Influence of gonadotropin-releasing hormone and timing of insemination relative to estrus on pregnancy rates of dairy cattle at first service

The objective was to determine the influence of gonadotropin-releasing hormone on pregnancy rates of dairy cattle at first services, when both the timing of hormone injection and insemination were altered relative to the onset of estrus. Cows (n = 325) were assigned randomly to six groups making up a 2 X 2 X 2 incomplete factorial experiment; dose of GnRH (100 micrograms versus saline), timing [1 h (early) or 12 to 16 h (late) after first detected estrus] of AI, and timing of hormone injection (early versus late) were the three main effects. Cows were observed for estrus 4 times daily. Treatments and resulting pregnancy rates were: 1) hormone injection early plus AI early (35%), 2) hormone injection late plus AI early (34%), 3) saline injection early plus AI early (30%), 4) hormone injection late plus AI late (30%), 5) hormone injection early plus AI late (46%), and 6) saline injection late plus AI late (43%). Pregnancy rate in the first four groups (32%) was less than that in the latter two groups (44%). Concentrations of LH in serum were greater for cows given hormone or saline injections in early estrus than for cows injected with either hormone of saline during late estrus. Concentrations of LH in serum 2 h after GnRH were elevated above those of controls, whether GnRH was injected during early or late estrus. Neither concentrations of LH during estrus nor concentrations of progesterone 8 to 14 d after estrus explained the possible antifertility effect of GnRH given during late estrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of human chorionic gonadotropin with genotype and parity on fertility responses of lactating dairy cows

Fertility-promoting effects of treatment of lactating dairy cattle with human chorionic gonadotropin (hCG) after artificial insemination (AI) have been variable. Here, we tested whether fertility response to hCG in lactating Holstein cows interacts with genotype and parity. Primiparous (n = 538) and multiparous (n = 613) cows were treated with hCG (3,300 IU) or vehicle 5 d after AI. Pregnancy was diagnosed on d 32 and 60 after AI. A subset of cows (n = 593–701) was genotyped for 4 single nucleotide polymorphisms (SNP) previously associated with fertility. Treatment with hCG increased progesterone concentration on d 12 after AI regardless of genotype or parity. Pregnancy per AI was improved by hCG in primiparous cows but not in multiparous cows. Moreover, hCG treatment interacted with a SNP in coenzyme Q9 (COQ9) to affect fertility. Fertility of cows treated with vehicle was greatest for the AA allele, whereas fertility was lowest for the same genotype among cows treated with hCG. Pregnancy per AI was also affected by genotype for heat shock protein A1-like (HSPA1L) and progesterone receptor (PGR), but no interactions were observed with treatment. Genotype for a SNP in prostate androgen-regulated mucin-like protein 1 (PARM1) was not associated with fertility. Overall, results show that variation in response to hCG treatment on fertility depends on parity and interacts with a SNP in COQ9.     

Characteristics and retention of luteal structures, extended postinsemination cycle, progesterone, and pregnancy-specific protein B in serum after human chorionic gonadotropin treatment of dairy cows

Our objectives were to determine characteristics (size, number, and stayability) of luteal structures formed in response to human chorionic gonadotropin (hCG) administered on d 7 after timed artificial insemination (AI) and the influence of hCG on returns to estrus and pregnancy outcome. Holstein cows (n=328), milked 3 times daily, previously inseminated at first service were assigned randomly to a completely randomized design consisting of 2 treatments when at least 1 corpus luteum (CL) was detected on d 7 after AI. Treatment consisted of 1,000 IU hCG or 1 mL of saline (control) administered i.m. Blood was collected and luteal structures were mapped and sized by transrectal ultrasonography on d 7, 14, 21, 28, and 32 after AI. Blood also was collected on d 60 in all pregnant cows. Treatment with hCG induced new luteal structures in 70% of cows, regardless of pregnancy status or number of pretreatment CL. Cows producing greater than the median 46 kg of energy-corrected milk per day were less likely to respond to hCG. The number of total luteal structures per cow, original CL volume, and total luteal volume (original CL + new luteal structures) were increased by hCG. Progesterone concentration was greater in pregnant than nonpregnant cows on d 14 unless cows responded to hCG by forming new luteal structures. Concentrations of progesterone were greatest in pregnant, hCG-treated cows. Pregnancy per AI at d 32 or 60 after first AI was less in hCG- than saline-treated cows because pregnancy outcome for hCG cows that had only 1 pretreatment CL and failed to respond to hCG was only 55 to 61% of that observed in controls. Proportions of cows returning to estrus from 18 to 25 d after AI were less in hCG than control cows but greater for cows returning >25 d. Regardless of treatment, 25% of cows in both treatments retained at least 1 original CL to d 28 after AI and were not pregnant on d 32. Progesterone concentrations in these nonpregnant cows with retained CL between d 14 and 28 after AI were intermediate between nonpregnant cows that returned to estrus by d 25 and all pregnant cows. Concentrations of pregnancy-specific protein B were elevated in some of these nonpregnant, CL-retained cows, indicating early pregnancy loss. Retention of original luteal tissue in nonpregnant cows to d 28 after AI indicated that pregnancy had been initiated but failed, as verified by concentrations of progesterone and pregnancy-specific protein B.     

Effect of nerve growth factor-β administered at insemination for lactating Holstein dairy cows bred after timed-artificial insemination protocol

The objectives of this study were to determine the effects of nerve growth factor-β (NGF), purified from bulls' seminal plasma and administered at the time of artificial insemination (AI), on progesterone post-AI, interferon-stimulated genes (ISG), and pregnancy per AI (P/AI) for lactating Holstein dairy cows enrolled in a timed-AI protocol. We hypothesized that administration of NGF at the time of AI would increase plasma progesterone post-AI, upregulate relative abundance of ISG, and improve P/AI in lactating dairy cows. Holstein cows (n = 557) from a single commercial dairy farm were blocked by parity and randomly assigned to receive an intramuscular injection containing 296 µg of bovine purified NGF at the time of AI, diluted in 2 mL of phosphate-buffered saline (NGF: n = 275), or receive only the 2 mL of phosphate-buffered saline (control: n = 282). Plasma progesterone and corpus luteum size were assessed in a subset of cows (NGF: n = 32; control: n = 36) at d 7, 14, and 19 post-AI. Relative mRNA abundance of ISG (ISG15, MX1, MX2, and RTP4) was assessed in peripheral blood leukocytes on d 19 post-AI. Pregnancy diagnosis was performed at 37 and 65 d post-AI. There was an interaction effect between treatment and parity for plasma progesterone; however, plasma progesterone and ISG did not differ between treatments. There were no effects of NGF for P/AI at 37 d post-AI (NGF = 40.0% vs. control = 41.6%), 65 d post-AI (NGF = 36.0% vs. control = 38.1%), and for pregnancy loss (NGF = 8.4% vs. control = 7.7%). The current study revealed that effects to NGF in lactating Holstein cows were minor and contingent with parity for progesterone, and no improvement in ISG relative abundance and P/AI were observed.     

Bayesian estimation of sensitivity and specificity of a milk pregnancy-associated glycoprotein ELISA test for pregnancy diagnosis between 23 and 27 days after insemination in Holstein dairy cows

Pregnancy diagnosis using pregnancy-associated glycoprotein (PAG) ELISA technology in blood or milk samples is validated from 28 d after insemination in dairy cows. The objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of a commercial milk PAG-based ELISA in Holstein dairy cows between 23 and 27 d after insemination. Milk samples (n = 268) from 257 Holstein dairy cows 23 to 27 d after AI were submitted for PAG ELISA testing. Pregnancy status was confirmed by either a second milk PAG ELISA test conducted between 28 and 50 d after insemination (n = 200) or transrectal ultrasonography performed between 28 and 59 d after insemination (n = 68). A Bayesian latent class model was used to compare the paired results from the test at 23 to 27 d after AI test to the reference test. The latent class model typically used for comparing 2 or more imperfect tests was extended to include the possibility of pregnancy loss between the 23 to 27 d test and the reference test. Informative priors for the probability of pregnancy loss, and for the Se and Sp of the PAG and ultrasonography reference tests were obtained from the scientific literature. Estimated median Se and Sp of the PAG ELISA test conducted between 23 and 27 d after AI were 0.98 (95% credible interval 0.93 to 1.0) and 0.98 (0.89 to 1.0), respectively, when using a standardized corrected optical density threshold of 0.15. Although the accuracy of the test under investigation was excellent, more data will be needed to confirm the optimal diagnostic cut point for PAG in milk for early pregnancy diagnosis in this time window. The optimal timing of pregnancy diagnosis will depend on herd-specific logistics and the action to be taken to re-inseminate nonpregnant cows.     

Maintenance of pregnancy in dairy cattle after treatment with human chorionic gonadotropin or gonadotropin-releasing hormone

The objectives were to determine whether a single injection of either human chorionic gonadotropin (hCG) or GnRH would: 1) increase ancillary formation of new luteal structures, 2) increase serum concentrations of progesterone, and 3) increase pregnancy survival in dairy females treated once between 26 and 71 d of pregnancy. A total of 421 cows were enrolled between January and November 2001, with 92, 106, and 223 females (included 68 nulliparous heifers at 1 location) treated at the 3 locations. Upon diagnosis of pregnancy, females were allocated randomly to receive 100 μg of GnRH, 1,000 IU of hCG, or 2 mL of saline. Blood samples were collected at 0, 1, 2, and 4 wk after treatment, and pregnancy status was reassessed at 1, 2, and 4 wk. New luteal structures were formed in 23.8% of cattle, with hCG (50%) and GnRH (26%) being more effective than saline (7%). Treatment had no effect on the proportion of females forming 2 new luteal structures (7.6%), and 36.2% of all induced structures regressed during the 4-wk study period. Pregnancy losses were unaffected by treatment, stage of pregnancy, or number of induced luteal structures but were nearly 9-fold greater in females in which induced luteal structures regressed. No loss occurred in females having 2 new luteal structures. Pregnancy losses decreased quadratically from 30 to 42 d. Serum progesterone did not differ among treatments, but among females forming new luteal structures, progesterone was greater at 1 (7.2 ± 0.3 vs. 6.3 ± 0.2 ng/ mL) and 2 wk (7.0 ± 0.3 vs. 6.1 ± 0.2 ng/mL) after treatment. Progesterone at the first pregnancy diagnosis was predictive of imminent pregnancy loss; the lower the initial progesterone, the sooner subsequent loss was observed. The right ovary was dominant in the location of new luteal structures. Regression of new luteal structures occurred more often on the left ovary and contra-lateral to the corpus luteum of pregnancy (53.2 vs. 22%). In conclusion, treatment of dairy cattle with either GnRH or hCG failed to prevent pregnancy loss, but concentrations of progesterone were predictive of subsequent pregnancy loss.     

Time to increase in pregnancy-specific protein B following artificial insemination is a direct determinant of subsequent pregnancy loss in lactating dairy cows

Increasing progesterone (P4) during early conceptus development may be crucial for establishment of pregnancy in dairy cattle. The objective of this study was to determine if human chorionic gonadotropin (hCG) at various times after ovulation will increase serum P4 during elongation and increase the chances for, and reduce variability to, initial increase in pregnancy-specific protein B (PSPB) following artificial insemination (AI). Time to PSPB increase was defined as the first day of increase in concentrations of PSPB between d 18 and 28 after ovulation in cows with ≥12.5% increases for 3 consecutive days compared with baseline. Lactating cows (n = 368) synchronized to Double-Ovsynch (first service) or Ovsynch (second or greater service) received one of 4 treatments: no hCG (control), or 3,000 IU of hCG on d 2 (D2), 2 and 5 (D2+5), or 5 (D5) after ovulation. All cows were examined via ultrasound on d 5 and 10 postovulation to determine percentage of cows with hCG-induced accessory CL (aCL) and to quantify and measure all luteal structures. Samples for serum P4 were collected on d 0, 5, 19, and 20 postovulation. The P4 was increased in D2, D2+5, and D5 groups compared with control. The D2+5 and D5 treatments increased aCL and P4 compared with D2 and control. The D2 treatment increased P4 on d 5 after ovulation compared with control. Serum PSPB samples were collected daily from all cows on d 18 through 28 after ovulation for determination of d of PSPB increase. Pregnancy diagnoses were performed via ultrasound examination on d 35, 63, and 100 after ovulation and AI. The D5 treatment reduced percentage of cows with, and increased the time to, PSPB increase. Primiparous cows with ipsilateral aCL had reduced pregnancy loss before d 100 postovulation compared with cows with contralateral aCL. Cows that had PSPB increase >21 d postovulation had 4× greater chances of pregnancy loss compared with cows that had PSPB increase on d 20 or 21. The highest quartile of P4 on d 5, but not on d 19 and 20, was associated with reduced time to PSPB increase. Time to PSPB increase appears to be an important measurement to understand reasons for pregnancy loss in lactating dairy cows. Increasing P4 utilizing hCG after ovulation did not enhance early pregnancy or reduce pregnancy losses in lactating dairy cows.     

Evaluation of methods of resynchronization for insemination in cows of unknown pregnancy status

The objectives of this study were to evaluate the effect of 3 methods of resynchronization of estrus and ovulation for lactating dairy cows of unknown pregnancy status on conception rate and time to pregnancy. Holstein cows (n = 495) were randomly assigned to 1 of 3 treatments: 1) control (n = 167), resynchronization with a timed AI protocol upon diagnosis of nonpregnancy on d 31 after preenrollment AI (PAI); 2) CIDR-G (n = 159), use of an intravaginal progesterone insert from d 14 to 21 after AI, with AI at estrus from d 21 to 24 and initiation of a timed AI protocol on d 24 after AI in cows not reinseminated; 3) CIDR-G + ECP (n = 169), the same treatment as CIDR-G but with an injection of 1 mg of estradiol cypionate at the time of progesterone insert removal. Cows were continuously reenrolled in the same treatment until diagnosed as pregnant, which resulted in a total of 1,148 AI (495 PAI and 653 resynchronized AI; RAI). Blood was collected from 1,001 cows on d 14, 21, and 24 after each AI for analysis of progesterone, and ovaries were scanned on d 21, 24, and 31 after AI. The presence of an active corpus luteum was presumed based on progesterone ≥1 ng/mL. Pregnancy was diagnosed by ultrasonography on d 31 and 61 after AI. The presence of an active corpus luteum and the incidence of luteolysis were similar for all treatments from d 14 to 24; however, luteolysis increased in the CIDR-G + ECP treatment from d 21 to 24. Conception rates for the PAI and all AI were similar on d 31 and 61 after insemination. Conception rates at 31 and 61 d after the RAI were also similar among treatments. Overall pregnancy loss for the PAI, RAI, and all AI were similar for all treatments. The accuracy of estrous detection, based on progesterone concentration within 2 d of detection of estrus, was similar for all treatments for the RAI and averaged 95.3%. The estrus-detection rate (EDR) decreased for the CIDR-G and CIDR-G + ECP treatments from d 14 to 21, but increased from d 21 to 24 compared with control cows; however, the EDR was smaller for cows in the CIDR-G treatment during the entire resynchronization period compared with those in the CIDR-G + ECP and control groups. The reinsemination interval was reduced in cows receiving the CIDR-G + ECP treatment compared with control cows because of increased EDR after removal of the intravaginal insert; however, the interval from study enrollment to pregnancy was not different among treatments. These results indicate that the reproductive performance of dairy cows did not differ among the 3 resynchronization treatments evaluated.     

Effect of equine chorionic gonadotropin treatment during a progesterone-based timed artificial insemination program on reproductive performance in seasonal-calving lactating dairy cows

The aim of this study was to investigate the effect of progesterone (P4)-based timed artificial insemination (TAI) programs on fertility in seasonal-calving, pasture-based dairy herds. A total of 1,421 lactating dairy cows on 4 spring-calving farms were stratified based on days in milk (DIM) and parity and randomly allocated to 1 of 3 treatments: (1) control: no hormonal treatment; cows inseminated at detected estrus; (2) P4-Ovsynch: cows received a 7-d P4-releasing intravaginal device (PRID Delta; CEVA Santé Animale, Libourne, France) with 100 μg of a gonadotropin-releasing hormone (GnRH) analog (Ovarelin; CEVA Santé Animale) at PRID insertion, a 25-mg injection of PGF_2α (Enzaprost; CEVA Santé Animale) at PRID removal, GnRH at 56 h after device removal and TAI 16 h later; (3) P4-Ovsynch+eCG: the same as P4-Ovsynch, but cows received 500 IU of equine chorionic gonadotropin (eCG; Syncrostim; CEVA Santé Animale) at PRID removal. At 10 d before mating start date (MSD), all cows that were ≥35 DIM were examined by transrectal ultrasound to assess presence or absence of a corpus luteum; body condition score (BCS) was also recorded. Pregnancy diagnosis was performed by transrectal ultrasonography 30 to 35 d after insemination. Overall pregnancy/AI (P/AI) was not different between groups (50.9, 49.8, and 46.3% for control, P4-Ovsynch, and P4-Ovsynch+eCG, respectively) but the 21-d pregnancy rate was increased by the use of synchronization (35.0, 51.7, and 47.2%, respectively). Compared with the control group, synchronization significantly reduced the interval from MSD to conception (34.6, 23.0, and 26.5 d, respectively) and consequently reduced the average days open (98.0, 86.0, and 89.0 d). Across all treatment groups, DIM at the start of synchronization affected P/AI (42.3, 49.5, and 53.9% for <60, 60–80, and >80 DIM, respectively), but neither parity (46.5, 50.4, and 48.4% for parity 1, 2, and ≥3, respectively) nor BCS (44.0, 49.4, and 58.6% for ≤2.50, 2.75–3.25, and ≥3.50, respectively) affected the likelihood of P/AI. Two-way interactions between treatment and DIM, parity, or BCS were not detected. In conclusion, the use of TAI accelerated pregnancy establishment in cows in a pasture-based system by reducing days open, but eCG administration at PRID removal did not affect P/AI.     

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.     

Fertility of lactating dairy cows treated with Ovsynch after presynchronization injections of PGF2 alpha and GnRH

Synchronization of ovulation (Ovsynch) using GnRH and PGF2 alpha allows control of follicle growth, corpus luteum regression, and ovulation, but resulting pregnancy rates vary. This study examined whether presynchronization to allow initiation of Ovsynch during diestrus would improve pregnancy rates at timed artificial insemination (AI). Lactating dairy cows (n = 427), 69 to 75 d postpartum, were randomly assigned to two groups by parity. Control cows received Ovsynch (GnRH, d 0; PGF2 alpha, d 7; GnRH, d 9; timed AI 16 h after second GnRH). Treated cows received presynchronization injections of PGF2 alpha and GnRH, 10 and 7 d, respectively, before starting Ovsynch. Pregnancy diagnoses were performed 36 d after AI. Progesterone (P4) concentrations from a subset of cows (n = 84) were determined in serum samples collected on d 0, 3, and 7 of Ovsynch. Presynchronization increased the percentages of cows with > or = 1 ng/ml serum P4 compared with control cows at first injection of GnRH (d 0; 93 vs. 56%) and on d 3 (90.7 vs. 51.2%) during Ovsynch. On day of PGF2 alpha, d 7 during Ovsynch, percentages of cows with > or = 1 ng/ml serum P4 were similar (95.3%, treated vs. 82.9%, control) but more treated cows had > or = 2 ng/ml serum P4 (95.3 vs. 63.4%). However, pregnancy to timed AI was similar between treated (41.5%) and control cows (38.3%). Cows with above-average milk production had greater pregnancy rate (45.8 vs. 33.8%) compared with lower producing cows. Although presynchrony increased the proportion of cows with luteal function at onset of Ovsynch, pregnancy rate to timed AI was not improved. Cows with above-average milk production had greater fertility at timed AI than herdmates with lower milk production.     

Relationship of plasma beta-carotene and vitamin A to luteal function in postpartum cattle

The influence of plasma concentrations of beta-carotene and vitamin A on in vivo progesterone production by bovine corpora lutea after gonadotropin-releasing hormone-induced LH release was assessed in 39 postpartum dairy cows. Thirty Holsteins and nine Jerseys were given 100 micrograms gonadotropin-releasing hormone on d 12 of an estrous cycle, which began from 30 to 49 d postpartum. Concentrations of beta-carotene and vitamin A in plasma and progesterone and LH in serum were determined prior to gonadotropin-releasing hormone injection (0 h); serum progesterone and LH concentrations were also determined 1, 2, and 3 h after injection of gonadotropin-releasing hormone. Serum concentrations of progesterone and LH were increased by gonadotropin-releasing hormone. Incremental progesterone production in an analysis of covariance was influenced by breed as well as the interactions of breed with vitamin A, of season with beta-carotene, and of season with vitamin A. The regression coefficients were positive for beta-carotene and negative for vitamin A in all cases. In conclusion, luteal function in the postpartum cow appears to be related to plasma concentrations of beta-carotene and vitamin A.     

Effect of treatment with human chorionic gonadotropin 7 days after artificial insemination or at the time of embryo transfer on reproductive outcomes in nulliparous Holstein heifers

Our objective was to assess the effect of treatment with human chorionic gonadotropin (hCG) 7 d after artificial insemination (AI) or at the time of in vitro-fertilized (IVF) embryo transfer on reproductive outcomes, including progesterone (P4), interferon-tau stimulated gene 15 (ISG15), pregnancy-specific protein B (PSPB), and pregnancies per AI (P/AI) or pregnancies per embryo transfer (P/ET), in nulliparous Holstein heifers. Heifers in experiment 1 were randomly assigned to receive no treatment (control; n = 129) or 2,000 IU of hCG 7 d after AI to a detected estrus (estrus = experimental d 0; hCG; n = 132). Heifers in experiment 2 were randomly assigned to receive no treatment (control; n = 143) or 2,000 IU of hCG (hCG; n = 148) at transfer of an IVF embryo 7 d after the last GnRH treatment of a 5-d controlled internal drug release-synch protocol (last GnRH = experimental d 0). Blood samples were collected from a subgroup of heifers (experiment 1, n = 82; experiment 2, n = 104) at d 7, 11, 18, 20, 25, 28, and 32, and blood samples from heifers diagnosed pregnant were collected on d 35, 39, 46, 53, 60, and 67. Blood samples were assayed for P4 by RIA and for PSPB by ELISA, and expression of ISG15 was assessed in mRNA isolated from blood leukocytes on d 18 and 20. Data were analyzed by ANOVA and logistic regression using the MIXED and GLIMMIX procedures. In both experiments, treatment with hCG increased P4 concentrations from d 11 to 32; however, treatment did not affect P/AI or P/ET at d 32 or 67, PSPB concentrations from d 11 to 67 of pregnancy, or relative ISG15 mRNA concentrations on d 18 or 20. Heifers diagnosed not pregnant at d 32 in experiment 2 with an extended luteal phase (>20 d) and treated with hCG had greater relative ISG15 mRNA concentrations on d 20 than control heifers. Treatment with hCG did not affect pregnancy loss in experiment 1, whereas heifers treated with hCG at the time of IVF embryo transfer had fewer pregnancy losses from d 32 to 67 than control heifers. We concluded that treatment with 2,000 IU of hCG 7 d after AI or at the time of embryo transfer increased P4 concentrations without affecting P/AI or P/ET in nulliparous Holstein heifers.     

Pregnancy rates after timed AI of heifers following removal of intravaginal progesterone inserts

Reproductive performance of dairy heifers was compared for each of 2 synchronization protocols: The first group of 54 heifers was synchronized using intravaginal progesterone inserts (CIDR) plus estradiol cypionate (ECP) on d 0, PGF(2alpha) on d 7, and ECP again on d 8 (CIDR-ECP); a second group of 56 heifers was synchronized using CIDR and ECP on d 0, PGF(2alpha) on d 7, and GnRH on d 9 (CIDR-GnRH). All heifers received timed artificial insemination (TAI) at 48, 56, or 72 h after CIDR removal on d 7. Pregnancy diagnosis was conducted by ultrasonography 32 +/- 1 d post AI to confirm pregnancy and at 60 +/- 1 d post AI to determine embryo survival. Ovaries were monitored by ultrasonography daily from d 0 to 7 and twice daily from d 8 to ovulation to examine emergence of a new wave of follicles, size of the ovulatory follicle, and timing of ovulation on 15 heifers per protocol. New follicular development was detected 3.7 +/- 0.2 d after CIDR insertion. Heifers receiving CIDR-ECP had a shorter interval from CIDR removal to ovulation than heifers receiving CIDR-GnRH (63.8 +/- 3.0 vs. 71.6 +/- 2.3 h, respectively); however, ovulation occurred 39.8 +/- 3.0 h after ECP or 23.6 +/- 2.3 h after GnRH. Diameters of ovulatory follicles did not differ between treatments. Overall pregnancy rate for synchronized heifers was 60.1%, and embryo survival was 98%. Pregnancy rate for heifers synchronized with CIDR-ECP was 63.0% and similar to that in heifers synchronized with CIDR-GnRH (57.1%). Pregnancy rate was affected by time of AI for heifers synchronized using CIDR-ECP but not for those synchronized with CIDR-GnRH. Heifers in the CIDR-ECP group that were inseminated 56 h after CIDR removal had a higher pregnancy rate (81.0%) compared with heifers inseminated 48 (66.7%) or 72 h (50.0%) after CIDR removal. Either ECP or GnRH used in a CIDR-based TAI program in dairy heifers can achieve acceptable reproductive performance.     

Effect of human chorionic gonadotrophin administration 2 days after insemination on progesterone concentration and pregnancy per artificial insemination in lactating dairy cows

The aim of this study was to examine the effect of a single administration of human chorionic gonadotrophin (hCG) during the establishment of the corpus luteum (CL) on progesterone (P4) concentration and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Postpartum spring-calving lactating dairy cows (n = 800; mean ± SD days in milk and parity were 78.5 ± 16.7 and 2.3 ± 0.8, respectively) on 3 farms were enrolled on the study. All cows underwent the same fixed-time AI (FTAI) protocol involving a 7-d progesterone-releasing intravaginal device with gonadotrophin-releasing hormone (GnRH) administration at device insertion, prostaglandin at device removal followed by GnRH 56 h later, and AI 16 h after the second GnRH injection. Cows were blocked on days postpartum, body condition score, and parity and randomly assigned to receive either 3,000 IU of hCG 2 d after FTAI or no further treatment (control). Blood samples were collected on d 7 and 14 postestrus by coccygeal venipuncture on a subset of 204 cows to measure serum P4 concentration, and pregnancy was diagnosed by ultrasonography approximately 30 and 70 d after FTAI. Administration of hCG caused an increase in circulating P4 concentrations compared with the control treatment on d 7 (+22.2%) and d 14 (+25.7%). The P/AI at 30 d after FTAI was affected by treatment, farm, body condition score, and calving to service interval. Overall, administration of hCG decreased P/AI (46.3% vs. 55.1% for the control). Among cows that did not become pregnant following AI, a greater proportion of control cows exhibited a short repeat interval (≤17 d) compared with cows treated with hCG (8.6% vs. 2.8%, respectively). In addition, the percentages of cows pregnant at d 21 (59.6% vs. 52.0%) and d 42 (78.3% vs. 71.9%) were greater in control than in hCG-treated cows. The overall incidence of embryo loss was 10.7% and was not affected by treatment. There was a tendency for an interaction between treatment and CL status at synchronization protocol initiation for both P4 concentration and P/AI. In conclusion, administration of hCG 2 d after FTAI increased circulating P4 concentrations. Unexpectedly, cows treated with hCG had lower fertility; however, this negative effect on fertility was manifested primarily in cows lacking a CL at the onset of the synchronization protocol.     

Timed artificial insemination with estradiol cypionate or insemination at estrus in high-producing dairy cows

A total of 799 Holstein cows from 3 herds were randomly assigned at 37 +/- 3 d in milk (DIM) to timed artificial insemination (AI) or insemination at detected estrus. Cows were presynchronized with injections of PGF(2alpha) at 37 and 51 DIM. At 65 DIM, cows received an injection of GnRH, followed 7 d later by PGF(2alpha). Cows in the estrus-detected group were inseminated after being observed in estrus during the 7 d after the last PGF(2alpha). Cows in the timed AI group received an injection of 1 mg of estradiol cypionate (ECP) 24 h after the last PGF(2alpha). If detected in estrus or=1 ng/mL; L = <1 ng/mL), resulting in 8 combinations (LLL, LHL, LLH, LHH, HHH, HHL, HLH, and HLL). Conception rates and pregnancy rates were higher for cows in the timed AI group than in the estrus-detected group at 30, 44, and 58 d (e.g., at 58 d, pregnancy rates were 42.2% for multiparous cows or 34.4% for primiparous cows in the group receiving ECP and timed AI compared with only 20.8 or 18.8% for respective parity subgroups for the treatment group inseminated only at detected estrus). Pregnancy losses were 11.5% from 30 to 58 d and did not differ between treatments. Cyclic cows within both treatments had higher estrous responses, conception rates, and pregnancy rates. Cows that responded to presynchronization and to luteolysis (HHL) had the highest conception and pregnancy rates, followed by cows classified as LHL. Use of 1 mg of ECP to induce ovulation as part of a synchrony regimen improved reproduction at first postpartum insemination in dairy cows.     

Changes of luteinizing hormone and progesterone for dairy cows after gonadotropin-releasing hormone at first postpartum breeding

Gonadotropin-releasing hormone administered at breeding enhances fertility of dairy cows, so a study was designed to evaluate the mechanism for enhanced fertility following administration of gonadotropin-releasing hormone at first postpartum breeding. Twenty-four cows were assigned randomly to one of two treatments, 100 micrograms of gonadotropin-releasing hormone intramuscular or saline vehicle intramuscular at insemination. Blood samples for luteinizing hormone assay were taken at 2-h intervals prior to breeding and .5-h intervals for 3 h after insemination. Composite morning milk samples for progesterone assay were collected for 30 days after insemination or until next estrus. Cows given gonadotropin-releasing hormone had higher luteinizing hormone concentrations in blood serum following treatment than cows given saline, 13.2 versus 3.0 ng/ml. There was no relationship between luteinizing hormone and subsequent conception. Progesterone for cows that became pregnant was higher throughout sampling days. Mean progesterone concentrations were 4.6 versus 2.2 ng/ml in pregnant and nonpregnant cows during the first 4 days after insemination. Cows treated with gonadotropin-releasing hormone that conceived had higher progesterone than other cows, and that was evident at the first 4 days postbreeding.     

Fertility of holstein dairy heifers after synchronization of ovulation and timed AI or AI after removed tail chalk

Nonlactating Holstein dairy heifers (n=352) 13 mo of age were managed using a 42-d artificial insemination (AI) breeding period in which they received AI after removed tail chalk evaluated once daily. At AI breeding period onset (d 0), heifers were randomly assigned to receive synchronization of ovulation (100 microg of GnRH, d 0; 25 mg of PGF2alpha, d 6; 100 microg of GnRH, d 8) and timed AI (TAI; d 8) and AI after removed tail chalk for the entire AI breeding period (GPG; n=175), or AI after removed tail chalk for the entire AI breeding period (TC; n=177). As expected, 17.7% (31/175) of GPG heifers received AI after removed tail chalk before scheduled TAI. Pregnancy rate per artificial insemination (PR/AI) at approximately 30 d after first AI tended to be greater for TC (46.5%) than for GPG (38.3%) heifers. No treatment x inseminator interaction was detected; however, overall PR/AI was low for heifers in both treatments due to variation among the 3 inseminators (24.8, 30.0, and 58.0%). Pregnancy loss from approximately 30 to approximately 75 d after first AI was 10% and did not differ between treatments. Based on survival analysis, days to first AI was greater for TC than for GPG heifers, whereas days to pregnancy across the 42-d AI breeding period did not differ between treatments. Overall, 81.2% of GPG heifers receiving TAI synchronized luteal regression and ovulated within 48 h after the second GnRH injection. We conclude that this synchronization protocol can yield acceptable fertility in dairy heifers if AI to estrus is conducted between treatment with GnRH and PGF2alpha and AI efficiency is optimized.     

Double insemination and gonadotropin-releasing hormone treatment of repeat-breeding dairy cattle

Our objective was to determine if double inseminations during the same estrous period of dairy cattle eligible for their third or fourth service (repeat breeders) would improve pregnancy rates equivalent to injections of GnRH given at the time of AI. Repeat-breeding, lactating cows from six herds (five herds in the San Joaquin Valley of central California and one herd in northeast Kansas) were assigned randomly to four treatment groups when detected in estrus: 1) single AI plus no injection, 2) single AI plus 100 micrograms GnRH at AI, 3) double AI plus no injection, or 4) double AI plus 100 micrograms of GnRH at AI. Inseminations were performed according to the a.m.-p.m. rule. The second AI for the double AI treatment was given 12 to 16 h after the first AI. Injections of GnRH were given intramuscularly immediately following the single AI or the first AI of the double AI. Pregnancy rates of cows given a single AI and hormone injection were numerically higher in all six herds than those of their herdmates given only a single AI. In five of six herds, the pregnancy rates of cows given a double AI and hormone injection were numerically higher than pregnancy rates of their herdmates given only a double AI. Overall pregnancy rates for the four treatments were 1) 112/353 (32.1%), 2) 165/406 (41.6%), 3) 119/364 (33.5%), and 4) 135/359 (37.5%). Gonadotropin-releasing hormone increased pregnancy rates of repeat breeders compared with controls given only a single AI. No further benefit beyond the single AI was accrued from the double AI treatment, with or without concurrent hormone administration.     

Initiation of the breeding season in a grazing-based dairy by synchronization of ovulation

Lactating dairy cows (n = 228) in a semiseasonal, grazing-based dairy were subjected to artificial insemination (AI) to start the 23-d breeding season (d 0 to 22) followed by natural service (d 23 to 120). Cows were randomly assigned to: 1) Ovsynch (GnRH, d -10; PGF2,, d -3; GnRH, d -1; timed AI, d 0) followed by AI at estrus (tail paint removal) on d 1 to 22 (Ovsynch; n = 114); or 2) AI at estrus (tail paint removal) throughout 23 d of AI breeding (tail paint; n = 114). Days to first AI service were greater and the 23-d AI service rate was less for tail paint vs. Ovsynch cows (12.0 +/- 0.6 d vs. 0 d; and 84.2 vs. 100%, respectively). However, conception to first AI was greater for tail paint vs. Ovsynch cows (47.3 vs. 27.3%, respectively). Cows in the tail paint group received only one AI, during 23 d of AI, but 46.4% of Ovsynch cows received a second AI, with similar conception (43.1%) to that of tail paint cows at first AI (47.3%). Based on serum progesterone, incomplete luteal regression after PGF2alpha, and poor ovulatory responses to GnRH contributed to lower conception to timed AI in the Ovsynch group. Cumulative pregnancy rates for tail paint and Ovsynch cows did not differ after 23 d of AI breeding (47.3 vs. 46.3%, respectively) nor after 120 d of AI/ natural service breeding (80.5 vs. 83.3%, respectively). Lactating cows in this grazing-based dairy synchronized poorly to Ovsynch resulting in reduced conception to timed AI compared with AI after tail paint removal.