Short communication: Optimization of a timed artificial insemination program for reproductive management of heifers in Canadian dairy herds.

Pregnancy per AI (P/AI) following the use of 1 of 2 timed AI (TAI) protocols and 2 different intervals between TAI and resynchronization were compared in heifers that were inseminated with either conventional or sex-sorted semen. Holstein heifers (n = 317; 527 inseminations) were submitted to a 5-d Cosynch protocol with (+) or without (-) GnRH at the time of controlled internal drug release (CIDR) insertion on d 0, CIDR removal and a single PGF2α treatment on d 5, and TAI plus GnRH on d 8 (72 h later). Visual estrus detection (ED) was conducted on d 6 in the afternoon and d 7 in the morning and heifers observed in estrus were artificially inseminated on d 7 in the afternoon. Heifers were alternately assigned conventional or sex-sorted semen. Pregnancy was diagnosed by ultrasound 27 and 42 d after AI, and heifers diagnosed as nonpregnant were resynchronized, up to 3 times, starting on d 27 or 34 to provide an interbreeding interval of 35 or 42 d. Overall, TAI protocol had no effect on P/AI at 27 or 42 d after artificial insemination or on pregnancy loss, but P/AI following the first service tended to be higher in the -GnRH TAI group (66.3 vs. 56.8%). Pregnancy per AI at 27 d (61.9 vs. 55.5%) tended to differ between conventional and sex-sorted semen. Heifers artificially inseminated based on ED tended to have a greater P/AI (67.6 vs. 58.2%) and had decreased pregnancy loss (0.0 vs. 4.1%) than those submitted to TAI. A greater number of heifers in the -GnRH TAI protocol were artificially inseminated on ED than the +GnRH TAI protocol (21.5 vs. 13.7%). No difference in P/AI was observed between the 35- and 42-d interbreeding intervals; however, more heifers in the 42-d group were artificially inseminated based on ED than in the 35-d group (22.7 vs. 7.8%). A 5-d Cosynch+CIDR TAI protocol without the initial GnRH and with a single PGF2α at CIDR removal is an acceptable alternative to achieve high P/AI when either conventional or sex-sorted semen is used in Holstein heifers. Breeding heifers based on detected estrus increases labor, but has the potential to increase fertility.

Effect of vehicle and route of administration of letrozole on ovarian function in a bovine model.

The objective of this study was to determine the effects of vehicle and route of administration of letrozole on ovarian function in sexually mature beef heifers. On Day 3 (Day 0=ovulation), heifers were assigned randomly to four treatment groups and given 1mgkg(-1) letrozole intravenously (iv, n=10) or intramuscularly (im, n=10) or given a placebo iv (control iv, n=5) or im (control im, n=5). The interwave interval was longer in heifers treated with letrozole im than in im and iv controls (11.7±0.30 vs 9.5±0.50 and 10±0.43, respectively; P<0.05). Corpus luteum diameter profiles and plasma progesterone concentrations were greater (P<0.03 and P<0.05, respectively) in heifers treated with letrozole im compared with control im. Plasma oestradiol concentrations were lower in both letrozole-treated groups compared with controls (P≤0.03). Plasma LH concentrations tended to be elevated at the time of wave emergence in heifers treated with letrozole im compared with other groups (group-by-day interaction, P=0.06) and plasma FSH concentrations tended to be greater (P<0.09) in heifers treated with letrozole by either route compared with a single control group. We conclude that intramuscular administration of letrozole in oil is a feasible route and vehicle for the development of a letrozole-based treatment protocol for herd synchronisation in cattle.

Effect of duration of the growing phase of ovulatory follicles on oocyte competence in superstimulated cattle.

In the present study, we tested the hypotheses that oocyte competence is compromised by a longer duration of follicular growth and that it is not affected by FSH starvation. Cows were allocated to short FSH (n=14), FSH starvation (n=13) and long FSH (n=13) groups. The first two groups were given eight doses of FSH, whereas the third group was given 14 doses of FSH, starting from the day of wave emergence (Day 0). A progesterone-releasing device (controlled internal drug release; CIDR) was placed intravaginally at the start of the experiment in all groups. The short FSH group was given prostaglandin (PG) F2α on Day 3, whereas the two other groups received PGF2α on Day 6. In all cows, the CIDR was removed at the time of PGF treatment; porcine (p) LH was given 24h after CIDR removal and cows were inseminated 24 and 36 h later. Reproductive tracts were collected 4 days after insemination and ova and/or embryos were cultured for ≥6 days. The FSH starvation group had fewer ovulations (P=0.001), and ova and/or embryos (P<0.05). No difference in embryo quality was detected between long and short FSH groups at 7, 9 or 10 days after artificial insemination. In conclusion, oocyte competence was not altered by the duration of the follicular growth phase in superstimulated cows, whereas FSH starvation substantially reduced the ability of superstimulated follicles to ovulate.

The use of equine chorionic gonadotropin in the treatment of anestrous dairy cows in gonadotropin-releasing hormone/progesterone protocols of 6 or 7 days.

In seasonally calving, pasture-based dairy farm systems, the interval from calving to first estrus is a critical factor affecting reproductive efficiency. This study evaluated the effects of equine chorionic gonadotropin (eCG) on the reproductive response of lactating, seasonally calving dairy cows diagnosed with anovulatory anestrus by rectal palpation. Cows on 15 commercial dairy farms were selected for initial inclusion based on nonobserved estrus by 7 d before the planned start of mating. All cows were palpated rectally and evaluated for body condition score and ovary score, and were included for treatment according to the trial protocol if diagnosed with anovulatory anestrus. All cows received a standard anestrous treatment protocol consisting of insertion of a progesterone device, injection of 100 µg of GnRH at the time of device insertion, and injection of PGF(2α) at device removal (GPG/P4). Cows were randomly assigned to 1 of 2 groups (6 d or 7 d) for duration of progesterone device insertion. Within each of these groups, cows were further randomly assigned to receive either 400 IU of eCG at device removal or to remain untreated as controls, resulting in a 2×2 arrangement of treatment groups: (1) 6-d device and no eCG (n=484); (2) 6-d device and eCG (n=462); (3) 7-d device and no eCG (n=546); and (4) 7-d device and eCG (n=499). Cows were detected for estrus from the time of progesterone device removal and were inseminated; those not detected in estrus within 60 h after progesterone device removal received 100 µg of GnRH and were inseminated at 72 h. The primary outcomes considered were proportion of cows conceiving within 7 d of the beginning of breeding (7-d conception rate; 7-d CR), proportion pregnant within 28 d (28-d in calf rate; 28-d ICR), and days to conception (DTC). We found no significant differences between the 6- and 7-d insertion periods and found no 6- or 7-d insertion period × eCG treatment interactions. Inclusion of eCG into either length of GPG/P4 protocol increased 7-d CR (36.0 vs. 30.6%) and 28-d ICR (58.6 vs. 52.3%) and decreased median days to conception. The use of eCG in GPG/P4 breeding protocols will improve reproductive efficiency in seasonally calving, anestrous dairy cattle.

Estrous activity and pregnancy outcomes in Holstein heifers subjected to a progesterone based 5-d CO-Synch protocol with or without administration of initial GnRH.

The objectives of this study were to investigate whether estrous activity and its impact on pregnancy outcomes would differ in heifers subjected to a 5 d CO-Synch plus progesterone releasing intravaginal device (PRID) protocol with or without an initial GnRH treatment. Holstein Heifers (n = 308) were fitted with a collar-mounted automated activity monitoring system approximately 1 week prior to the initiation of the synchronization protocol (Day -7). Heifers were assigned randomly to a 5 d CO-Synch plus PRID protocol either with (GnRH; n = 154) or without (NGnRH; n = 154) an initial administration of 100 µg of GnRH at the time of PRID insertion (Day 0). Heifers received a single administration of 500 µg of cloprostenol (PGF) at the time of PRID removal (Day 5) and again 24 h later (Day 6). Approximately 72 h after PRID removal (Day 8), heifers were timed-inseminated (TAI) and concurrently 100 µg of GnRH was administered to those not exhibiting estrus. All inseminations were done by one of two technicians using either sex-sorted (n = 252) or conventional (n = 56) frozen-thawed semen. Transrectal ultrasonography was done on Day 0 to determine ovarian cyclicity and normalcy of the reproductive tract and 30 and 45 d post-TAI to determine and confirm pregnancy, respectively. The percentage of heifers determined to be in estrus following PRID removal was greater in the GnRH than in the NGnRH group (94 vs. 82%, respectively; P < 0.01). The mean interval from PRID removal to the onset of estrus was shorter for GnRH- than NGnRH-treated heifers (50.8 vs. 59.2 h, respectively; P < 0.01). Pregnancy per AI (P/AI) at 30 d post-TAI tended to be greater for GnRH than NGnRH heifers (68 vs. 59%, respectively; P = 0.1). However, P/AI at 45 d post-TAI (65 vs. 57%, respectively) and pregnancy loss between 30 and 45 d post-TAI (6 vs. 4.5%, respectively) did not differ. The association between the interval from PRID removal to the onset of estrus and P/AI at 30 d post-TAI was linearly negative for GnRH heifers; for every 1 h increase in the interval from PRID removal to the onset of estrus, the predicted probability of P/AI at 30 d post-TAI tended (P = 0.08) to be reduced by 2.7%. The association between the interval from PRID removal to the onset of estrus and P/AI at 30 d post-TAI was not significant for NGnRH heifers. In addition, the interval from TAI to subsequent estrus, in non-pregnant heifers, was approximately 3 d longer for the GnRH than the NGnRH group (20.7 vs. 17.5 d, respectively). In summary, the initial GnRH treatment in a 5 d CO-Synch plus PRID protocol increased estrus expression and reduced the interval from PRID removal to the onset of estrus in Holstein heifers and tended to increase P/AI at 30 d, but did not affect P/AI at 45 d post-TAI.

A high dose of initial GnRH increased ovulatory response and fertility in Holstein heifers subjected to a progesterone based 5-d CO-Synch protocol.

The objectives of this study were to investigate the effect of presynchronization or dose of GnRH on ovulatory response to initial GnRH, estrus expression and reproductive outcomes in Holstein heifers subjected to a progesterone based 5-d CO-Synch protocol. Heifers were fitted with a collar-mounted automated activity monitoring system on Day -5 and were assigned randomly to one of three groups: 1) G100 (n = 111), a 5-d CO-Synch plus PRID protocol with administration of 100 µg of GnRH at the time of PRID insertion (Day 0), 500 µg of cloprostenol (PGF) at the time of PRID removal (Day 5) and again 24 h later (Day 6); 2) G200 (n = 112), same as G100 except 200 µg GnRH was administered on Day 0; 3) P10 (n = 111), PRID + PGF on Day -5 followed by a 5 d CO-Synch with administration of 100 µg of GnRH on Day 0, PGF at the time of PRID removal (Day 5) and again 24 h later (Day 6). Approximately 72 h after PRID removal (Day 8), all heifers were timed-inseminated (TAI) and concurrently 100 µg of GnRH was administered to those not exhibiting estrus. All inseminations were done by one technician using either sex-sorted (n = 265) or conventional (n = 69) frozen-thawed semen. Transrectal ultrasonography was done to determine ovarian cyclicity and normalcy of the reproductive tract, ovarian dynamics and pregnancy. All heifers were cyclic and ovulatory response to initial GnRH was greater (P < 0.01) in G200 (51.8%) and P10 (47.7%) compared to G100 (27.9%). Estrus expression rate tended to be greater (P = 0.08) in G100 (93.7%) compared to G200 (85.7%) and intermediate in P10 (89.2%). Expression of estrus was associated positively with P/AI at 45 d post-TAI (P < 0.01; 70.2 vs. 31.4% for heifers expressing or not expressing estrus). Heifers in the G200 group had greater P/AI at 30 (P < 0.01) and 45 (P = 0.01) d post-TAI (79.5 and 75.9%, respectively) compared to those in G100 (63.1 and 60.4%) or P10 (64.0 and 62.2%) groups. Pregnancy loss did not differ among treatment groups (overall 3.9%). Results indicate that increasing the dose of the initial GnRH from 100 to 200 µg resulted in increased ovulatory response and improved P/AI in Holstein heifers subjected to a progesterone based 5-d CO-Synch protocol. Although heifers presynchronized with a combination of PRID and PGF had an increased ovulatory response to the initial GnRH, P/AI did not differ from the standard progesterone based 5-d CO-Synch protocol.

Predictors of the ovarian superstimulatory response and oocyte collection in prepubertal heifers.

The objectives were to investigate the relationships between antral follicle counts and plasma AMH and FSH at the time of follicular wave emergence in prepubertal calves, and to determine the effects of age and duration of gonadotropin treatment on the ovarian superstimulatory response in pre- and post-pubertal heifers. Hereford crossbred prepubertal (Replicate 1 and 2, n = 20) and post-pubertal heifers (Replicates 1, n = 8; Replicate 2, n = 8) were assigned randomly to 2 treatment groups and given FSH for either 4 or 7 d (25 mg pFSH im at 12-h intervals). Prepubertal heifers were first treated at 4 mo and again at 7 mo of age. Blood samples were collected immediately before the first FSH administration, that was initiated 36 h after follicular ablation. An LH treatment (12.5 mg im) was given 12 h after the last FSH injection. Follicular fluid and cumulus-oocyte complexes (COC) were collected 24 h after LH treatment. At wave emergence, the number of follicles ≥1 mm (AFC, 31.1 ± 4.0 vs 16.2 ± 1.8; P < 0.001) and the plasma concentrations of AMH (606.4 ± 90.5 vs 279.6 ± 28.3 pg/mL; P = 0.001) were higher at 4 than at 7 mo of age, while plasma FSH concentrations did not differ between ages. At oocyte collection, a higher number of follicles ≥6 mm were observed in prepubertal calves at 4 mo of age and post-pubertal heifers than in calves at 7 mo of age (32.4 ± 5.4 and 22.0 ± 2.3 vs 14.9 ± 2.0, respectively; P = 0.003). Intrafollicular concentrations of estradiol were lower (23.7 ± 4.5 vs 144.0 ± 29.5 ng/mL; P < 0.0001) and of progesterone tended to be higher (217.5 ± 29.3 vs 157.0 ± 33.9 ng/mL; P = 0.07) in the 7- than in the 4-d groups. A greater number of COC was collected from calves at 4 mo of age and heifers than the 7-mo-old calves (13.4 ± 2.6 and 6.0 ± 1.0 vs 5.8 ± 1.1, respectively; P = 0.008). Overall, the 7-d FSH treatment tended to result in a greater proportion of expanded COC than the 4-d treatment in calves (50.1 ± 7.7 vs 31.9 ± 6.8%; P = 0.07). In summary, there was a positive relationship between AFC and plasma AMH concentrations at the time of wave emergence. A higher AFC was observed in calves at 4- than 7-mo of age, which resulted in greater ovarian response to gonadotropin treatment. Following an exogenous LH stimulus, COC maturation rates were greater in the 7-d than in the 4-d FSH treatment groups, resulting in collection of a higher proportion of fully expanded COC.

Treatments for the synchronisation of bovine recipients for fixed-time embryo transfer and improvement of pregnancy rates.

Although embryo transfer technology has been used commercially in cattle for many years, the inefficiency of oestrus detection, especially in recipients, has limited the widespread application of this technology. The most useful alternative to increase the number of recipients utilised in an embryo transfer program is the use of protocols that allow for embryo transfer without the need for oestrus detection, usually called fixed-time embryo transfer (FTET). Most current FTET protocols are based on progestin-releasing devices combined with oestradiol or GnRH, which control and synchronise follicular wave dynamics and ovulation. Conception rates to a single FTET have been reported to be similar to those after detection of oestrus, but pregnancy rates are higher because these treatments have increased the proportion of recipients that receive an embryo. Recent changes to treatments for FTET, such as the administration of eCG, have resulted in increased pregnancy rates and provide opportunities to make these treatments easier to perform on farm.

The effect of altering the timing of GnRH administration and artificial insemination in a modified 5-d CO-Synch protocol using sex-sorted semen in dairy heifers.

The objective was to optimize fertility in a modified 5-d CO-Synch protocol by altering the timing of GnRH administration and AI. Holstein heifers (14-16 mo) received a controlled internal drug releasing device (CIDR) on d 0 and on d 5, CIDR were removed, prostaglandin F2α was administered and estrus detection patches were applied. Estrus was detected at 36, 48, 56 and 72 h after CIDR removal. In Experiment 1, control heifers (n = 195) received GnRH concurrent with timed-AI (TAI) 72 h after CIDR removal, regardless of expression of estrus. Treatment heifers expressing estrus at 36 or 48 h were AI at 56 h (n = 101) and the remaining heifers were randomly assigned to one of two groups: GnRH administration at 56 h and TAI at 72 h (GnRH56, n = 147) or GnRH administered concurrently with TAI at 72 h (GnRH72, n = 148). In Experiment 2, heifers expressing estrus at 36 or 48 h following CIDR removal were AI at 56 h (n = 118) and the remaining heifers were either TAI at 72 h (TAI72, n = 102) or 80 h (TAI80, n = 102), with only heifers not displaying estrus by TAI given GnRH concurrent with AI. All heifers were inseminated with sex-sorted semen and pregnancy diagnosis was performed at 28 d following TAI. In Experiment 1, estrus rate (P = 0.81) and pregnancy per AI (P/AI; P = 0.34) did not differ among control, GnRH56 and GnRH72 groups, so GnRH56 and GnRH72 groups were combined into one split-TAI (STAI) group. The P/AI was greater in heifers displaying estrus by 48 h compared to those not displaying estrus and receiving GnRH in both groups (69.5 vs. 31.3%; P < 0.01) and there was a tendency for P/AI to increase in STAI heifers displaying estrus compared to control heifers displaying estrus (73.3 vs. 62.1%; P = 0.10). In Experiment 2, the estrus rate was increased in TAI80 compared to TAI72 heifers (81.4 vs. 62.7%, P < 0.01); however, there was no difference in P/AI (P = 0.74). Heifers displaying estrus in the TAI72 group had a greater P/AI than heifers not displaying estrus in either group (P < 0.01) and tended to have a greater P/AI than heifers in estrus in the TAI80 group (87.5 vs. 69.9%, P = 0.07). Across both experiments, P/AI was increased in heifers that displayed estrus before AI compared with heifers that did not and performing a STAI tended to increase P/AI in heifers that displayed estrus before AI. Other attempts made to optimize P/AI in the modified 5-d CO-Synch protocol by altering the timing of GnRH administration and/or AI were unsuccessful.

Comparison of the effects of two shortened timed-AI protocols on pregnancy per AI in beef cattle.

The objective was to compare pregnancy per AI (P/AI) between two shortened timed-AI (TAI) protocols in beef cattle. This study also determined whether administration of eCG in heifers and timing of AI in cows would affect P/AI. Cattle were submitted at random to either a modified 5-d Co-synch protocol (Day 0 = progesterone releasing device (CIDR); Day 5 = CIDR removal and 500 µg of cloprostenol (PGF); Day 8 = 100 µg GnRH concurrent with AI) or J-synch protocol (Day 0 = CIDR insertion and 2 mg of estradiol benzoate i.m.; Day 6 = CIDR removal and 500 µg PGF; Day 9 = 100 µg GnRH concurrent with AI). In Experiment 1, 1135 heifers (13-15 mo of age) received an estrus detection patch (Estrotect™) on Day 5 and 579 were selected at random to receive 300 IU of equine chorionic gonadotropin (eCG) at the time of CIDR removal. Patches were scored from 0 to 3 based on color change between initial application and AI; 0 = unchanged, 1 = ≤ 50% change, 2 = > 50% change, 3 = missing. Estrus was defined to have occurred when the patch was scored 2 or 3. In Experiment 2, 399 cyclic, non-lactating beef cows from 1 location were submitted to either the modified 5-d Co-synch or J-synch protocol and within each protocol cows were TAI at either 66 ±â€¯1 (n = 199) or 72 ±â€¯1 h (n = 200) following CIDR removal. Transrectal ultrasonography was used in both experiments to determine presence of a corpus luteum (CL) on Day 0, and to diagnose pregnancy 35 d after TAI. In Experiment 1, eCG increased estrus rate only in heifers without a CL on day 0 that were submitted to the modified 5-d Co-synch protocol (41.9 vs. 69.6%). Heifers submitted to the J-synch protocol had greater (P = 0.03) P/AI compared with those in the modified 5-d Co-synch (48.7 vs. 41.1%) and heifers that expressed estrus before AI had increased (P < 0.0001) P/AI compared to those that did not (53.6 vs. 36.5%). Administration of eCG and presence of a CL tended to affect P/AI (P = 0.13). In Experiment 2, cows submitted to the J-synch protocol tended (P = 0.07) to have greater P/AI compared to those in the modified 5-d Co-synch (74.1 vs. 66.5%). There was no association between P/AI and timing of AI. In summary, the J-synch protocol resulted in greater P/AI than the modified 5-day Co-synch protocol in heifers and cows. Administration of eCG increased estrus rate in heifers without a CL at the start of the protocol and tended to improve P/AI in all heifers. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows subjected to either TAI protocol.

Effects of progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI in beef cattle.

Three experiments were conducted to determine the effects of low-dose progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI (TAI) in beef cattle (GnRH on Day 0, PGF(2alpha) on Day 7, with GnRH and TAI on Day 9, 54-56 h after PGF(2alpha)). Experiments 1 and 2 were 2 x 2 factorials with presynchronization (with or without a once-used CIDR; Days -15 to 0 in Experiment 1 and Days -7 to 0, with PGF(2alpha) at insertion, in Experiment 2), and with or without 400 IU eCG on Day 7 in suckled cows. In Experiment 3, suckled cows and nulliparous heifers were either presynchronized with a twice-used CIDR (Days -5 to 0) and PGF(2alpha) at insertion, or no treatment prior to insertion of a new CIDR (Days 0-7). Presynchronization increased (P<0.05) ovulation rate to GnRH on Day 0 (75.0% vs 48.7%, 76.7% vs 55.0%, and 60.0% vs 36.1% for Experiments 1, 2, and 3, respectively), increased the diameter of the preovulatory follicle in Experiments 1 and 2, and increased the response to PGF(2alpha) (regardless of parity) in Experiment 1 (P<0.01), and in primiparous cows in Experiment 2 (P<0.01). Effects of presynchronization on pregnancy rates (53.4% vs 54.1%, 57.7% vs 45.3%, and 54.3% vs 44.4% for Experiments 1, 2, and 3, respectively) were influenced by parity and eCG (P<0.05). Treatment with eCG had no effect (P>0.05) on the diameter of the preovulatory follicle (Experiment 1), or the response to PGF(2alpha) (Experiments 1 and 2), but tended (P=0.08) to improve pregnancy rates, especially in primiparous cows that were not presynchronized (P<0.01). However, the effects of eCG and presynchronization were not additive.

Follicle deviation and ovulatory capacity in Bos indicus heifers.

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.

Effect of initial GnRH and time of insemination on reproductive performance in cyclic and acyclic beef heifers subjected to a 5-d Co-synch plus progesterone protocol.

This study evaluated the effect of initial GnRH and timing of AI in a 5-d Co-synch plus CIDR (device containing 1.38 g of progesterone) protocol on pregnancy per AI (P/AI) and pregnancy loss in beef heifers. A secondary objective was to determine if the effect of initial GnRH on reproductive performance was influenced by cyclicity. Crossbred beef heifers (n = 1068; 301-514 kg of body weight, and 13-15 mo of age) at three locations were assigned to either a 5-d Co-synch plus CIDR protocol with (CIDR5G) or without (CIDR5NG) an initial injection of 100 µg of GnRH at CIDR insertion (Day 0). All heifers received a single dose of 500 µg of cloprostenol at CIDR removal (Day 5) and were divided into two groups to receive GnRH and TAI at either 66 or 72 h (Day 8) after CIDR removal. All heifers were inseminated by one technician with frozen-thawed semen from 1 of 4 sires available commercially. Transrectal ultrasonography was performed on Day 0 to determine cyclicity (presence of CL) and normalcy of the reproductive track, and 27 d after TAI to determine pregnancy status. Non-pregnant heifers (n = 470) were assigned to either a CIDR5G or a CIDR5NG protocol with TAI at 72 h after CIDR removal. Twelve days after second AI, heifers were exposure to bulls for 20 d and pregnancy diagnoses were performed approximately 30 d after second TAI and 60 d after bulls were removed to diagnose bull pregnancies and determine pregnancy loss rate. The percentage of acyclic heifers was 20.3%. Overall P/AI after first TAI was 55.6% (594/1068) and did not differ between CIDR5G and CIDR5NG (56.1 vs. 55.1%), or between TAI66 and TAI72 (55.8 vs. 55.4%). However, cyclic heifers were more likely to become pregnant than acyclic ones (59.3 vs. 41.2%; P < 0.01). Moreover, acyclic heifers subjected to the CIDR5NG had fewer P/AI than those subjected to CIDR5G (P < 0.01). Overall P/AI after resynchronization was 55.1% and did not differ between CIDR5G and CIDR5NG (51.3 vs. 59.0%). Overall pregnancy loss after first and second TAI were 3.0% (18/594) and 3.9% (8/205), respectively. When pregnancy loss data were combined, synchronization protocol (4.1 vs. 2.3% for CIDR5NG and CIDR5G; P = 0.01), cyclicity (5.8 vs. 2.9% for acyclic and cyclic; P = 0.03) and the interaction between synchronization protocol and cyclicity (P = 0.04) were significant. The overall cumulative pregnancy at the end of the breeding season was 94.2% (1006/1068); acyclic heifers were less likely to be pregnant at the end of the breeding season (88.4 vs. 95.8%; P < 0.01). In summary, the initial GnRH administration in a 5-d Co-synch plus CIDR protocol that includes a single PGF treatment is necessary in acyclic beef heifers to optimize P/AI, but not in cyclic heifers. Moreover, omission of initial GnRH was associated to greater pregnancy losses, particularly in acyclic heifers. Timing of AI did not affect P/AI.

Evaluation of a modified GnRH-based timed-AI protocol associated with estrus detection in beef heifers inseminated with sex-selected or conventional semen.

The main objective was to compare pregnancy per AI (P/AI) between sex-selected and conventional semen in cyclic beef heifers subjected to a 5-day Co-synch plus CIDR protocol and evaluated the usefulness of an estrus detection (ED) aid to identify heifers that were most likely to conceive. This study also determined if the expression of estrus before timed-AI (TAI) would be associated with increased P/AI in acyclic heifers inseminated with conventional semen. Heifers (n = 1690; 320-523 kg of body weight, and 13-15 mo of age) at three locations over 2 years were scanned by ultrasonography to determine cyclicity (presence of luteal tissue) and reproductive tract normalcy. Cyclic heifers (n = 1331) received a progesterone releasing device (CIDR) on Day 0, CIDR removal and 500 µg of cloprostenol (PGF) on Day 5, and 100 µg of GnRH along with TAI on Day 8. Acyclic heifers (n = 275) received the same treatment with the addition of GnRH on Day 0. On Day 5, all heifers received ED patches (Estrotect™) that were scored from 0 to 3, based on color change between initial application and Day 8; 0 = unchanged, 1 = ≤ 50% color change, 2 = > 50% color change, 3 = missing. Estrus was defined to have occurred when an ED patch was scored 2 or 3. Cyclic heifers were inseminated with either frozen-thawed sex-selected or conventional semen from either of three sires available commercially (two per year). Acyclic heifers were inseminated with conventional semen. Pregnancy diagnosis was performed by transrectal ultrasonography 28 or 48 d after TAI, depending on management. The percentage of cyclic heifers was 83.9% and the average estrus response was 63.8%. P/AI was greater (P < 0.01) in cyclic compared to acyclic heifers (53.3 vs. 36.0%) and tended to be greater (P = 0.07) for conventional semen (52.3 vs. 47.6%), despite all acyclic heifers being inseminated with conventional semen. Heifers with an ED patch scored 2 (61.1%) or 3 (58.6%) had greater (P < 0.01) P/AI than those scored 0 (31.8%) or 1 (33.1%), regardless of semen type. Pregnancy per AI was greater (P < 0.01) for heifers detected in estrus (60.6 vs. 32.3%). In cyclic heifers that did not exhibit estrus, P/AI was lower (P < 0.01) in those inseminated with sex-selected semen (27.8 vs. 45.9%), while in heifers that exhibited estrus, P/AI only tended to be lower (P = 0.08; 56.7 vs. 65.5%). In summary, P/AI was greater in cyclic heifers, in those inseminated with conventional semen and in those exhibiting estrus before TAI. The ED patches were considered useful to identify animals for TAI with sex-selected semen and could be used to increase the adoption of this technology in beef herds.

Effects of estradiol on gonadotrophin release, estrus and ovulation in CIDR-treated beef cattle.

The effects of estradiol-17beta (E-17beta) or estradiol benzoate (EB) on gonadotrophin release, estrus and ovulation in beef cattle were evaluated in two experiments. In experiment 1, 16 ovariectomized cows received a previously used CIDR insert from days 0 to 7 and 1mg of EB on day 8; they also received 5mg of E-17beta on days 0 or 1, or 5mg of E-17beta+100mg of progesterone on day 0. There was only an effect of time (P<0.0001) on plasma concentrations of progesterone, estradiol, FSH, and LH. Following treatment with E-17beta, plasma FSH concentrations were suppressed for approximately 36 h, whereas plasma LH concentrations were reduced (P<0.05) for 6 h, but surged within 24 h. Injecting 1mg of EB 24 h after CIDR removal decreased (P<0.02) plasma LH concentrations for 6h, followed by an LH surge at 18 h. In experiment 2, ovary-intact heifers (n=40) received a used CIDR and 5mg of E-17beta+100mg of progesterone on day 0. On day 7, CIDR were removed, PGF given, and heifers received nothing (control) or 1mg of EB 12, 24, or 36 h later. In these groups, plasma LH peaked (mean+/-SEM) 78.0+/-23.0, 37.8+/-8.5, 44.4+/-10.3, and 51.0+/-5.1 h after CIDR removal (means, P<0.001; variances, P<0.001) and intervals from CIDR removal to ovulation were 102.0+/-6.7, 63.6+/-3.6, 81.6+/-3.5, and 78.0+/-4.1h (P<0.05). The interval from CIDR removal to ovulation was shorter and less variable in EB-treated groups; the interval from EB to ovulation was shortest (P<0.05) in the 12-h group. In summary, E-17beta or EB decreased both FSH and LH, but LH increased after 6h (despite elevated progesterone concentrations). Following CIDR removal, 1mg of EB effectively synchronized LH release, and ovulation (in intact cattle), but the interval from CIDR removal to EB treatment affected the time of ovulation.

Relationship between circulating progesterone at timed-AI and fertility in dairy cows subjected to GnRH-based protocols.

The objectives of this retrospective study were: 1) to investigate the effect of plasma progesterone (P4) concentrations at the time of timed-AI (TAI) on fertility, and 2) to examine risk factors associated with plasma P4 concentrations that impair fertility in lactating dairy cows subjected to GnRH-based protocols. Data from 872 lactating Holstein cows that had, or had not been presynchronized prior to a 7-day GnRH-based TAI protocol were examined. However, data from only those cows (n = 697; 79.9%) that ovulated after second GnRH were analyzed. Plasma P4 concentrations were determined using a solid-phase radioimmunoassay. Transrectal ultrasonography was used to determine cyclicity at first GnRH treatment, ovulation after first and second GnRH treatments, and pregnancy status at 32 and 60 d after TAI. Parity, days in milk (DIM) and BCS were also recorded. Plasma P4 concentrations at TAI ranged from 0.0 to 9.94 ng/mL (overall mean ± SEM, 0.32 ± 0.02 ng/mL) and 41 (5.9%) cows had P4 ≥ 1.0 ng/mL at TAI. The percentage of cows diagnosed pregnant at 32 and 60 d after TAI was 45.1 and 41.6%, respectively, and pregnancy loss from 32 to 60 d after TAI was 7.6%. Plasma P4 concentrations at TAI affected (P < 0.01) P/AI at 32 and 60 d, but did not affect (P > 0.1) pregnancy loss. No cows with plasma P4 concentrations >0.80 ng/mL became pregnant. However, ROC curve analysis revealed that the optimal P4 threshold at TAI for P/AI at 32 d was ≤0.50 ng/mL, with a sensitivity and specificity of 94.9 and 20.9, respectively. The percentage of cows with plasma P4 concentrations >0.50 ng/mL was 15.8% (110/697). Furthermore, a significant (P < 0.05) quadratic relationship between plasma P4 concentrations at TAI and P/AI at 32 d was observed. The maximum predicted probability of pregnancy was 0.54 at a P4 concentration of 0.26 ng/mL. Based on the odds ratios (OR), cows with P4 ≤ 6.2 ng/mL at PGF were 2.3 times less likely to have P4 > 0.50 ng/mL at TAI compared to cows with P4 > 6.2 ng/mL (OR = 0.44; 95% CI = 0.27 to 0.71; P < 0.01). Also, cyclic cows subjected to presynchronization were less likely to undergo luteal regression than non-presynchronized or acyclic cows (OR = 5.3; 95% CI = 1.77 to 16.27; P < 0.01). In summary, plasma P4 concentrations >0.50 ng/mL at TAI resulted in significantly reduced fertility. As elevated plasma P4 concentrations at TAI were more frequent in cows with lower P4 at PGF or those subjected to presynchronization, both groups are most likely to benefit from an additional PGF treatment prior to TAI.

Resynchronization of previously timed-inseminated beef heifers with progestins.

The objective was to determine the efficacy of a previously used CIDR or melengestrol acetate (MGA; 0.5mg/head/day) for resynchronization of estrus in beef heifers not pregnant to timed-AI (TAI). In three experiments and a field trial, heifers were reinseminated 6-12 h after first detection of estrus. Pregnancy diagnosis was done from approximately 25-43 days after either TAI or reinsemination. In Experiment 1, 79 heifers received a once-used CIDR from 13 to 20 days after TAI and 80 heifers were untreated controls. For these two groups, there were 34 and 35 heifers, respectively, not pregnant to TAI; median +/- S.E. intervals from TAI to onset of estrus were 22 +/- 0.2 days versus 20 +/- 0.6 days (P < 0.001); estrus rates were 70.6% versus 85.7% (P = 0.1); conception rates were 62.5% versus 76.7% (P < 0.3); and pregnancy rates were 44.1% versus 65.7% (P = 0.07), for CIDR and untreated (control) groups, respectively. In Experiment 2, heifers (n = 651) were TAI (Day 0) and 13 days later randomly assigned to one of seven groups (n = 93 per group) to receive a once-used CIDR (three groups; Days 13-20), MGA (three groups; Days 13-19), or no treatment (control group). Groups given a CIDR or MGA also received: no further treatment (CIDR or MGA alone); 1.5mg estradiol-17beta (E-17beta) and 50 mg progesterone (P4) in 2 mL canola oil on Day 13; or E-17beta and P4 on Day 13 and 0.5 mg E-17beta on Day 21 (24 h after CIDR removal or 48 h after the last feeding of MGA). Pregnancy rate to TAI was lowest (P < 0.05) for the group given a CIDR plus E-17beta and P4 on Day 13 and E-17beta on Day 21. Variability in return to estrus was greater (P < 0.001) in the control and MGA groups than in CIDR groups. Conception and pregnancy rates in heifers given a CIDR (65.1 and 61.4%) were higher (P<0.01) than those fed MGA (49.6 and 40.4%), but not different from controls (62.2 and 54.9%, respectively). In Experiment 3, 616 heifers received a once- or twice-used CIDR for 7 days, beginning 13+/-1 days after TAI, with or without a concurrent injection of 150 mg of P4 (2 x 2 factorial design). Pregnancy rate to TAI was 47.2%. In heifers that returned to estrus, there was no significant difference between a once- or twice-used CIDR for rates of estrus (68.8%, P < 0.3), conception (65.9%, P < 0.6) and pregnancy (45.3%, P < 0.8). Injecting progesterone at CIDR insertion increased the median interval from CIDR removal to onset of estrus (P < 0.05) and reduced rates of estrus (63.8% versus 73.8%, P<0.05), conception (60.5% versus 70.6%, P = 0.1) and pregnancy (38.6% versus 52.2%, P < 0.02). In a field trial, 983 heifers received a once-used CIDR for 7 days, beginning 13 +/- 1 days after TAI. Pregnancy rate to TAI was 55.2%. The median (and mode) of the interval from CIDR removal to estrus was 2.5 days. Estrus, conception and pregnancy rates were 78.2, 70.3 and 55.0% (overall pregnancy rate to TAI and rebreeding, 78.7%). In summary, a once- or twice-used CIDR for 7 days, starting 13 +/- 1 days after TAI resulted in the majority of nonpregnant heifers detected in estrus over a 4-day interval, with acceptable conception rates; however, injecting progesterone at CIDR insertion significantly reduced both estrus and pregnancy rates, and estradiol treatment after CIDR removal was associated with a decreased pregnancy rate to TAI. Fertility was higher in heifers resynchronized with a once-used CIDR than with MGA.

Synchronization of ovulation in cattle with an aromatase inhibitor-based protocol.

A study was designed to determine the effect of stage of the estrous cycle on the proportion of animals that ovulated and the synchrony of ovulation of heifers treated with an aromatase inhibitor-based protocol. Forty-eight heifers were treated intramuscularly with 500 µg of cloprostenol (PGF) followed by 100 µg of GnRH 24 hours later to serve as control data for comparison of the ovulatory response to a subsequent aromatase inhibitor protocol. Daily ultrasound examinations were done to determine the incidence of and interval to ovulation. At the time of ovulation (Day 0), heifers were assigned randomly to five day-groups (n = 8-11/group) and given an intravaginal device containing 3 g of letrozole for 4 days starting on Day 0, 4, 8, 12, or 16. At the time of device removal, heifers were given PGF followed by GnRH 24 hours later. Ultrasound examinations were done daily from 2 days before device insertion to 9 days after the posttreatment ovulation. The preovulatory follicle diameter after letrozole treatment was larger in the Day 4 group compared to the Day 0 and 16 groups and intermediate in the Day 8 and 12 groups (P < 0.001). Compared to control data, the percentage of heifers that ovulated after letrozole treatment was greater (87.1% vs. 69.4%, respectively; P < 0.05) as was the synchrony of ovulation (residuals: 0.24 ± 0.07 vs. 0.68 ± 0.13; P < 0.01). The day on which letrozole treatment was initiated did not affect the proportion of heifers that ovulated or the interval to ovulation. Plasma estradiol concentrations at the time of removal of the letrozole device in the Day 0 and 4 groups was lower (P < 0.05) than in the corresponding controls. Estradiol concentrations in the Day 8 and 12 groups did not differ from already low concentrations in the respective controls. Corpus luteum diameter profiles and progesterone production were not affected by day-group although reduced luteal lifespan after letrozole treatment was observed and requires further investigation. In summary, a protocol involving a letrozole-impregnated intravaginal device for 4 days, PGF treatment at device removal, and GnRH 24 later resulted in a greater ovulation rate and greater synchrony of ovulation than in heifers not given letrozole. Results suggest that the protocol may be initiated effectively at random stages of the estrous cycle and may provide impetus for further studies to assess the efficacy of a letrozole-based synchronization protocol for fixed-time insemination.

Efficacy of a single intramuscular injection of porcine FSH in hyaluronan prior to ovum pick-up in Holstein cattle.

Plasma FSH profiles, in vitro embryo production (IVP) after ovum pickup (OPU), and establishment of pregnancy with IVP embryos were compared in untreated Holstein oocyte donors and those superstimulated with multiple injections or a single intramuscular (IM) injection of porcine FSH (pFSH) in hyaluronan (HA). Plasma FSH profiles were determined in 23 heifers randomly allocated to one of four groups. Controls received no treatment, whereas the F200 group received 200 mg of pFSH in four doses, 12 hours apart. The F200HA and F300HA groups received 200- or 300-mg pFSH in 5 mL or 7.5 mL, respectively of a 0.5% HA solution by a single IM injection. Plasma FSH levels were determined before the first pFSH treatment and every 6 hours over 96 hours. All data were analyzed by orthogonal contrasts. Circulating FSH area under curve (AUC) in pFSH-treated animals was greater than that in the control group (P = 0.02). Although the AUC did not differ among FSH-treated groups (P = 0.56), the total period with elevated plasma FSH was greater in the F200 group than in the HA groups (P < 0.0001). However, the F300HA group had a greater AUC than the F200HA group (P = 0.006), with a similar total period with elevated plasma FSH (P = 0.17). The IVP was performed in 90 nonlactating Holstein cows randomly allocated to one of the four treatment groups as in the first experiment. A greater proportion of medium-sized (6-10 mm) follicles was observed in cows receiving pFSH, regardless of the treatment group (P < 0.0001). Also, numbers of follicles (P = 0.01), cumulus-oocyte complexes (COCs) retrieved (P = 0.01) and matured (P = 0.02), cleavage rates (P = 0.002), and blastocysts produced per OPU session (P = 0.06) were greater in cows receiving pFSH, regardless of the treatment group. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COCs cultured (P = 0.04), and blastocysts produced per OPU session (P = 0.06) than cows in the F300HA group. Similar pregnancy rates were observed 50 to 60 days after transferring IVP embryos from donors in the different treatment groups (P > 0.05). In conclusion, a single IM injection of pFSH combined in 0.5% HA resulted in similar plasma FSH profiles as twice-daily pFSH treatments. Treatment of nonlactating donors with pFSH, with or without HA, resulted in increased IVP over untreated controls. A single dose of 200 mg of pFSH in 0.5% HA resulted in greater IVP than 300-mg pFSH in HA. Finally, pregnancy rates with IVP embryos were similar, regardless donor treatment.

Effects of oestradiol and some of its esters on gonadotrophin release and ovarian follicular dynamics in CIDR-treated beef cattle.

Three experiments were conducted to: (1) compare the effect of three oestradiol formulations on gonadotrophin release in ovariectomised cows; (2) compare the effects of either oestradiol-17beta (E-17beta) or oestradiol benzoate (EB), given at two doses, on the synchrony of ovarian follicular wave emergence in CIDR-treated beef cattle; and (3) determine the timing of ovulation of the dominant follicle of a synchronised follicular wave following administration of E-17beta or EB 24h after progesterone withdrawal. In Experiment 1, ovariectomised cows (n = 16) received a once-used CIDR on Day 0 (beginning of the experiment) and were allocated randomly to receive 5mg of E-17beta, EB or oestradiol valerate (EV) plus 100mg progesterone i.m. The CIDR inserts were removed on Day 7. There were effects of time, and a treatment-by-time interaction (P < 0.0001) for plasma concentrations of both oestradiol and FSH. Plasma oestradiol concentrations peaked 12h after treatment, with highest (P < 0.01) peak concentrations in cows given E-17beta; estradiol concentrations subsequently returned to baseline by 36 h in E-17beta-treated cows and by 96 h in EB- and EV-treated cows. Plasma FSH concentrations decreased by 12h after oestradiol treatment in all groups (P < 0.0001), reached a nadir at 24h, and increased by 60 h in all groups; plasma FSH reached higher (P < 0.02) concentrations in E-17beta-treated than in EB- or EV-treated cows. In Experiment 2, non-lactating Hereford cows (n = 29) received a new CIDR on Day 0 (beginning of the experiment), and were assigned randomly to receive 1 or 5mg of E-17beta or EB i.m. on Day 1. On Day 8, CIDR were removed and PGF was given. Transrectal ultrasonography was done once daily from 2 days before CIDR insertion to 2 days after CIDR removal, and then twice-daily to ovulation. Although there was no difference among groups in the interval from oestradiol treatment to follicular wave emergence (4.2 +/- 0.3 days; P = 0.5), 5mg of E-17beta resulted in the least variable interval to wave emergence (P < 0.005), compared with the other treatment groups which were not different (P = 0.1). For the interval from CIDR removal to ovulation, there were no differences among groups for either means (P = 0.5) or variances (P = 0.1). In Experiment 3, beef heifers (n = 32) received a once-used CIDR on Day 0 (beginning of the experiment) plus 100mg progesterone i.m. and were assigned randomly to receive 5mg E-17beta or 1mg EB i.m. On Day 7, CIDR were removed and all heifers received PGF. On Day 8 (24h after CIDR removal), each group was subdivided randomly to receive 1mg of either E-17beta or EB i.m. There was no effect of oestradiol formulation on interval from treatment to follicular wave emergence (4.1 +/- 0.2 days; P = 0.7) or on the median interval (76.6h; P = 0.7) or range (72-120 h; P = 0.08) from CIDR removal to ovulation. In summary, oestradiol treatments suppressed FSH in ovariectomised cows, with the duration of suppression dependent on the oestradiol formulation. Both E-17beta and EB effectively synchronised ovarian follicular wave emergence and ovulation in CIDR-treated cattle, and the interval from CIDR removal to ovulation did not differ in heifers given either E-17beta or EB 24h after CIDR removal.