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. 17.2 mm) 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.     

Follicular wave of the ovulatory follicle and not cyclic status influences fertility of dairy cows

Two experiments evaluated the influence of follicular wave at artificial insemination (AI) on fertility of dairy cows. In experiment 1, data from 5,607 lactating cows enrolled in estrous and ovulation synchronization programs for AI were evaluated. Cows’ blood was analyzed for progesterone 7 to 14 d apart, with the second sample collected on the day of the first GnRH (GnRH1) of the synchronization protocol. Cows were classified as cyclic if progesterone was ≥1 ng/mL in at least 1 of the 2 samples and as anovular if both samples were <1 ng/mL. Cyclic cows were categorized as low (CLOW; < 1 ng/mL) or high (CHIGH; ≥ 1 ng/mL) progesterone on the day of GnRH1, which would result in ovulation of the dominant follicle of the first (FW) and second (SW) follicular waves, respectively, at AI. Pregnancy per AI (P/AI) was determined 30 and 53 d after AI. In experiment 2, 220 cyclic Holstein cows received 2 injections of PGF_2α administered 14 d apart. The Ovsynch protocol (d 0 GnRH, d 7 PGF_2α, d 9 GnRH, d 9.5 timed AI) was initiated either 3 or 10 d after the second PGF_2α of the presynchronization to result in insemination to the FW or SW dominant follicles. Blood was analyzed for progesterone and ovaries were scanned to determine ovulatory responses and follicle diameter. Pregnancy was determined on d 32 and 67 after timed AI. In experiment 1, P/AI on d 30 was greater for CHIGH cows than for CLOW and anovular cows (43.0, 31.3, and 29.7%, respectively), but because of pregnancy loss, P/AI on d 53 was lowest for anovular cows. Proportions of cows with short reinsemination intervals differed among groups and were 7.1, 15.7, and 11.9% for CHIGH, CLOW, and anovular cows, respectively. Pregnancy loss was greater for anovular cows than for CLOW cows (15.0 vs. 10.0%) and was intermediate for CHIGH cows (13.5%). In experiment 2, 9.8 and 97.2% of the FW and SW cows, respectively, had progesterone ≥1 ng/mL at GnRH1. Concentrations of progesterone at the GnRH1 and PGF_2α injections of the Ovsynch protocol were greater for SW cows than FW cows. Pregnancy per AI was greater for SW cows than for FW cows (41.7 vs. 30.4%) despite less ovulation to GnRH1 in SW cows than in FW cows (78.7 vs. 88.4%). Collectively, these data indicate that follicular wave of the ovulatory follicle and not cyclic status caused the greatest reduction in P/AI in dairy cows. Whether the culprit is the follicle itself or the hormonal milieu characteristic of the first follicular wave and the early stage of the estrous cycle remains to be elucidated. Synchronization programs that induced ovulation of the FW follicle at AI reduced P/AI in lactating dairy cows, and ovulation of the FW follicle, or development of the ovulatory follicle under low progesterone concentrations, or both, might be mechanisms for reduced fertility in anovular cows.     

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-PGF_2α, and d 10-GnRH and AI) on the same day. Cows enrolled in the GGPG (GnRH-GnRH-PGF_2α-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.     

Effects of supplemental progesterone after artificial insemination on expression of interferon-stimulated genes and fertility in dairy cows

The objectives of the current study were to evaluate the effects of supplemental progesterone after artificial insemination (AI) on expression of IFN-stimulated genes (ISG) in blood leukocytes and fertility in lactating dairy cows. Weekly cohorts of Holstein cows were blocked by parity (575 primiparous and 923 multiparous) and method of insemination (timed AI or AI on estrus) and allocated randomly within each block to untreated controls, a controlled internal drug release (CIDR) containing 1.38 g of progesterone from d 4 to 18 after AI (CIDR4), or a CIDR on d 4 and another on d 7 after AI and both removed on d 18 (CIDR4+7). Blood was sampled to quantify progesterone concentrations in plasma and mRNA expression in leukocytes for the ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) and receptor transporter protein-4 (RTP4) genes. Pregnancy was diagnosed on d 34 ± 3 and 62 ± 3 after AI. Treatment increased progesterone concentrations between d 5 and 18 after AI in a dose-dependent manner (control = 3.42, CIDR4 = 4.97, and CIDR4+7 = 5.46 ng/mL). Cows supplemented with progesterone tended to have increased luteolysis by d 19 after AI (control = 17.2; CIDR4 = 29.1; CIDR4+7 = 30.2%), which resulted in a shorter AI interval for those reinseminated after study d 18. Pregnancy upregulated expression of ISG in leukocytes on d 19 of gestation, but supplementing progesterone did not increase mRNA abundance for ISG15 and RTP4 on d 16 after insemination and tended to reduce mRNA expression on d 19 after AI. For RTP4 on d 19, the negative effect of supplemental progesterone was observed only in the nonpregnant cows. No overall effect of treatment was observed on pregnancy per AI on d 62 after insemination and averaged 28.6, 32.7, and 29.5% for control, CIDR4, and CIDR4+7, respectively. Interestingly, an interaction between level of supplemental progesterone and method of AI was observed for pregnancy per AI. For cows receiving exogenous progesterone, the lower supplementation with CIDR4 increased pregnancy per AI on d 62 in cows inseminated following timed AI (CIDR4 = 39.2; CIDR4+7 = 27.5%); in those inseminated following detection of estrus, however, the use of a second insert on d 7 resulted in greater pregnancy per AI (CIDR4 = 26.9; CIDR4+7 = 31.5%). Pregnancy loss did not differ among treatments. Supplemental progesterone post-AI using a single intravaginal insert on d 4 was beneficial to pregnancy in cows inseminated following timed AI, but incremental progesterone with a second insert on d 7 did not improve fertility of dairy cows.     

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_2α 14 d apart, and cows observed in estrus following the second PGF_2α 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_2α, 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 ≥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_2α 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 ≥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.     

Diurnal variations of progesterone,testosterone, and androsta-1,4-diene-3,17-dione in the rumen and in vitro progesterone transformation by mixed rumen microorganisms of lactating dairy cows

Five Holstein lactating dairy cows fed 5 total mixed rations (TMR) with different forage combinations were used in a 5 × 5 Latin square design to investigate diurnal variations of progesterone (P4), testosterone, and androsta-1,4-diene-3,17-dione (ADD) concentrations in the rumen. Meanwhile, different P4 inclusion levels [0 (control), 2, 20, 40, 80, and 100 ng/mL in culture fluids] were incubated in vitro for 6, 12, 24, 36, 48, and 72 h together with rumen mixed microorganisms grown on a maize-rich feed mixture (maize meal:Chinese ryegrass hay = :1) with an aim to determine microbial P4 transformation into testosterone and ADD. Ruminal P4, testosterone, and ADD concentrations of lactating dairy cows were greater in the TMR with forage combination of corn silage plus alfalfa hay or Chinese wild ryegrass hay than the TMR with the corn stover-based forage combination. The diurnal fluctuation pattern showed that P4, testosterone, and ADD concentrations in the rumen were greater at nighttime than daytime and peaked 6 h after feeding in the morning or afternoon. The in vitro batch cultures showed that the P4 elimination rate was highest at the P4 addition of 20 ng/mL and declined with the further increased addition of P4. The treatments after dosing P4 exhibited a shorter time than the control group until half of the initial P4 inclusion was eliminated (i.e., half time), and the lowest half time (1.46 h) occurred at the P4 addition of 20 ng/mL. In summary, the ruminal steroids concentration was affected by forage type and quality, and the rumen microorganisms exhibited great ability to transform P4 into testosterone and ADD, depending on incubation time and initial P4 addition level, suggesting that the host might affect the metabolism of its rumen microorganisms via the endogenous steroids.     

Atypical progesterone profiles and fertility in Swedish dairy cows

The incidence of normal and atypical progesterone profiles in Swedish dairy cows was studied. Data were collected from an experimental herd over 15 yr, and included 1,049 postpartum periods from 183 Swedish Holstein and 326 Swedish Red and White dairy cows. Milk progesterone samples were taken twice weekly until initiation of cyclical ovarian activity and less frequently thereafter. Progesterone profiles were 1) normal profile: first rise in milk progesterone above the threshold value before d 56 postpartum, followed by regular cyclical ovarian activity (70.4%); 2) delayed onset of cyclical ovarian activity: low milk progesterone the first 56 d postpartum (15.6%); 3) cessation of cyclical ovarian activity: ovarian activity resumed within 56 d postpartum, but ceased for a period of 14 d or more (6.6%); and 4) prolonged luteal phase: ovarian activity resumed within 56 d postpartum, but milk progesterone remained elevated in the nonpregnant cow for a period of 20 d or more (7.3%). Swedish Holsteins had 1.5 times higher risk of atypical profile than Swedish Red and Whites. Risk of atypical profiles was 0.5 and 0.7 times lower for older cows compared with first-parity cows; 2.3 times higher for cows in tie-stalls compared with those in loose housing; 2.6 times higher for cows calving during winter compared with summer; 0.5 times lower for cows in earlier (1994-1999) calving-year groups compared with the most recent (2000-2002); 2.5 times higher for cows with planned extended calving interval compared with conventional calving interval; and 2.2 times higher for an atypical profile in previous lactation compared with a normal profile. Cows with atypical profiles had a 15-d increase in interval from calving to first artificial insemination and an 18-d increase in interval from calving to conception. Progesterone samples taken within the first 60 d postpartum were used to calculate the percentage of samples above the threshold value of luteal activity. This measure had a significantly different mean in profiles and can be used to separate delayed onset of cyclical ovarian activity profiles and prolonged luteal phase profiles from normal. Thereby, it may be a more effective tool than measurements based only on the onset of ovarian cyclical activity in genetic evaluation of early postpartum fertility in dairy cows.     

Concentrations of progesterone in milk of cows administered an intravaginal progesterone insert

Milk from pregnant cows contains concentrations of progesterone (P4) considered safe for human consumption. The objective of this study was to determine if concentrations of P4 in milk during administration of an intravaginal progesterone insert (CIDR insert) are less than concentrations of P4 in milk associated with pregnancy. Results have implications for human use of milk from cows receiving CIDR inserts. Holstein cows (N = 64; > 40 and < 150 d after calving) were administered 25 mg of PGF2alpha i.m. (study d 0) and 20 cows detected in estrus from 2 to 4 d later were assigned randomly to either control (N = 10; no further treatment) or CIDR insert (N = 10; 1.38 g of P4) inserted on study d 17 (14 +/- 1 d after estrus) and removed 7 d later. Composite milk samples were collected contemporaneously from each of the 20 estrous cycling cows and from 10 pregnant cows (> or = 60 and < or = 220 d of gestation) twice daily from study d 17 to 27. Concentrations of P4 in defatted milk samples were quantified using a validated radioimmunoassay. Mean logs of areas under the curve of concentrations of P4 from the afternoon on study d 17 through the afternoon on study d 27 were 3.05 ng day/ml for control, 3.33 ng day/ml for CIDR insert, and 3.81 ng day/ml for pregnant cows. Therefore, increased P4 due to pregnancy was 0.76 ng day/ml (3.81-3.05), whereas the increase in P4 due to CIDR insert was only 0.28 ng day/ml (3.33-3.05). Applying a 95% confidence interval to 0.28 ng day/ml provided an upper value of 0.70 ng day/ml, lower than the increase due to pregnancy. Because milk from pregnant cows is considered safe for human consumption, it follows that milk from cows administered CIDR inserts should also be considered safe, based on concentrations of P4.     

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, β-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 interval between induction of ovulation and artificial insemination (AI) and supplemental progesterone for resynchronization on fertility of dairy cows subjected to a 5-d timed AI program

Objectives were to investigate 2 intervals from induction of ovulation to artificial insemination (AI) and the effect of supplemental progesterone for resynchronization on fertility of lactating dairy cows subjected to a 5-d timed AI program. In experiment 1, 1,227 Holstein cows had their estrous cycles presynchronized with 2 injections of PGF_2α at 46 and 60 d in milk (DIM). The timed AI protocols were initiated with GnRH at 72 DIM, followed by 2 injections of PGF_2α at 77 and 78 DIM and a second injection of GnRH at either 56 (OVS56) or 72 h (COS72) after the first PGF_2α of the timed AI protocols. All cows were time-inseminated at 72 h after the first PGF_2α injection. Pregnancy was diagnosed on d 32 and 60 after AI. In experiment 2, 675 nonpregnant Holstein cows had their estrous cycles resynchronized starting at 34 d after the first AI. Cows received the OVS56 with (RCIDR) or without (RCON) supplemental progesterone, as an intravaginal insert, from the first GnRH to the first PGF_2α. Pregnancy diagnoses were performed on d 32 and 60 after AI. During experiment 2, subsets of cows had their ovaries scanned by ultrasonography at the first GnRH, the first PGF_2α, and second GnRH injections of the protocol. Blood was sampled on the day of AI and 7 d later, and concentrations of progesterone were determined in plasma. Cows were considered to have a synchronized ovulation if they had progesterone <1 and >2.26 ng/mL on the day of AI and 7 d later, respectively, and if no ovulation was detected between the first PGF_2α and second GnRH injections during resynchronization. In experiment 1, the proportion of cows detected in estrus at AI was greater for COS72 than OVS56 (40.6 vs. 32.4%). Pregnancy per AI (P/AI) did not differ between OVS56 (46.4%) and COS72 (45.5%). In experiment 2, cows supplemented with progesterone had greater P/AI compared with unsupplemented cows (51.3 vs. 43.1%). Premature ovulation tended to be greater for RCON than RCIDR cows (7.5 vs. 3.6%), although synchronization of the estrous cycle after timed AI was similar between treatments. Timing of induction of ovulation with GnRH relative to insemination did not affect P/AI of dairy cows enrolled in a 5-d timed AI program. Furthermore, during resynchronization starting on d 34 after the first AI, supplementation with progesterone improved P/AI in cows subjected to the 5-d timed AI protocol.     

Study of progesterone and cortisol concentrations in the Italian Friesian claw

The present research was conducted to study progesterone and cortisol concentrations in the claw of cattle and to verify whether the cattle claw could be considered an efficient matrix to provide retrospective information regarding progesterone and cortisol concentrations related to pregnancy and peripartum periods. These 2 steroids are involved in hoof growth. The study was performed on 32 calves and 24 pregnant milking cows of the Holstein breed, which were clinically healthy and lacking any claw disorders. Samples of at least 0.5 cm in thickness were taken from the sole. Progesterone and cortisol concentrations were determined by RIA. The cortisol concentration in the horny shoe of calves from 0 to 30 d of age was significantly higher than the concentration at 31 to 60 and 61 to 120 d of age. Conversely, the progesterone concentration showed no statistically significant difference in relation to age. The horn progesterone concentrations recorded in the milking dairy cows at 7 mo of pregnancy showed high variability in the horizontal sections of the sole (the individual coefficient of variation ranged between 0.09 and 1.11). In 6 cows, genuine extreme values (genuine outliers) of the progesterone level were found. Moreover, significant differences existed among the progesterone concentrations of the sole's transverse sections. We detected a significant positive correlation between the weight of the horn samples after freeze-drying and their weight after hydration. The cortisol and progesterone levels in soaked horn samples were found to be significantly lower than in the same dry samples. These results show that cortisol and progesterone can be measured in the cattle claw horn. The claws of mature dairy cows could not be used as a matrix to provide a retrospective measure of cumulative hormone secretion, whereas the analysis of the calves’ claw horns showed retrospective hormonal information similar to hair samples.     

Reproduction in dairy cows following progesterone insert presynchronization and resynchronization protocols

The objectives of this study were to evaluate the effects of an intravaginal insert containing progesterone (CIDR, controlled internal drug releasing) administered in presynchronization and resynchronization protocols on cyclicity, detection of estrus, pregnancy rate, and pregnancy loss to first AI; reinsemination patterns; and pregnancy rates to second postpartum AI before and after the time of first-service pregnancy diagnosis in dairy cows. Holstein cows (n = 1,052) were blocked by parity and BCS at 3 ± 3 d in milk (study d 0 = day of calving) and assigned randomly to 1 of 3 presynchronization treatments. During the presynchronization programs, all cows received 2 injections of PGF_2α, on study d 35 and 49. Cows enrolled in the control presynchronization treatment received AI after detected estrus from study d 49 to 62. Cows enrolled in the CIDR estrus-detection (CED) presynchronization treatment received a CIDR insert from study d 42 to 49 and AI on detection of estrus from d 49 to 62. Cows enrolled in the CIDR timed AI (CTAI) presynchronization treatment received the same treatment as CED, but were subjected to timed AI on study d 72 after the Ovsynch (GnRH, 7 d PGF_2α, 2 d GnRH, 24 h timed AI) protocol. The control and CED cows not inseminated by study d 62 were enrolled in the Ovsynch protocol on the same day and received timed AI on study d 72. After first AI, cows were assigned to no resynchronization (RCON) or resynchronization with a CIDR insert (RCIDR) between 14 and 21 d after AI. Blood samples collected on study d 35, 49, and 62 were analyzed for concentrations of progesterone and cows were classified as anestrous when progesterone was <1.0 ng/mL in the first 2 samples. On study d 62, anestrous cows with progesterone ≥ 1.0 ng/mL were classified as having resumed cyclicity. Pregnancy was diagnosed at 31 and 60 d after first AI and at 42 d after second AI. A greater proportion of anestrous cows in CED and CTAI became cyclic by d 62 postpartum than control cows. Resynchronization with the CIDR insert increased the pregnancy rate at 31 d after first AI in CED and CTAI, and at 60 d after AI in all cows because of reduced pregnancy loss. These results indicate that presynchronization with the CIDR insert increased induction of cyclicity in anestrous cows and that resynchronization with the CIDR insert did not affect the reinsemination rate but did reduce pregnancy loss and increased the pregnancy rate at 60 d after first AI.     

Short communication: Effects of the progesterone receptor variants on fertility traits in cattle

The progesterone receptor (PGR) gene is a key factor in the initiation and maintenance of pregnancy and in embryo development. Currently, it is unknown what variants of the PGR gene are related to fertility traits in cattle. Identification of such variants would allow the implementation of marker-assisted selection in breeding schemes. The objective of this study was to investigate the association of single nucleotide polymorphisms (SNP) of PGR with fertility traits in Holstein dairy cattle. An in vitro fertilization system was used to maximize the efficiency of the identification of genetic factors affecting fertility. This in vitro fertilization system would allow the assessment of fertilization and embryonic survival rates independently of influences from the uterine environment. A total of 5,566 fertilization attempts were performed, and a total of 3,679 embryos were produced using oocytes from 324 Holstein cows and semen from 10 Holstein bulls. Sequencing of pooled DNA samples from ovaries revealed an SNP (G/C) in intron 3 of PGR. A generalized linear model was used to analyze the association of this SNP with fertilization and embryonic survival rates for each ovary. Oocytes obtained from CC ovaries showed a 61% fertilization rate, compared with 68 and 69% for GC and GG ovaries, respectively. The survival rate of embryos produced from GG ovaries was 5 and 6% higher than that of GC and CC ovaries. These results indicate that the PGR SNP could be used in marker-assisted selection breeding programs in Holstein dairy cattle.     

Effect of bovine somatotropin (500 mg) administered at ten-day intervals on ovulatory responses, expression of estrus, and fertility in dairy cows

The objectives of this study were to evaluate the effect of administering 500 mg of recombinant bovine somatotropin (bST) every 10 d on ovulatory responses, estrous behavior, and fertility of lactating Holstein cows. Lactating dairy cows were assigned to 1 of 2 treatments: a control with no administration of bST (73 primiparous and 120 multiparous cows) or 6 consecutive administrations of 500 mg of bST (83 primiparous and 123 multiparous cows) given subcutaneously at 10-d intervals starting 61 ± 3 d postpartum (study d 0), concurrent with the initiation of the timed artificial insemination (AI). Blood samples were collected thrice weekly from 61 ± 3 to 124 ± 3 d in milk (DIM), and plasma samples were analyzed for concentrations of estradiol, glucose, insulin, insulin-like growth factor 1, and progesterone. The estrous cycle of cows was presynchronized with 2 injections of PGF_2α at 37 ± 3 and 51 ± 3 DIM, and the Ovsynch timed AI protocol was initiated at 61 ± 3 DIM. Ovaries were scanned to determine ovulatory responses during the Ovsynch protocol. Pregnancy was diagnosed at 33 and 66 d after AI. Body condition was scored on study d 0, 10, 42, and 76. Sixty-four cows were fitted with a pressure mounting sensor with radiotelemetric transmitters to monitor estrous behavior. Treatment of lactating dairy cows with 500 mg of bST at 10-d intervals increased yields of milk and milk components in the first 2 mo after treatment. Body condition of bST-treated cows remained unaltered, whereas control cows gained BCS. Treatment with bST increased concentrations of insulin-like growth factor 1 chronically, but concentrations of insulin and glucose increased only transiently in the first 7 d after the first injection of bST. Concentrations of progesterone during and after the Ovsynch protocol remained unaltered after treatment with bST; likewise, ovulatory responses during the Ovsynch protocol were mostly unaltered by treatment. Concentration of estradiol tended to be greater for bST cows than for control cows immediately before induction of ovulation in the Ovsynch protocol. Similarly, the mean and the peak concentrations of estradiol were greater for bST cows than for control cows when monitored during spontaneous estrus. Nevertheless, duration of estrus and the median number of standing events were less for bST cows than for control cows. Pregnancies per AI after the first and second postpartum inseminations were not affected by bST treatment. Treatment of lactating dairy cows with 500 mg of bST every 10 d improved lactation performance, but it did not affect pregnancies per AI and it reduced expression of estrus.     

Characterization of progesterone profiles in fall-calving Norwegian Red cows

Progesterone profiles in Norwegian Red cows were categorized, and associations between the occurrence of irregularities in the profiles and the commencement of luteal activity were investigated. The cows were managed in 3 feeding trials from 1994 to 2001 and from 2005 to 2008 at the Norwegian University of Life Sciences. The cows were followed from calving, and the milk samples collected represented 502 lactations from 302 cows. Milk samples for progesterone analysis were taken 3 times weekly from 1994 throughout 1998 and from 2005 to 2008 and 2 times weekly from 1999 to 2001. Commencement of luteal activity was defined as the first day of 2 consecutive measurements of progesterone concentration ≥3 ng/mL not earlier than 10 d after calving. Delayed ovulation type I was defined as consistently low progesterone concentration, <3 ng/mL for ≥50 d postpartum. Delayed ovulation type II was defined as prolonged interluteal interval with milk progesterone measurements <3 ng/mL for ≥12 d between 2 luteal phases. Persistent corpus luteum (PCL) type I was defined as delayed luteolysis with milk progesterone ≥3 ng/mL for ≥19 d during the first estrous cycle postpartum. Persistent corpus luteum type II was defined as delayed luteolysis with milk progesterone ≥3 ng/mL for ≥19 d during subsequent estrous cycles before first artificial insemination. Delayed ovulation type I was present in 14.7%, delayed ovulation type II in 2.8%, PCL type I in 6.7%, and PCL type II in 3.3% of the profiles. Commencement of luteal activity was related to milk yield, parity, PCL type I, and the summated occurrence of PCL type I and II. The least squares means for the interval to commencement of luteal activity were 24.2 d when PCL type I and II were present and 29.5 d when PCL type I and II were absent. The likelihood of pregnancy to first service was not affected in cows with a history of PCL when artificial insemination was carried out at progesterone concentrations <3 ng/mL (i.e., during estrus); however, cows that had experienced PCL were more likely to be inseminated during a luteal phase. The occurrence of delayed ovulation and PCL in Norwegian Red cows was less than that reported in most other dairy populations.     

Relationships among body condition score,milk constituents,and postpartum luteal function in Norwegian dairy cows

Relationships among body condition score (BCS), milk constituents, and resumption of postpartum luteal function were studied in 162 lactations of first- and second-parity Norwegian dairy cows. Milk components included acetone, lactose, fat, protein, urea, and ratios of fat to protein and fat to lactose. Milk progesterone concentrations were used to determine intervals from calving to first luteal response (> 5 ng/ml). Intervals to first luteal response were divided into categories of early (< or = 24 d) or late (> 24 d) responses. Higher BCS were observed during wk 4 and 5 postpartum among both first- and second-parity cows with early compared with delayed luteal responses. Second-parity cows with early onset of luteal function also had higher BCS from wk 6 through 12, whereas first-parity cows with early onset of luteal function had higher BCS from wk 13 through 15. Higher acetone levels from wk 2 through 4 postpartum were associated with late luteal response in second-parity cows. Greater milk lactose content during wk 1, 2, 3, 6, 7, and 8 postpartum and higher fat fractions during wk 4 postpartum were related to early luteal response in second-parity cows. Relationships between milk constituents and onset of luteal function were less evident and occurred later postpartum among first-parity cows than among second-parity cows. Measures of weekly milk composites obtained during the early postpartum period and BCS were closely associated with postpartum resumption of luteal function. Acetone and lactose values in milk from the first 4 wk postpartum predicted postpartum luteal function in second-parity cows at a sensitivity of 0.84 and specificity of 0.86.     

Pregnancy in dairy cows after synchronized ovulation regimens with or without presynchronization and progesterone

Two experiments examined pregnancy after synchronized ovulation (Ovsynch) with or without progesterone (P4) administered via controlled internal drug release (CIDR) intravaginal inserts. In experiment 1, 262 lactating cows in one herd were in 3 treatments: Ovsynch (n = 91), Ovsynch + CIDR (n = 91), and control (n = 80). The Ovsynch protocol included injections of GnRH 7 d before and 48 h after an injection of PGF20. Timed artificial insemination (TAI; 57 to 77 d postpartum) was 16 to 20 h after the second GnRH injection. Cows in the Ovsynch + CIDR group also received a CIDR (1.9 g of P4) insert for 7 d starting at first GnRH injection. Control cows received A-I when estrus was detected using an electronic estrus detection system. Based on serum P4, 44.1% of cows were cyclic before Ovsynch. Pregnancy rates at 29 d (59.3 vs. 36.3%) and 57 d (45.1 vs. 19.8%) after TAI and embryo survival (75.9 vs. 54.5%) from 29 to 57 d were greater for Ovsynch + CIDR than for Ovsynch alone. In experiment 2, 630 cows in 2 herds received TAI at 59 to 79 d postpartum after 6 treatments. Estrous cycles were either presynchronized (2 injections of PGF2alpha 14 d apart; n = 318) or not presynchronized (n = 312). Within those groups, Ovsynch was initiated 12 d after second presynchronization PGF2alpha, and used alone (n = 318) or with CIDR inserts for 7 d (1.38 g of P4/insert, n = 124 or 1.9 g of P4/insert, n = 188). Before Ovsynch, 80% of cows were cyclic. Presynchronization increased pregnancy (46.8 vs. 37.5%) at 29 d after TAI, but CIDR inserts had no effect on pregnancy in experiment 2. Overall embryonic survival between 29 and 57 d in experiment 2 was 57.7%. Use of CIDR inserts with Ovsynch improved conception and embryo survival in experiment 1 but not in experiment 2, in part due to differing proportions of cyclic cows at the outset. Presynchronization before Ovsynch enhanced pregnancy rate.     

Pharmacokinetics of progesterone in lactating dairy cows: Gaining some insights into the metabolism from kinetic modeling

Progesterone pharmacokinetics were analyzed for plasma hormone concentrations ranging from linear to saturated metabolism in lactating Holstein cows with differing daily milk yields. The adequacy of 2-coupled first-order (bi-exponential equation), hyperbolic (Michaelis-Menten equation), and sigmoidal (Hill equation) kinetic models to describe the experimental progesterone pharmacokinetic profiles was examined on a statistical basis. After nonlinear regression and statistical analysis of the data-fitting capability, a simple one-compartment model based on Hill equation proved to be most adequate. This model indicates an enzyme-catalyzed metabolism of progesterone involving cooperative substrate-binding sites, resulting from allosteric effects that yield a sigmoidal saturation rate curve. Kinetic parameters were estimated for 2 groups of lactating Holstein cows with different daily milk yields. We found, for the first time, a remarkable quantitative agreement of the Hill coefficient value with that reported in pharmacokinetic studies involving cytochrome P450, family 3, subfamily A (CYP3A)-mediated reactions in other mammals, humans included. It seems that positive cooperativity makes enzymes much more sensitive to plasma progesterone concentration, and their activities can undergo significant changes in a narrow range of concentration as characteristic of sigmoidal behavior. Therefore, the values of classical pharmacokinetic parameters, such as the elimination constant, half-life, and clearance rate, were found to be highly dependent on the plasma progesterone concentration.     

Estimating probability of insemination success using milk progesterone measurements

The aim of this study was to quantify the effects of progesterone profile features and other cow-level factors on insemination success to provide a real-time predictor equation of probability of insemination success. Progesterone profiles from 26 dairy herds were analyzed and the effects of profile features (progesterone slope, cycle length, and cycle height) and cow traits (milk yield, parity, insemination during the previous estrus) on likelihood of artificial insemination success were estimated. The equation was fitted on a training data set containing data from 16 herds (6,246 estrous cycles from 3,404 lactations). The equation was tested on a testing data set containing data from 10 herds (8,105 estrous cycles from 3,038 lactations). Predictors were selected to be implemented in the final equation if adding them to a base model correcting for timing of insemination and parity decreased the overall likelihood distance of the model. Selected variables (cycle length, milk yield, cycle height, and insemination during the previous estrus) were used to build the final model using a stepwise approach. Predictors were added 1 by 1 in different order, and the model that had the smallest likelihood distance was selected. The final equation included the variables timing of insemination, parity, milk yield, cycle length, cycle height, and insemination during the previous estrus, respectively. The final model was applied to the testing data set and area under the curve (AUC) was calculated. On the testing data set, the final model had an AUC of 58%. When the farm effect was taken into account, the AUC increased to 63%. This equation can be implemented on farms that monitor progesterone and can support the farmer in deciding when to inseminate a cow. This can be the first step in moving the focus away from the current paradigm associated with poorer estrus detection, where each detected estrus is automatically inseminated, to near perfect estrus detection, where the question is which estrous cycle is worth inseminating?