Ovarian responses and embryo survival in recipient lactating Holstein cows treated with equine chorionic gonadotropin

The objectives of Experiment 1 were to determine a dose of eCG that would increase total luteal volume and plasma progesterone (P4) concentration on estrous cycle Day 7 in cows. The objectives of Experiment 2 were to determine the effects of treating embryo recipient lactating Holstein cows with eCG on pregnancy per embryo transfer (P/ET). In Experiment 1, lactating dairy cows at 63 ± 3 d postpartum (DIM) received no treatment (control, n = 10), or 600 (eCG6, n = 19), or 800 (eCG8, n = 19) IU of eCG 2 d after the start of the ovulation-synchronization protocol, Day -8 (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH). Blood was sampled on Days -10, -8, -3, 0, 7, and 14 for P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, 0, and 7. In Experiment 2, lactating dairy cows were paired according to parity and previous insemination (0 or > 1 insemination) and assigned to receive 800 IU of eCG (eCG8, n = 152) 2 d after the start of the ovulation-synchronization protocol (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH) or to receive no treatment (control, n = 162). Blood was sampled on Days -10, -3, 0, 7, and 14 for determination of P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, and 7, and cows with a CL > 20 mm in diameter on Day 7 received an embryo. In Experiment 1, P4 concentration on Day 7 was higher (P < 0.05) for eCG8 cows (2.3 ± 0.3 ng/mL) compared with control (1.2 ± 0.3 ng/mL) and eCG6 (1.1 ± 0.3 ng/mL) cows. In Experiment 2, eCG8 primiparous cows had more (P < 0.01) follicles > 10 mm on Day -3 compared with control primiparous cows (2.5 ± 0.9 vs 1.7 ± 0.5 mm), but multiparous control and eCG8 cows did not differ. A larger (P = 0.03) percentage of control cows received an embryo (87.5 vs 79.1%) compared with eCG8 cows. Among cows that received an embryo, total luteal volume on Day 7 was affected (P = 0.05) by treatment (eCG8 = 8.3 ± 0.4 cm³, control = 6.2 ± 0.4 cm³), but P4 concentration on Day 7 did not differ significantly between treatments. The percentage of cows pregnant 53 d after ET (overall, 24.2%) was not significantly different between control and eCG8 cows. In the current study, no differences in P/ET were observed between control and eCG8 cows and treatment with eCG increased the percentage of cows with asynchronous estrous cycle.     

Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows

Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.     

Pharmacokinetics of eCG and induction of fertile estrus in bitches using eCG followed by hCG

The aim was to design a protocol combining eCG followed by hCG for estrus induction in the bitch. In Experiment 1, three ovariohysterectomized bitches received 10 000 IU of eCG iv, and 15 days later 10 000 IU of eCG im. Blood samples were taken up to 144 h after each injection to measure eCG concentrations. In Experiment 2, 25 healthy, intact late anestrous bitches were assigned to one of five doses of eCG (5, 10, 15, 20, 44, or 50 IU/kg eCG im; [TRT5-TRT50]). Sexual behavior (SB), clinical signs of estrus (CSE) and vaginal cytology (VC) samples were obtained and scored before eCG administration and every other day until onset of estrus, or for 14 days. In Experiment 3, intact late anestrous bitches were assigned to a treatment group (TRT; n = 16) and received eCG (50 IU/kg im) followed by hCG (500 IU im) 7 days later; or to a placebo group (PLA; n = 8) where they received 1 mL saline solution im. All bitches that were induced in estrus were mated or AI with fresh semen. In Experiment 1, maximum observed concentration (C_max) eCG were similar between im and iv routes (6.1 ± 0.9 vs. 8.6 ± 0.5 IU/mL, P > 0.08), whereas time for maximum observed concentration (T_max.) was longer for im compared to iv routes (17.5 ± 0.5 vs. 11.6 ± 0.3 h, P < 0.01). The area under the curve (AUC) was similar for im and iv routes (P > 0.48), and eCG was detectable in serum for at least 144 h for both routes. In Experiment 2, 3 days or 3 to 5 days after treatment, all bitches in TRT50 had higher scores compared to TRT5-44 animals (P < 0.01). In TRT50, the mean interval from treatment to estrus was 4.0 ± 0.4 days. In Experiment 3, the mean interval from treatment to estrus was shorter in the TRT group compared to the PLA group (4.1 ± 3.3 vs. 68.5 ± 4.4 days, P < 0.01). The previous interestrus interval was similar for TRT and PLA groups (199.6 ± 7.2 vs. 197.5 ± 10.2 days), but the new interestrus interval was shorter for the TRT compared to the PLA group (164.0 ± 7.2 vs. 212.2 ± 10.2 days; treatment by interval interaction, P < 0.007). Serum P₄ concentrations increased on the first day of cytologic diestrus after treatment in bitches in TRT (0.7 ± 0.3 vs. 22.8 ± 4.2 ng/mL; P < 0.01); but did not change in PLA (P > 0.84). Ninety-four percent of animals were bred (15/16; AI, n = 7; natural mating, n = 8), and 80% (12/15) became pregnant. None of the bitches had any side effects from the eCG and hCG therapy. We concluded that 50 IU/kg of eCG combined 7 days later with 500 IU of hCG was effective to induce normal and fertile estrus in bitches at 164 days post estrus, with an 80% pregnancy rate, with no side effects, and with a reduction of 48 days of the interestrus interval.     

Evaluation of the effectiveness of intrauterine treatment with formosulphathiazole of clinical endometritis in postpartum dairy cows

In cattle, elimination of bacterial contamination from the uterine lumen after parturition is often delayed or compromised, and pathogenic bacteria can persist, causing uterine disease and infertility. The aim of this study was to compare the clinical and bacteriologic recovery following a single intrauterine administration of formosulphatiazole, cephapirin or placebo in cows with clinical endometritis. Cows (n = 80), no less than 28 days postpartum, with clinical endometritis were enrolled in the study. Endometritis was diagnosed by a complete reproductive examination, including rectal palpation, ultrasonography, vaginoscopy and uterine swab. All cows were randomly assigned to receive one of three intrauterine treatments (T0): 2500 mg of formosulphatiazole (Group A); 500 mg of cephapirin (Group B); placebo (4250 mg of propylene glycol; Group C). Cows were examined at the first estrus after treatment or no more than 30 days after (T1). Bacteria isolated were E. coli, A. pyogenes, Pasteurella spp. and Streptococcus spp. After treatment, in Group A and B only 6/30 (20.0%) and 6/24 (25.0%) cows showed a positive bacteriologic culture (P > 0.05), while in Group C the number of positive animals was significantly higher (19/26; 73.1%; P < 0.05). At T0, total clinical scores were similar between the three groups (Group A: 5.84 ± 1.07; Group B: 5.91 ± 1.0; Group C: 5.62 ± 1.17; P > 0.05) and indicative of clinical endometritis. At T1, endometritis scores were significantly lower than those reported before uterine infusion (P < 0.05); however, Group A and B score, 0.4 ± 0.9 and 1.0 ± 2.1, respectively, correspond to no and slight endometritis, while animals in Group C reported a total endometritis score significantly higher (4.6 ± 3.5; P < 0.05) corresponding to endometritis. In the present study, a commercial formosulphatiazole preparation was as effective as cephapirin and more effective than placebo for the treatment of clinical endometritis.     

Does supplementing dairy cows with β-carotene during the dry period affect postpartum ovarian activity, progesterone, and cervical and uterine involution?

β-carotene is the main natural precursor of vitamin A and plays an important role in reproductive efficiency and immune function in dairy cows. The objective of this study was to investigate whether a supplement of β-carotene given during the dry period is able to 1) increase blood concentrations of β-carotene postpartum, 2) improve ovarian function and progesterone production, and 3) enhance uterine involution and uterine health. This study was conducted using 40 Holstein cows. On the day of drying-off, cows were allocated to one of two dietary treatments: control diet (C, n = 20) or control diet plus 1g/d β-carotene (BC, n = 20). The β-carotene supplement was given individually to the cows until calving. Blood samples were obtained regularly before and after calving from the cows to measure the concentrations of β-carotene. The diameters of the cervix and uterine horns were measured regularly using ultrasonography. Endometrial cytology samples were acquired from the cervix and uterus to determine uterine health. Milk samples were obtained three times per week for progesterone assay. Additional blood samples were taken on the day of calving, 7 and 21 days postpartum to determine the plasma concentrations of amino acids. Blood concentrations of β-carotene were not different before the start of the experiment (C, 3.03 ± 0.22 mg/L vs BC, 3.12 ± 0.22 mg/L, P > 0.05). Blood concentrations of β-carotene in the BC group peaked (7.45 ± 0.24 mg/L) 1 month after drying-off while the concentrations in the C group remained constant. β-carotene concentrations then decreased in both groups. The difference in blood concentrations of β-carotene between groups became significant 2 weeks after the start of the supplement until 2 weeks postpartum. There was no significant difference in the interval from calving to ovulation between groups (C, 27.8 ± 3.46 d vs BC, 35.8 ± 3.55 d, P > 0.05). The dietary supplement of β-carotene during the dry period had no effect on ovarian activity, progesterone production, cervix and uterine horn diameters. Plasma concentrations of hydroxyproline in the BC group were higher than in the C group on day 21 postpartum (BC, 20.8 ± 1.33 μmol/L vs C, 15.0 ± 1.33 μmol/L; P < 0.01). On day 28 postpartum the percentage of neutrophils in the BC group was lower than in the C group (cervical smear; C, 21.0 ± 3.22% vs BC, 9.7 ± 3.14%, P < 0.05 and uterine smear; C, 32.0 ± 3.86% vs BC, 20.9 ± 3.76%, P < 0.05). In the present experiment a dietary supplement of β-carotene had no effect on ovarian activity. However, due to effects of β-carotene on hydroxyproline profiles and their potential relationship with uterine function we speculate that uterine involution may have been more complete and that uterine inflammation may have been reduced in cows which received the β-carotene compared to controls.     

Effect of postpartum suppression of ovulation on uterine involution in dairy cows

The objective of this study was to investigate the effect of time of first postpartum ovulation after calving on uterine involution in dairy cows with and without uterine puerperal disease. Transvaginal follicular puncture (FP) of follicles >6 mm suppressed ovulation and development of a CL until Day 42 after calving. Fifty-three lactating Holstein Friesian cows (3.4 ± 1.2 years old, parity 2.5 ± 1.0 [median ± mean absolute deviation]) were divided into groups on the basis of the presence (UD+) or absence (UD−) of uterine disease and whether FP was carried out (FP+) or not (FP−). Uterine disease was defined as the occurrence of retained fetal membranes and/or metritis. This resulted in the following groups: UD−FP− (n = 15), UD−FP+ (n = 13), UD+FP− (n = 13), and UD+FP+ (n = 12). A general examination, vaginoscopy, transrectal palpation, and transrectal B-mode sonography of the reproductive organs were conducted on Days 8, 11, 18, and 25 and then every 10 days until Day 65 after calving. After hormonal synchronization of ovulation (cloprostenol between Days 55 and 60 postpartum and GnRH 2 days later), cows were inseminated in the next spontaneous estrus. On average, the cows ovulated on Day 21.0 ± 6.0 (UD−FP−), 50.0 ± 4.0 (UD−FP+), 16.0 ± 3.0 (UD+FP−), and 48.0 ± 2.0 (UD+FP+) postpartum. Calving-to-conception interval and first-service conception rates were not affected by FP (P > 0.05). Healthy cows with FP had smaller (P < 0.05) uterine horn and cervical diameters assessed sonographically than cows without FP. FP reduced the prevalence of purulent vaginal discharge and uterine size assessed transrectally in UD+ cows (P < 0.05). The results showed that suppression of an early ovulation by transvaginal FP improved uterine involution in cows with and without uterine disease.     

Reproductive function in beef cows calving in the spring or fall

Plasma progesterone was determined twice weekly for approximately 100 days postpartum in suckled purebred (Hereford) and crossbred (beef x dairy) cows that calved in the spring or late summer/fall season. The progestèrone profiles and occurrence of estrus were used to determine ovulation times and to monitor ovarian function. Postpartum ovulations occurred significantly earlier in crossbred than in purebred cows (38.1 ± 18.5 vs. 58.1 ± 21.8, P<0.01) and in fall compared to spring calving cows (32 ± 13.9 vs. 59.1 ± 20.3, P<0.001). Rations providing either 70 or 100% of requirements for metabolizable energy were fed from 30 days prepartum until the end of the subsequent rebreeding period. Cows receiving the 70% energy ration ovulated slightly earlier but there was no effect of ration on days to pregnancy. The minimal effect of energy ration was not unexpected in this trial since many of the cows were overconditioned during late gestation.The correlation between calendar date of calving and interval to first ovulation was significant for spring (r = ?0.38, P<0.01) but not for fall calving animals. Since cows were confined and received a balanced ration of stored feeds throughout the year, photoperiod and/or temperature rather than nutritional factors would be the probable cause of delayed ovarian activity in spring calvers.Reproductive performance was assessed during the period when samples were collected and the response for purebred (n=105) and crossbreds (n=142), respectively were: ovulated by day 60 postpartum, 57 vs. 87%; mated by day 100 without conception, 17 vs. 26%; ovulated before day 100 without detection and mating, 10 vs. 4%; anovulatory to day 100, 7 vs. 0%; pregnant by day 100, 67 vs. 70%. Similar comparisons for spring (n=133) and summer/fall (n=144) calvers, respectively, were: ovulated by 60 days postpartum, 56 vs. 96%; mated by day 100 without conception, 27 vs. 17%; ovulated by day 100 without detection and mating, 8 vs. 5%; anovulatory to day 100, 5 vs. 0%; pregnant by day 100, 60 vs. 78%. The difference with the greatest practical significance is that a higher proportion of the late summer/fall calving animals were pregnant by day 100 postpartum (P<0.01) which indicates that reproductive performance is superior in fall calving beef cows.     

Production of cloned calves by combination treatment of both donor cells and early cloned embryos with 5-aza-2/-deoxycytidine and trichostatin A

We previously reported that treatment of both donor cells and early cloned embryos with a combination of 0.01 μM 5-aza-2^/-Deoxycytidine (5-aza-dC) and 0.05 μM trichostatin A (TSA) significantly improved development of cloned bovine embryos in vitro. In the present study, we investigated the effect of this combination treatment on the in vivo development potency and postnatal survivability of cloned calves. Blastocysts (77 and 82 blastocysts derived from untreated (control) and treated groups, respectively) were individually transferred to recipient cows. Relative to the control group, the combination treatment of both donor cells and early embryos with 5-aza-dC and TSA dramatically increased the cleavage rate (49.2 vs 63.6%, P < 0.05) at 24 h of culture, and blastocyst development rate on Days 6 and 7 of culture (18.8 vs 33.9% and 27.1 vs 38.5% respectively, P < 0.05). Although pregnancy rate did not differ 40 d after transfer, it was lower in the treated than control group 90 d after transfer (7.8 vs 29.3%, P < 0.05). In the control group, there were three calves born to 77 recipients (only two survived beyond 60 d), whereas in the treated group, 17 calves were born to 82 recipients, and 11 survived beyond 60 d. In conclusion, a combination treatment of donor cells and early cloned embryos with 5-aza-dC and TSA significantly enhanced development of somatic cell cloned bovine embryos in vivo; cloning efficiency (number of surviving calves at 60 d of birth/number of recipient cows) was increased from 2.6 to 13.4%.     

Effects of resynchronization strategies for lactating Holstein cows on pattern of reinsemination, fertility, and economic outcome

The objectives were to evaluate the pattern of re-insemination, pregnancy outcomes to re-insemination in estrus and at fixed time, and economic outcomes of lactating Holstein cows submitted to three resynchronization protocols. Cows were enrolled in the Experiment at 32 ± 3 d after pre-enrollment Artificial Insemination (AI), 7 d before pregnancy diagnosis, and randomly assigned to three resynchronization protocols. All cows diagnosed not pregnant at 39 ± 3 d after pre-enrollment AI were submitted to the Cosynch72 (Day 0 GnRH, Day 7 prostaglandin F_2α, and Day 10 GnRH and fixed time AI). Cows assigned to the control treatment received no further treatment, cows assigned to the GGPG treatment received a GnRH injection on Day −7, and cows assigned to the CIDR treatment received a controlled internal drug release (CIDR) insert containing 1.38 g of progesterone from Days 0-7. Cows observed in estrus were re-inseminated on the same day. Pregnancy was diagnosed at 39 ± 3 and 67 ± 3 d after re-insemination. Costs of the resynchronization protocols were calculated for individual cows enrolled in the study and pregnancies generated were given a value of $275. The GGPG treatment resulted in the slowest (P ≤ 0.06) rate of re-insemination. Overall pregnancy per AI (P/AI) at 39 ± 3 (P = 0.50) and 67 ± 3 (P = 0.49) d after re-insemination were not affected by treatment. Although cost of the control protocol was (P < 0.01) the smallest, return per cow resynchronized was (P < 0.01) greater for GGPG and CIDR protocols. We concluded that presynchronizing the estrous cycle of cows with GnRH or treating cows with a CIDR insert during resynchronization altered the pattern of re-insemination and improved the economic return of resynchronized cows.     

Effect of early postpartum PGF2α treatment on reproductive performance in dairy cows with calving and puerperal traits

The objective of this study was to evaluate the effects of early postpartum PGF two alpha treatment on reproductive performance in dairy cows with calving and puerperal traits. A total of 363 Holstein cows (128 primiparous and 235 multiparous) were selected based on the presence of at least one of calving and puerperal traits (dystocia, retained placenta, twin, abortion, and postpartum uterine infections) and were assigned to two groups (treatment and control) irrespective of presence or absence of luteal tissue. Cows in the treatment group were treated twice with 25 mg dinoprost 8 h apart on day 20 postpartum, and for the control group saline placebo was administered. As it was speculated that the timing of a second dose would mimic the release of endogenous PGF2α from the uterus, our hypothesis was that two doses of PGF2α 8 h apart may increase the duration of elevated plasma prostaglandin F2α metabolite concentration in these cows. Recorded reproductive variables included days to first estrus, days to first AI, first service conception rate, pregnancy by 150 days in milk, service per conception, open days, and the percentage of repeat breeder animals. The data were analyzed using SPSS (Version 15) (IBM North America, New York, NY, USA) and Minitab (Version 14) (Minitab, State College, PA, USA). Although early postpartum PGF2α treatment had no effect on days to first estrus (36.7 days vs. 34.9 days, P = 0.056) and days to first AI (70.5 days vs. 72.2 days, P = 0.537), it increased first service conception rate (47.1% vs. 27.6%, P < 0.001); and this was more remarkable in primiparous cows (64.7% vs. 25%, P < 0.001). PGF2α treatment reduced the mean service per conception (1.92 vs. 2.72, P < 0.001) and the mean open days (112 days vs. 144 days, P < 0.001), and increased pregnancy by 150 days in milk (DIM) (80% vs. 66%, P = 0.004). The prevalence of repeat breeder syndrome in cows with calving and puerperal traits was reduced by PGF2α treatment (10% vs. 29.8%, P < 0.001). In conclusion, treatment of cows with calving and puerperal traits twice with a luteolytic dose of PGF2α 8 h apart on Day 20 postpartum improved reproductive performance and reduced the prevalence of repeat breeder syndrome.     

Luteal function,largest follicle,and fertility in postpartum dairy cows treated with 14dCIDR-PGF2α versus 2xPGF2α-Ovsynch for timed AI

A method for timed artificial insemination (AI) that is used for beef cows, beef heifers, and dairy heifers employs progesterone-releasing inserts, such as the controlled internal drug release (CIDR; Zoetis, New York, NY, USA) that are left in place for 14 days. The 14-day CIDR treatment is a method of presynchronization that ensures that cattle are in the late luteal phase of the estrous cycle when PGF_2α is administered before timed AI. The objective of this study was to test the effectiveness of the 14dCIDR-PGF_2α program in postpartum dairy cows by comparing it with the traditional “Presynch-Ovsynch” (2xPGF_2α-Ovsynch) program. The 14dCIDR-PGF_2α cows (n = 132) were treated with a CIDR insert on Day 0 for 14 days. At 19 days after CIDR removal (Day 33), the cows were treated with a luteolytic dose of PGF_2α, 56 hours later were treated with an ovulatory dose of GnRH (Day 35), and 16 hours later were inseminated. The 2xPGF_2α-Ovsynch cows were treated with a luteolytic dose of PGF_2α on Day 0 and again on Day 14. At 12 days after the second PGF_2α treatment (Day 26), the cows were treated with GnRH. At 7 days after GnRH, the cows were treated with PGF_2α (Day 33), then 56 hours later treated with GnRH (Day 35), and then 16 hours later were inseminated. There was no effect of treatment or treatment by parity interaction on pregnancies per AI (P/AI) when pregnancy diagnosis was performed on Day 32 (115/263; 43.7%) or Days 60 to 90 (99/263; 37.6%) after insemination. There was an effect of parity (P < 0.05) on P/AI because primiparous cows had lesser P/AI (35/98; 35.7%) than multiparous cows (80/165; 48.5%) on Day 32. Cows observed in estrus after the presynchronization step (within 5 days after CIDR removal or within 5 days after the second PGF_2α treatment) had greater P/AI than those not observed in estrus (55/103; 53.4% vs. 60/160; 37.5%; observed vs. not observed; P < 0.01; d 32 pregnancy diagnosis). When progesterone data were examined in a subset of cows (n = 208), 55.3% of cows had a “prototypical” response to treatment (i.e., the cow had an estrous cycle that was synchronized by the presynchronization treatment and then the cow responded appropriately to the subsequent PGF_2α and GnRH treatments before timed AI). Collectively, cows with a prototypical response to either treatment had 52.2% P/AI that was greater (P < 0.001) than the P/AI for cows that had a nonprototypical response (19%) (P/AI determined at 60–90 days of pregnancy). In conclusion, we did not detect a difference in P/AI when postpartum dairy cows were treated with 14dCIDR-PGF_2α or 2xPGF2α-Ovsynch before timed AI. The primary limitation to the success of either program was the failure of the cow to respond appropriately to the sequence of treatments.     

Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows II: Strategies and factors affecting fertility

In Experiments 1, 2, and 3, we evaluated the effects of temporary weaning (TW), equine chorionic gonadotropin (eCG), and follicle-stimulating hormone (FSH) treatments on results of a fixed-time artificial insemination (TAI) protocol in postpartum Bos indicus cows. In Experiment 1, treatment with 400 IU eCG or with TW for 48 h consistently improved pregnancy rates (PRs) at TAI, but, in Experiment 2, FSH treatment was less effective than eCG or TW. In Experiment 3, the inclusion of eCG treatment in cows subjected to TW did not improve PRs. We concluded that TW or 400 IU eCG should be included in the TAI protocol in postpartum Bos indicus cows to enhance fertility. In Experiment 4, we used records from heifers and cows treated with the proposed protocol during the 2006-2007 (n = 27,195) and 2007-2008 (n = 36,838) breeding seasons from multiple locations in Brazil to evaluate factors potentially affecting PRs. Overall PR at TAI was 49.6% (31,786 of 64,033). Pregnancy rate differed (P < 0.01) among farm within location (results ranging between 26.8% and 68.0%; P < 0.01), cow group within farm, by breed (Bos indicus, 48.3% [26,123 of 54,145]; Bos taurus, 61.7% [3652 of 5922]; and crossbred Bos indicus × Bos taurus, 50.7% [2011 of 3966]), category (nulliparous, 39.6% [2095 of 5290]; suckled primiparous, 45.2% [3924 of 8677]; suckled multiparous, 51.8% [24,245 of 46,767]; and nonsuckled multiparous, 46.1% [1522 of 3299]), body condition score at TAI (≤2.5, 43.0% [3409 of 7923]; 3.0, 49.6% [18,958 of 38,229]; and ≥3.5, 52.7% [9419 of 17,881]). Days postpartum at beginning of protocol did not affect PR (30 to 60 d, 47.6% [4228 of 8881]; 61 to 90 d, 51.7% [16,325 to 31,572]; and 91 to 150 d, 50.8% [7616 to 14,991]; P > 0.1). Pregnancy rate was also consistently affected (P < 0.01) by sire (results ranging from 7.2% to 77.3%) and artificial insemination technician (results ranging from 15.1% to 81.8%).     

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone,equine chorionic gonadotropin,or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n = 36) and anestrous (n = 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P = 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows.     

Comparison of progesterone-based protocols with gonadotropin-releasing hormone or estradiol benzoate for timed artificial insemination or embryo transfer in lactating dairy cows

The objective was to compare two protocols for synchronizing ovulation in lactating Holstein cows submitted to timed AI (TAI) or timed ET (TET). Within each farm (n = 8), cows (n = 883; mean ± SEM 166.24 ± 3.27 d postpartum, yielding 36.8 ± 0.34 kg of milk/d) were randomly assigned to receive either: 1) an intravaginal progesterone insert (CIDR^®) with 1.9 g of progesterone + GnRH on Day -10, CIDR^® withdrawal + PGF2α on Day -3, and 1 mg estradiol cypionate on Day -2 (treatment GP-P-E; n_TAI = 180; n_TET = 260); or 2) a CIDR^® insert + 2 mg estradiol benzoate on Day -10, PGF2α on Day -3, CIDR^® withdrawal + 1 mg estradiol cypionate on Day -2 (treatment EP-P-E; n_TAI = 174; n_TET = 269). Cows were subsequently randomly assigned to receive either TAI on Day 0 or TET on Day 7. Serum progesterone concentration on Day -3 was greater in GP-P-E than in EP-P-E (2.89 ± 0.15 vs 2.29 ± 0.15 ng/mL; P < 0.01), with no significant effect of group on serum progesterone on Day 7. Compared to cows submitted to TAI, those submitted to TET had greater pregnancy rates on Day 28 (44.0% [233/529] vs 29.7% [105/354]; P < 0.001) and on Day 60 (37.6% [199/529] vs 26.5 [94/354]; P < 0.001). However, there were no effects of treatments (GP-P-E vs EP-P-E; P > 0.10) on synchronization (87.0% [383/440] vs 85.3% [378/443]), conception (TAI: 35.3% [55/156] vs 33.8% [50/148]; TET: 50.7% [115/227] vs 51.3% [118/230]) and pregnancy rates on Days 28 (TAI: 30.5% [55/180] vs 28.7% [50/174]; TET: 44.2% [115/260] vs 43.9% [118/269]) and 60 (TAI: 27.2% [49/80] vs 25.9% [45/174]; TET: 38.8% [101/260] vs 36.4% [98/269]). In conclusion, GP-P-E increased serum progesterone concentrations on Day -3, but rates of synchronization, conception, and pregnancy were not significantly different between cows submitted to GP-P-E and EP-P-E protocols, regardless of whether they were inseminated or received an embryo.     

Inferences of body energy reserves on conception rate of suckled Zebu beef cows subjected to timed artificial insemination followed by natural mating

The influence of body condition score (BCS), rump fat thickness (RFAT), and live weight (LW), and the changes in these parameters during the interval from 165 of prepartum (i.e., 125 days of prior gestation) to 112 postpartum on first service conception and pregnancy rates were investigated in suckled Zebu (Bos indicus) beef cows (n = 266) subjected to timed artificial insemination (TAI) followed by natural mating. The aforementioned parameters were recorded at 165 ± 14 days (mean ± standard error) prepartum (concurrent with the weaning of previous calf), at parturition, and at 42 ± 7 days (at the onset of the synchronization of ovulation protocol), 82 ± 7 days (30 days after TAI), and 112 ± 7 days (60 days after TAI) postpartum. At the start of the breeding season (BS), cows were subjected to a synchronization of ovulation program for TAI. Bulls were placed with cows 10 days after TAI and remained until the end of the study (112 days postpartum). Cows with the highest BCS at parturition had an increased probability of first service conception rate at 60 days after TAI (P = 0.02) and a reduced probability of occurrence of pregnancy loss (P = 0.05). Also, cows had a greater likelihood of conceiving postpartum if they had greater RFAT and BCS at 165 ± 14 days prepartum (P = 0.01 and P = 0.03, respectively) and at parturition (P = 0.0007 and P = 0.003, respectively). Cows that had an increase in RFAT and BCS during the dry period (i.e., interval from weaning of the previous calf to parturition) also had a greater likelihood of conceiving (P = 0.03 and P = 0.06, respectively) during the BS. Among the different time points, RFAT and BCS at parturition had the largest impact on risk of conception during the BS. The LW was a poor predictor of conception during the BS (P = 0.11–0.68) except for LW at 165 ± 14 days prepartum (P = 0.01). Collectively, the findings indicated that the likelihood of conception during the BS was highest in cows that had an improvement in RFAT and BCS during the dry period. Therefore, assuring a good nutritional status in the dry period (BCS ≥ 3.0 at 165 ± 14 days prepartum and ≥3.25 at parturition) is an important aim to optimize the postpartum conception rate of suckled Zebu beef cows subjected to TAI followed by natural mating.     

Superstimulation prior to the ovum pick-up to improve in vitro embryo production in lactating and non-lactating Holstein cows

The present study evaluated the efficacy of superstimulation with p-FSH (Folltropin) before the ovum pick-up (OPU) on IVP in lactating and nonlactating Holstein donors. A total of 30 Holstein cows (15 lactating and 15 nonlactating) were blocked by lactation status to one of two groups (control or p-FSH), in a cross-over design. On a random day of the estrous cycle, all cows received an intravaginal progesterone device and 2.0 mg IM of estradiol benzoate (Day 0). Cows in the control group received no further treatment, whereas cows in the p-FSH group received a total dosage of 200 mg of p-FSH on Days 4 and 5 in four decreasing doses 12 hours apart (57, 57, 43, and 43 mg). On Day 7, the progesterone device was removed, and OPU was conducted in both groups (40 hours after the last p-FSH injection in the p-FSH–treated group). There was no difference between groups (P = 0.92) in the numbers of follicles that were aspirated per OPU session (17.2 ± 1.3 vs. 17.1 ± 1.1 in control and p-FSH-treated cows, respectively); however, p-FSH-treated cows had a higher (P < 0.001) percentage of medium-sized follicles (6–10 mm) at the time of the OPU (55.1%; 285/517) than control cows (20.8%; 107/514). Although recovery rate was lower (60.0%, 310/517 vs. 69.8%, 359/514; P = 0.002), p-FSH-treated cows had a higher blastocyst production rate (34.5%, 89/258 vs. 19.8%, 55/278; P < 0.001) and more transferable embryos per OPU session were produced in the p-FSH group (3.0 ± 0.5 vs. 1.8 ± 0.4; P = 0.02). Regardless of treatment, non-lactating cows had a higher blastocyst rate (41.9%, 106/253 vs. 13.4%, 38/283; P = 0.001) and produced more transferable embryos per OPU session (3.5 ± 0.5 vs. 1.3 ± 0.3; P = 0.003) than lactating cows. Thus, superstimulation of Holstein donors with p-FSH before OPU increased the efficiency of IVP. In addition, non-lactating donors had higher percentage of in vitro blastocyst development and produced more embryos per OPU session than lactating cows.     

Exogenous progesterone enhances ova and embryo quality following superstimulation of the first follicular wave in Nelore (Bos indicus) donors

Three experiments were conducted to evaluate the effects of exogenous progesterone on superovulatory response and ova/embryo quality in Bos indicus donors superstimulated during the first follicular wave (FFW). We hypothesized that exogenous progesterone during gonadotropin treatments would improve ova and embryo quality. In Experiment 1, 18 Nelore cows were randomly allocated to three groups: (1) FFW; (2) FFW plus a progesterone-releasing device (FFW+P4); and (3) control (E2+P4). Cows in the FFW groups were superstimulated beginning at synchronized ovulation, whereas cows in the control group were superstimulated after synchronization of follicular wave emergence with estradiol plus progesterone (E2+P4). There were no differences in mean (± SD) numbers of transferable embryos between FFW+P4 (8.0 ± 4.5) and control (6.7 ± 4.8) groups, but both were higher (P = 0.006) than the FFW group (0.2 ± 0.4). In Experiment 2, FFW and FFW+P4 were compared in 20 Nelore donors; exogenous progesterone increased the number of transferable embryos (3.9 ± 3.4 vs. 1.3 ± 4.1, P = 0.003). In Experiment 3, FFW and FFW+P4 were compared in 10 Nelore donors except that cows were slaughtered 12 h after pLH (Lutropin-V^®, Bioniche Animal Health, Belleville, ON, Canada) treatment. More mature cumulus oocyte complex (COC) (expanded cumulus cell layers) were collected in the FFW+P4 group than in the FFW group (21.8 ± 13.1 vs. 10.8±14.7; P = 0.003). In summary, superovulatory response was satisfactory when FSH (Folltropin-V^®, Bioniche Animal Health) treatment was initiated at emergence of the first follicular wave in Nelore (Bos indicus) donors, and the hypothesis that administration of exogenous progesterone during the treatment will improve oocyte and embryo quality was supported.     

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

Ovsynch-type synchronization of ovulation protocols have suboptimal synchronization rates due to reduced ovulation to the first GnRH treatment and inadequate luteolysis to the prostaglandin F_2α (PGF_2α) treatment before timed artificial insemination (TAI). Our objective was to determine whether increasing the dose of the first GnRH or the PGF_2α treatment during the Breeding-Ovsynch portion of Double-Ovsynch could improve the rates of ovulation and luteolysis and therefore increase pregnancies per artificial insemination (P/AI). In experiment 1, cows were randomly assigned to a two-by-two factorial design to receive either a low (L) or high (H) doses of GnRH (Gonadorelin; 100 vs. 200 μg) and a PGF_2α analogue (cloprostenol; 500 vs. 750 μg) resulting in the following treatments: LL (n = 263), HL (n = 277), LH (n = 270), and HH (n = 274). Transrectal ultrasonography and serum progesterone (P4) were used to assess ovulation to GnRH1, GnRH2, and luteal regression after PGF_2α during Breeding-Ovsynch in a subgroup of cows (n = 651 at each evaluation). Pregnancy status was assessed 29, 39, and 74 days after TAI. In experiment 2, cows were randomly assigned to LL (n = 220) or HH (n = 226) treatment as described for experiment 1. For experiment 1, ovulation to GnRH1 was greater (P = 0.01) for cows receiving H versus L GnRH (66.6% [217/326] vs. 57.5% [187/325]) treatment, but only for cows with elevated P4 at GnRH1. Cows that ovulated to GnRH1 had increased (P < 0.001) fertility compared with cows that did not ovulate (52.2% vs. 38.5%); however, no effect of higher dose of GnRH on fertility was detected. The greater PGF_2α dose increased luteal regression primarily in multiparous cows (P = 0.03) and tended to increase fertility (P = 0.05) only at the pregnancy diagnosis 39 days after TAI. Overall, P/AI was 47.0% at 29 days and 39.7% at 74 days after TAI; P/AI did not differ (P = 0.10) among treatments at 74 days (LL, 34.6%; HL, 40.8%; LH, 42.2%; HH, 40.9%) and was greater (P < 0.001) for primiparous cows than for multiparous cows (46.1% vs. 33.8%). For experiment 2, P/AI did not differ (P = 0.21) between H versus L treatments (44.2% [100/226] vs. 40.5% [89/220]). Thus, despite an increase in ovulatory response to GnRH1 and luteal regression to PGF_2α, there were only marginal effects of increasing dose of GnRH or PGF_2α on fertility to TAI after Double-Ovsynch.     

Association between evaluation of the reproductive tract by various diagnostic tests and restoration of ovarian cyclicity in high-producing dairy cows

The uterine condition of clinically normal postpartum Holstein-Friesian dairy cows (n = 45) was evaluated once weekly (Weeks 3 to 7) by endometrial cytology, vaginal mucus collection device (VMCD), vaginoscopy, and ultrasonography to establish a relationship with postpartum resumption of ovulatory cycles. The time of first detection of the corpus luteum (CL) by ultrasonography and plasma progesterone concentration ≥1 ng/mL was recorded. By 49 d postpartum, 78% of the cows (n = 35) had resumed ovarian function (CL group), whereas the remainder (n = 10) had no CL (NCL group). There was a positive correlation between VMCD score and presence of fluid in the uterus in cows with a CL (P < 0.01) during Week 3 postpartum but no significant correlation in cows without a CL. Percentage of polymorphonuclear leukocytes (PMN%) was higher in the NCL group (mean ± SEM, 24.6 ± 9.4%) than in the CL group (11.7 ± 2.2%) during Week 5 postpartum (P < 0.05). The PMN% (4.5 ± 6.5%) and VMCD (0.5 ± 0.5) scores during Week 5 in cows ovulating by Day 28 were lower (P < 0.01) than the PMN% (15.0 ± 14.3%) and VMCD (1.1 ± 0.9) scores in those ovulating by Day 49. In conclusion, higher PMN% at 5 wk postpartum was associated with delayed resumption of ovarian cyclicity in high-producing dairy cows.     

Enhanced histone acetylation in somatic cells induced by a histone deacetylase inhibitor improved inter-generic cloned leopard cat blastocysts

The objective was to determine whether alterations of histone acetylation status in donor cells affected inter-generic SCNT (igSCNT)-cloned embryo development. Leopard cat cells were treated with trichostatin A (TSA; a histone deacetylase inhibitor) for 48 h, and then donor cells were transferred into enucleated oocytes from domestic cats. Compared to non-treated cells, the acetylated histone 3 at lysine 9 (AcH3K9) and histone 4 at lysine 5 (AcH4K5) in the TSA group increased for up to 48 h (P < 0.05). The AcH3K9 signal ratios of igSCNT group was higher than control group 3 h after activation (P < 0.05). Treatment with TSA significantly increased total cell number of blastocysts (109.1 ± 6.9 vs. 71.8 ± 2.9, mean ± SEM), with no significant effects on rates of cleavage or blastocyst development (71.1 ± 2.8 vs. 67.6 ± 2.9 and 12.2 ± 2.6 vs. 11.0 ± 2.6, respectively). When igSCNT cloned embryos were transferred into a domestic cat oviduct and recovered after 8 d, blastocyst development rates and total cell numbers were greater in the TSA-igSCNT group (20.7 ± 3.0% and 2847.6 ± 37.2) than in the control igSCNT group (5.7 ± 2.2% and 652.1 ± 17.6, P < 0.05). Average total cell numbers of blastocysts were approximately 4.4-fold higher in the TSA-igSCNT group (2847.6 ± 37.2, n = 10) than in the control group (652.1 ± 17.6, n = 8; P < 0.05), but were ∼2.9-fold lower than in vivo cat blastocysts produced by intrauterine insemination (8203.8 ± 29.6, n = 5; P < 0.001). Enhanced histone acetylation levels of donor cells improved in vivo developmental competence and quality of inter-generic cloned embryos; however, fewer cells in blastocysts derived from igSCNT than blastocysts produced by insemination may reduce development potential following intergeneric cloning (none of the cloned embryos were maintained to term).