Antimicrobial prophylaxis post-amniocentesis procedures in cattle: A randomized controlled equivalence study.

Amniocentesis is a routine procedure utilized on several species including human, equine, and bovine patients. Early assessment and discovery of new genetic traits in the cattle industry are highly desirable in order to accelerate genetic gain by shortening generational intervals. One of the main concerns from this procedure is the introduction of pathogenic bacterial contamination into the amniotic cavity thereby increasing the risks of spontaneous pregnancy losses post procedure. In this randomized controlled equivalence study, we have tested the effect of antimicrobial prophylaxis on the incidence of spontaneous abortions and contrasted it to untreated individuals post amniocentesis. On the treated group (n = 67) all heifers remained pregnant whereas 1 of the untreated group (n = 65) resulted in a spontaneous abortion during the study period. The latter represents 1.54% of pregnancy losses attributed to the risk associated to the amniocentesis procedure. However, the probability of inducing spontaneous abortion from the technique itself is not different to that of the contemporaneous population (n = 694) not undergoing amniocentesis viz., 1.59%. Following a two-tailed distribution, statistical analyses showed no significant differences across treatments (Fisher's exact test P = 0.49). The current prospective study indicates that performing amniocenteses on cattle have resulted in similar spontaneous pregnancy losses comparable to those of pregnant heifers without undergoing amniocentesis and regardless of antimicrobial use. In conclusion, prophylactic antimicrobials may not be applicable within the cattle amniocentesis framework.

Optimization of a 5-day fixed-time embryo transfer (FTET) protocol in heifers I. Manipulation of circulating progesterone through reutilization of intravaginal progesterone devices during FTET.

The objectives of the present study were to: 1) compare the reproductive efficiency of embryo transfer (ET) recipients after synchronization of estrus or a 5-day synchronization of ovulation protocol for fixed time ET (FTET), and 2) determine the effect of reutilization of intravaginal P4 devices (CIDRs), up to four times, in a 5-day FTET protocol. In Experiment 1, 817 dairy heifers were assigned to one of three groups: PGF + estrus detection, 5-d FTET protocol with new (1.38 g P4) or 2nd use CIDR (previously used once for 5 d). Fresh in vitro produced embryos were transferred 7 ± 1 day after estrus (PGF + estrus) or GnRH (5-day FTET). Utilization rate (transferred/treated) was greater (P < 0.001) in heifers submitted to FTET compared to ET after estrus, however pregnancies per ET (P/ET) were not different (P > 0.10). As a result, pregnancy per treated (P/treated) recipient was greater (P < 0.05) in heifers in the 5-day FTET protocol. In Experiment 2, 40 dairy heifers without a corpus luteum (CL) were randomly allocated into one of four groups using new, 2nd use, 3rd use (previously used twice for 5 d/each), or 4th use (previously used thrice for 5 d/each) CIDRs. Circulating P4 was reduced (P < 0.01) with each reutilization. In Experiment 3, ovarian follicular dynamics were evaluated in 238 dairy heifers submitted to a 5-day protocol with either new, 2nd use, 3rd use or 4th use CIDRs at random stages of the estrous cycle. Prostaglandin F2α (PGF) was administered at CIDR removal and again 24 h later. Ovulation was induced by GnRH treatment 72 h after CIDR removal. Preovulatory follicle diameter increased (P < 0.001) progressively with increasing CIDR reutilization. Ovulation rate did not differ between treatments, however, interval from CIDR removal to ovulation decreased (P < 0.001) in heifers receiving 3rd and 4th use CIDRs compared to new or 2nd use. Finally, in Experiments 4 and 5, 1203 heifers submitted to a 5-day FTET protocol were randomly assigned to receive either a new CIDR, a 3rd use CIDR (Experiment 4) or a 4th use CIDR (Experiment 5). Despite the increase in CL volume on D5 in heifers treated with 3rd use (P = 0.03) or 4th use CIDRs (P < 0.01), there were no differences (P > 0.05) in utilization rate, P/ET, or P/treated. Thus, use of a 5-day FTET synchronization protocol improves reproductive efficiency by increasing recipient utilization, and reutilization of CIDRs up to four times in recipient dairy heifers does not compromise reproductive performance.

Hepatic mRNA expression of enzymes associated with progesterone metabolism and its impact on ovarian and endocrine responses in Nelore (Bos indicus) and Holstein (Bos taurus) heifers with differing feed intakes.

The aim of this study was to evaluate circulating progesterone concentration (P4), LH pulsatility and ovarian follicular dynamics in Nelore (B. indicus) and Holstein (B. taurus) heifers under high (HDMI) and low (LDMI) dry matter/energy intakes. In addition, the effects of dry matter/energy intake and breed on hepatic expression of six genes associated with P4 metabolism (AKR1C4, AKR1D1, CYP3A4, CYP2C19, SRD5A1, and SRD5A3) was evaluated. Heifers received an intravaginal P4 device (1 g), 2 mg of estradiol benzoate (EB) i.m. and 500 µg of PGF2α at the begging of the synchronization protocol (D0). Eight days later, the P4 device was removed and all heifers received 1 mg of EB 24h later. Regardless of dry matter/energy intake, the number of recruited follicles was greater in Nelore than in Holstein heifers. In contrast, the maximum diameter of the dominant follicle was greater in Holstein than in Nelore heifers. Circulating P4 concentrations were greater in Nelore than in Holstein from D2 to D9, and in heifers receiving LDMI than those receiving HDMI from D1 to D8 of hormonal protocol. In addition, Holstein heifers had greater LH pulsatility and area under the curve of LH peaks compared to Nelore heifers. However, no effects were observed for LH values between feed intake levels. Interestingly, Holstein heifers had higher expression of SRD5A1, AKR1C4, AKR1D1 than Nelore heifers; whereas, for Nelore heifers, only the expression of CYP3A4 was higher compared to Holstein heifers. In conclusion, there are important differences in the follicular dynamics, circulating P4 and LH pulsatility concentrations that need to be considered during synchronization protocols for Nelore and Holstein breeds. More importantly, these differences appear to be at least partially modulated by the level of feed intake and the contrasting enzyme system in the liver involved with P4 metabolism between these cattle breeds.

Inducing ovulation with oestradiol cypionate allows flexibility in the timing of insemination and removes the need for gonadotrophin-releasing hormone in timed AI protocols for dairy cows.

The effects of addition of gonadotrophin-releasing hormone (GnRH) to a progesterone plus oestradiol-based protocol and timing of insemination in Holstein cows treated for timed AI (TAI) were evaluated. Cows (n=481) received a progesterone device and 2mg oestradiol benzoate. After 8 days, the device was removed and 25mg dinoprost was administered. Cows were allocated to one of three (Study 1; n=57) or four (Study 2; n=424) groups, accordingly to ovulation inducer alone (Study 1; oestradiol cypionate (EC), GnRH or both) or ovulation inducer (EC alone or combined with GnRH) and timing of insemination (48 or 54h after device removal; Study 2). In Study 1, the diameter of the ovulatory follicle was greater for GnRH than EC. Oestrus and ovulation rates were similar regardless of ovulatory stimuli. However, time to ovulation was delayed when GnRH only was used. In Study 2, cows treated with GnRH or not had similar pregnancy per AI (P/AI) 30 days (41.5% vs 37.3%; P=0.28) and 60 days (35.9% vs 33.0%; P=0.61) after TAI. TAI 48 and 54h after device removal resulted similar P/AI at 30 days (40.3% vs 38.5%; P=0.63) and 60 days (33.8% vs 35.1%; P=0.72). Thus, adding GnRH at TAI does not improve pregnancy rates in dairy cows receiving EC. The flexibility of time to insemination enables TAI of a large number of cows using the same protocol and splitting the time of AI.

Plasma antimullerian hormone as a predictor of ovarian antral follicular population in Bos indicus (Nelore) and Bos taurus (Holstein) heifers.

In Bos taurus cattle, antimullerian hormone (AMH) has been demonstrated to have a high degree of correlation with ovarian antral follicle count and the number of healthy follicles and oocytes. To document the correlation between the plasma concentration of AMH and follicular number in Bos indicus and Bos taurus heifers, Nelore (Bos indicus, n = 16) and Holstein heifers (Bos taurus, n = 16) had their ovarian follicular waves synchronized. After synchronization, ovarian antral follicular population (AFP) was evaluated three times at 60-day (d) intervals (T-120 d, 120 days before plasma AMH determination; T-60 d, 60 days before; and T0, at the time of plasma AMH determination). The plasma AMH concentration was positively correlated with the number of ovarian follicles on the day of the follicular wave emergence in Bos indicus (Nelore) and Bos taurus (Holstein) heifers at each evaluation time (p < 0.05). The AFP was higher in Bos indicus (Nelore) than in Bos taurus (Holstein) heifers (p < 0.05). Similarly, the AMH concentration was higher in Bos indicus (Nelore) than in Bos taurus (Holstein) heifers (p < 0.0001). When heifers were classified as to present high or low AFP according to the mean of the AFP within each genetic group, high-AFP heifers presented a greater (p < 0.0001) AMH concentration than low-AFP heifers, regardless of the genetic group. In conclusion, the AFP is positively correlated with plasma AMH concentration in both Bos indicus (Nelore) and Bos taurus (Holstein) heifers. Furthermore, Bos indicus (Nelore) heifers presented both greater plasma AMH concentrations and AFP than Bos taurus (Holstein) heifers.

Resynchronization with unknown pregnancy status using progestin-based timed artificial insemination protocol in beef cattle.

Two experiments were designed to evaluate the use of resynchronization (RESYNCH) protocols using a progestin-based timed artificial insemination (TAI) protocol in beef cattle. In experiment 1, 475 cyclic Nelore heifers were resynchronized 22 days after the first TAI using two different inducers of new follicular wave emergence (estradiol benzoate [EB; n = 241] or GnRH [n = 234]) with the insertion of a norgestomet ear implant. At ear implant removal (7 days later), a pregnancy test was performed, and nonpregnant heifers received a dose of prostaglandin plus 0.5 mg of estradiol cypionate, with a timed insemination 48 hours later. The pregnancy rate after the first TAI was similar (P = 0.97) between treatments (EB [41.9%] vs. GnRH [41.5%]). However, EB-treated heifers (49.3%) had a greater (P = 0.04) pregnancy per AI (P/AI) after the resynchronization than the GnRH-treated heifers (37.2%). In experiment 2, the pregnancy loss in 664 zebu females (344 nonlactating cows and 320 cyclic heifers) between 30 and 60 days after resynchronization was evaluated. Females were randomly assigned to one of two groups (RESYNCH 22 days after the first TAI [n = 317] or submitted only to natural mating [NM; n = 347]). Females from the NM group were maintained with bulls from 15 to 30 days after the first TAI. The RESYNC-treated females were resynchronized 22 days after the first TAI using 1 mg of EB on the first day of the resynchronization, similar to experiment 1. No difference was found in P/AI (NM [57.1%] vs. RESYNC [61.5%]; P = 0.32) or pregnancy loss (NM [2.0%] vs. RESYNC [4.1%]; P = 0.21) after the first TAI. Moreover, the overall P/AI after the RESYNCH protocol was 47.5%. Thus, the administration of 1 mg of EB on day 22 after the first TAI, when the pregnancy status was undetermined, promotes a higher P/AI in the resynchronized TAI than the use of GnRH. Also, the administration of 1 mg of EB 22 days after the TAI did not affect the preestablished pregnancy.

Use of sex-sorted sperm in lactating dairy cows upon estrus detection or following timed artificial insemination.

The present study evaluated the use of sex-sorted sperm upon estrus detection (ED) or following timed artificial insemination (TAI) in lactating dairy cows. Additionally, the effect of the presence of a corpus luteum (CL) at the beginning of the TAI protocol was verified. Cows (539 crossbred Gir × Holstein and 87 Holstein) were classified according to the presence or absence of CL by ultrasonography exam. Cows with a CL were randomly assigned into one of two groups (CL-ED/AI or CL-TAI), and cows without a CL (NoCL-TAI) received TAI. Cows from the CL-ED/AI group received 500mg of cloprostenol intramuscularly and were inseminated 12h after ED in the following five days. Cows from the TAI groups (CL or NoCL) received TAI. Cows receiving CL-ED/AI had a lower (P<0.0001) service rate (45.1%, 101/224) than TAI groups (CL-TAI=94.2%, 180/191 and NoCL-TAI=97.2%, 205/211). However, cows receiving AI upon ED (CL-ED/AI=31.7%, 32/101) presented higher (P=0.03) pregnancy per AI (P/AI) than cows bred following TAI (CL-TAI=19.4%, 35/180 and NoCL-TAI=23.9%, 49/205). Despite the lower P/AI, cows receiving TAI presented greater (P=0.07) proportion of pregnant cows at the end of the reproductive program (CL-TAI=18.3%, 35/191 and NoCL-TAI=23.2%, 49/211) than those inseminated upon ED (14.3%, 32/224). There was no effect (P=0.45) of the presence of a CL at the beginning of the synchronization protocol on P/AI. Thus, the use of TAI programs, regardless of the presence of CL in the beginning of the synchronization protocol, increases the service and pregnancy rates but reduces the P/AI when compared to the use of sex-sorted sperm upon ED.

Optimizing the use of sex-sorted sperm in timed artificial insemination programs for suckled beef cows.

Three experiments were designed to evaluate methods to optimize the use of sex-sorted sperm in timed AI (TAI) programs for suckled beef cows. In all 3 experiments, suckled Bos indicus cows were synchronized using an intravaginal progesterone (P4) device during 8 d and a 2.0-mg injection of intramuscular estradiol benzoate (EB) at device insertion. The females received PG and eCG (300 IU) at P4 device removal and 1.0 mg of EB 24 h later. The cows were inseminated 60 to 64 h after P4 device withdrawal. All cows had their ovaries scanned by transrectal ultrasound at TAI to indentify and to measure the largest follicle (LF) present. In Exp. 1, a total of 853 cows had their LF classified as <9 mm or ≥9 mm at the time of TAI; these cows were then randomly assigned to 4 groups according to their LF diameter (<9 mm or ≥9 mm) and the type of sperm used (sex-sorted or non-sex-sorted). There was an interaction (P = 0.02) between the type of sperm and LF diameter beginning at TAI[non-sex-sorted ≥9 mm = 58.9%a (126/214); non-sex-sorted <9 mm = 49.5%b (106/214);sex-sorted ≥9 mm = 56.8%ab (134/236); and sex-sorted <9 mm = 31.2%c (59/189), a≠b≠c = P < 0.05]. In Exp. 2, suckled cows (n = 491) were classified immediately before TAI as having displayed estrus or not (estrus or no estrus) between P4 device removal and TAI. These cows were randomly assigned to 4 groups according to the occurrence of estrus and the type of sperm (sex-sorted or non-sex-sorted). There were effects of the occurrence of estrus (P = 0.0003) and the type of sperm (P = 0.05) on pregnancy per AI [P/AI; no estrus, non-sex-sorted = 43.6% (27/62); estrus, non-sex-sorted = 58.5%; (107/183); no estrus, sex-sorted = 33.9% (21/62), and estrus, sex-sorted = 50.0% (92/184)]; however, no interaction between the occurrence of estrus and type of sperm was observed (P = 0.87). In Exp. 3, a total of 200 suckled cows presenting LF ≥9 mm at TAI were randomly assigned to receive sex-sorted sperm deposited into the uterine body (n = 100) or into the uterine horn ipsilateral to the recorded LF (n = 100). No effect of deeper AI on P/AI was found (P = 0.57). Therefore, the LF diameter at TAI and the occurrence of estrus can be used as selection criteria to identify cows with greater odds of pregnancy to receive sex-sorted sperm in TAI programs. In addition, performing TAI with sex-sorted sperm deeper into the uterus did not alter the pregnancy results.

Timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm.

The objective was to evaluate the effects of timing of insemination and type of semen in cattle subjected to timed artificial insemination (TAI). In Experiment 1, 420 cyclic Jersey heifers were bred at either 54 or 60 h after P4-device removal, using either sex-sorted (2.1 × 10(6) sperm/straw) or non-sorted sperm (20 × 10(6) sperm/straw) from three sires (2 × 2 factorial design). There was an interaction (P = 0.06) between time of AI and type of semen on pregnancy per AI (P/AI, at 30 to 42 d after TAI); it was greater when sex-sorted sperm (P < 0.01) was used at 60 h (31.4%; 32/102) than at 54 h (16.2%; 17/105). In contrast, altering the timing of AI did not affect conception results with non-sorted sperm (54 h = 50.5%; 51/101 versus 60 h = 51.8%; 58/112; P = 0.95). There was an effect of sire (P < 0.01) on P/AI, but no interaction between sire and time of AI (P = 0.88). In Experiment 2, 389 suckled Bos indicus beef cows were enrolled in the same treatment groups used in Experiment 1. Sex-sorted sperm resulted in lower P/AI (41.8%; 82/196; P = 0.05) than non-sorted sperm (51.8%; 100/193). In addition, there was a tendency for greater P/AI (P = 0.11) when TAI was performed 60 h (50.8%; 99/195) versus 54 h (42.8%; 83/194) after removing the progestin implant. In Experiment 3, 339 suckled B. indicus cows were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ultrasonographic examinations were performed twice daily in all cows to confirm ovulation. On average, ovulation occurred 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. The P/AI was greatest (37.9%) for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly less for TAI performed between 12.1 and 24 h (19.4%) or >24 h (5.8%) before ovulation. In conclusion, sex-sorted sperm resulted in a lesser P/AI than non-sorted sperm following TAI. However, improvements in P/AI with delayed time of AI were possible (Experiments 1 and 3), and seemed achievable when breeding at 60 h following progestin implant removal, compared to the standard 54 h normally used in TAI protocols.