments 1 and 2, LP cows had lower plasma concentrations of progesterone and greater concentrations of estradiol, and had larger ovulatory follicle diameter (20.4 vs. 17.2mm) at the end of the synchronization protocol than HP cows. Concentration of LH tended to be greater for LP than HP cows (0.98 vs. 0.84 ng/mL). The dominant follicle of LP cows had greater concentration of estradiol (387.5 vs. 330.9 ng/mL) and a lower concentration of total IGF-1 (40.9 vs. 51.7 ng/mL) than that of HP cows. In experiment 2, estradiol and progesterone concentrations did not differ between treatments from d 0 to 16; however, the proportion of cows with a short luteal phase tended to increase in LP than HP (25 vs. 0%). Concentrations of PGFM were greater for LP than HP. Uterine biopsies had a greater abundance of progesterone receptor, and tended to have less estrogen receptor-α abundance on d 12 compared with d 16. An interaction between treatment and day of collection was detected for estrogen receptor-α because of an earlier increase in protein abundance on d 12. Reduced concentrations of progesterone during the development of the ovulatory follicle altered follicular dynamics and follicular fluid composition, increased basal LH concentrations, and prematurely increased estrogen receptor-α abundance and exacerbated PGF(2α) release in the subsequent estrous cycle.

Effect of resynchronization with GnRH on day 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy cows

The objectives of the present study were to determine the effects of resynchronization with GnRH on Day 21 after artificial insemination (AI) on pregnancy rate and losses of pregnancy in lactating dairy cows. Holstein cows (n=585) on two dairy farms were assigned to one of two treatments in a randomized complete block design. On Day 21 after a pre-enrollment AI, animals assigned to the resynchronization (RES) group received 100 microg of GnRH i.m., whereas animals in the control (CON) group received no treatment. All animals were examined ultrasonographically on Days 21 and 28 after AI, and blood samples were taken for progesterone measurement on Day 21. Pregnancy was diagnosed on Day 28 and reconfirmed 14 days later. Nonpregnant cows on Day 28 were inseminated using timed AI after the completion of the Ovsynch protocol 10 and 17 days after enrollment in the study for RES and CON groups, respectively. Progesterone concentration > or =2.35 ng/ml was used as an indicator of pregnancy on Day 21. For RES and CON cows, pregnancy rate at Days 21 (70.9% versus 73.0%, P<0.56), 28 (33.1% versus 33.6%; P<0.80) and 42 (27.0% versus 26.8%; P<0.98) after the pre-enrollment AI did not differ. Administration of GnRH on Day 21 after AI had no effect on pregnancy loss in RES and CON groups from days 21 to 28 (53.2% versus 53.5%; P<0.94) and days 28 to 42 (17.9%; P<0.74) after AI. Pregnancy rate after the resynchronization period was similar for both treatment groups. Resynchronization with GnRH given on Day 21 after AI for initiation of a timed AI protocol prior to pregnancy diagnosis does not affect pregnancy rate and pregnancy loss in lactating dairy cows.

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.

Low progesterone concentration during the development of the first follicular wave reduces pregnancy per insemination of lactating dairy cows.

Objectives were to determine the effect of progesterone (P4) concentration on fertility of lactating dairy cows induced to ovulate follicles of the first follicular wave. Lactating dairy cows (n=989) at 38±3d postpartum were balanced by parity and body condition score and randomly assigned to 3 treatments: first follicular wave (FFW), first follicular wave with exogenous P4 (FFWP), or second follicular wave (SFW). All cows had their estrous cycle presynchronized with 2 injections of prostaglandin (PG) F(2α) given 14 d apart. Cows in the FFW and FFWP treatments started the ovulation synchronization protocol 3 d after the last PGF(2α) of the presynchronization protocol, whereas SFW cows received a GnRH injection (100 μg of gonadorelin diacetate; Cystorelin, Merial Ltd., Duluth, GA) 3 d after the last PGF(2α) of the presynchronization protocol and started the synchronization protocol 7 d later. The synchronization protocol consisted of GnRH on d -10, PGF(2α) on d -3, and GnRH concurrent with timed artificial insemination (AI) on d 0. Cows in the FFWP treatment received 2 controlled internal drug release inserts containing 1.38 g of P4 from d -8 to -3. Progesterone concentration was determined on d -10, -8, -6, -3, and 0 from all cows and at 7, 14, and 21 d after AI from a subsample of cows (n=170). Cows (n=715) had their ovaries scanned by ultrasound on d -10, -3, and 7 d. Pregnancy was diagnosed at 38 and 66 d after AI. Concentration of P4 from study d -8 to -3 was lowest for FFW cows (1.4±0.1 ng/mL) and similar between SFW (3.7±0.2 ng/mL) and FFWP (3.7±0.1 ng/mL) cows. Diameter of the dominant follicle on study d -3 was greater for FFW cows (16.5±0.3 mm) than for SFW cows (15.4±0.3 mm), but diameter of the dominant follicle of FFWP cows was not different (15.9±0.3 mm) compared with that of SFW and FFW cows. The incidence of multiple ovulation was largest for FFW cows (SFW=19.5, FFW=33.6, FFWP=19.0%), but pregnancy per AI (P/AI) at 66 d was smallest for FFW cows (SFW=38.9, FFW=22.3, FFWP=32.0%). Anovular cows in the SFW (19.4 vs. 42.8%) and FFWP (22.1 vs. 37.2%) treatments had reduced P/AI compared with cyclic cows, despite having similar or greater P4 concentration from study d -8 to -3, respectively. Estrus and ovulation synchronization protocols for lactating dairy cows must result in growth of ovulatory follicle under P4 concentration >2 ng/mL to ensure high P/AI.

Effect of increasing amounts of supplemental progesterone in a timed artificial insemination protocol on fertility of lactating dairy cows.

The objectives were to evaluate the effect of supplemental progesterone during a timed artificial insemination (TAI) protocol on pregnancy per insemination and pregnancy loss. Lactating dairy cows from 2 dairy herds were presynchronized with 2 injections of PGF(2alpha) 14 d apart, and cows observed in estrus following the second PGF(2alpha) injection were inseminated (n = 1,301). Cows not inseminated by 11 d after the end of the presynchronization were submitted to the TAI protocol (d 0 GnRH, d 7 PGF(2alpha), d 8 estradiol cypionate, and d 10 TAI). On the day of the GnRH of the TAI protocol (study d 0), cows were assigned randomly to receive no exogenous progesterone (control = 432), one controlled internal drug-release (CIDR) insert (CIDR1 = 440), or 2 CIDR inserts (CIDR2 = 440) containing 1.38 g of progesterone each from study d 0 to 7. Blood was sampled on study d 0 before insertion of CIDR for determination of progesterone concentration in plasma, and cows with concentration <1.0 ng/mL were classified as low progesterone (LP) and those with concentration > or =1.0 ng/mL were classified as high progesterone (HP). From a subgroup of 240 cows, blood was sampled on study d 3, 7, 17 and 24 and ovaries were examined by ultrasonography on study d 0 and 7. Pregnancy was diagnosed at 38 +/- 3 and 66 +/- 3 d after AI. Data were analyzed including only cows randomly assigned to treatments and excluding cows that were inseminated after the second PGF(2alpha) injection. The proportion of cows classified as HP at the beginning of the TAI protocol was similar among treatments, but differed between herds. Concentrations of progesterone in plasma during the TAI protocol increased linearly with number of CIDR used, and the increment was 0.9 ng/mL per CIDR. The proportion of cows with plasma progesterone > or =1.0 ng/mL on study d 17 was not affected by treatment, but a greater proportion of control than CIDR-treated cows had asynchronous estrous cycles following the TAI protocol. Treatment with CIDR inserts, however, did not affect pregnancy at 38 +/- 3 and 66 +/- 3 d after AI or pregnancy loss.

Resynchronization strategies to improve fertility in lactating dairy cows utilizing a presynchronization injection of GnRH or supplemental progesterone: I. Pregnancy rates and ovarian responses.

Objectives were to evaluate 3 resynchronization protocols for lactating dairy cows. At 32+/-3 d after pre-enrollment artificial insemination (AI; study d -7), 1 wk before pregnancy diagnosis, cows from 2 farms were enrolled and randomly assigned to 1 of 3 resynchronization protocols after balancing for parity, days in milk, and number of previous AI. All cows were examined for pregnancy at 39+/-3 d after pre-enrollment AI (study d 0). Cows enrolled as controls (n=386) diagnosed not pregnant were submitted to a resynchronization protocol (d 0-GnRH, d 7-PGF2alpha, and d 10-GnRH and AI) on the same day. Cows enrolled in the GGPG (GnRH-GnRH-PGF2alpha-GnRH) treatment (n=357) received a GnRH injection at enrollment (d -7) and if diagnosed not pregnant were submitted to the resynchronization protocol for control cows on d 0. Cows enrolled in CIDR treatment (n=316) diagnosed not pregnant received the resynchronization protocol described for control cows with addition of a controlled internal drug release (CIDR) insert containing progesterone (P4) from d 0 to 7. In a subgroup of cows, ovaries were scanned and blood was sampled for P4 concentration on d 0 and 7. After resynchronized AI, cows were diagnosed for pregnancy at 39+/-3 and 67+/-3 d (California herds) or 120+/-3 d (Arizona herds). Cows in the GGPG treatment had more corpora lutea than CIDR and control cows on d 0 (1.30+/-0.11, 1.05+/-0.11, and 1.05+/-0.11, respectively) and d 7 (1.41+/-0.14, 0.97+/-0.13, and 1.03+/-0.14, respectively). A greater percentage of GGPG cows ovulated to GnRH given on d 0 compared with CIDR and control cows (48.4, 29.6, and 36.6%, respectively), but CIDR and control did not differ. At 39+/-3 d after resynchronized AI, pregnancy per AI (P/AI) was increased in GGPG (33.6%) and CIDR (31.3%) cows compared with control (24.6%) cows. At 67 or 120+/-3 d after resynchronized AI, P/AI of GGPG and CIDR cows was increased compared with control cows (31.2, 29.5, and 22.1%, respectively). Presynchronizing the estrous cycle of lactating dairy cows with a GnRH 7 d before the start of the resynchronization protocol or use of a CIDR insert within the resynchronization protocol resulted in greater P/AI after resynchronized AI compared with control cows.