The reliable detection of estrus is an important scientific and practical challenge in dairy cattle farming. Female vocalization may indicate reproductive status, and preliminary evidence suggests that this information can be used to detect estrus in dairy cattle. The aim of this study was to associate the changes in the vocalization rate of dairy heifers with behavioral estrus indicators as well as test the influence of the type of estrus (natural estrus vs. superovulation-induced estrus). We analyzed 6 predefined estrus-related behavior patterns (standing to be mounted, head-side mounting, active mounting, chin resting, being mounted while not standing, and active sniffing in the anogenital region) and vocalization rates in the peri-estrus period (day of estrus ± 1 d) of 12 German Holstein heifers using audio-visual recordings. Each heifer was observed under natural estrus and a consecutive superovulation induced by FSH and cloprostenol. Estrus was determined by behavioral patterns and confirmed by clinical examination (vaginoscopy and ultrasound imaging of the ovaries) as well as by the concentration of peripheral progesterone. Estrus behavior and vocalization rates were analyzed in 3-h intervals (an average of 19 intervals for each heifer), and an estrus score was calculated based on the 6 behaviors. The interval with the highest estrus score (I0) was considered the estrus climax. We demonstrated similar time courses for the estrus score and vocalization rate independent of estrus type. However, in natural estrus, the maximum vocalization rate (±SE) occurred in the interval before estrus climax (I-1; 42.58 ± 21.89) and was significantly higher than that in any other interval except estrus climax (I0; 27.58 ± 9.76). During natural estrus, the vocalization rate was significantly higher within the interval before estrus climax (I-1; 42.58 ± 21.89 vs. 11.58 ± 5.51) than under superovulation. The results underscore the potential use of vocalization rate as a suitable indicator of estrus climax in automated estrus detection devices. Further studies and technical development are required to record and process individual vocalization rates.

Induction of ovulation by administration of gonadotropin-releasing hormone (GnRH) is commonly practiced in cattle to treat repeat breeders or cows exhibiting long estrous periods. This treatment may, however, disturb normal reproductive functions if timing is incorrect. The objective of the present study was to investigate the effect of exogenous GnRH on estradiol secretion of the ovulatory follicle, occurrence of ovulation, development and function of the corpus luteum (CL) and growth of a dominant follicle after ovulation in the bovine, when GnRH treatment was given before the expected physiological LH-surge. Luteolysis was induced by cloprostenol (PG) in three cows and six heifers. Every animal was assigned once to each of the following treatment or control manipulations, receiving either a single dose (0.1 mg) of GnRH (gonadorelin) at (1) 24 h (T1), (2) 48 h (T2), or (3) 72 h (T3) after PG, or (4) no gonadorelin (control manipulation, C). Ovaries were scanned by ultrasound and blood samples were collected for progesterone (P4) and estradiol-17beta (E-17beta) determination. Growth curves of dominant follicles between treatment I and the control differed significantly (P < 0.01). One day after ovulation, the diameter of the dominant follicle was almost 1 mm larger in T1. This difference remained almost unchanged during the entire follow-up period. The recruitment of a new follicular wave after ovulation seemed to occur earlier. Development of CL and levels and profiles of P4-production remained unaffected. When GnRH was given 1 day after PG injection, two animals showed significantly different development of CL (P < 0.05) and of P4-production (both in concentrations [P < 0.05] and profile [P < 0.01]). After normal ovulation and CL development, luteolysis took place on days 5 or 6 after ovulation, and animals ovulated on days 9 and 10. It is suggested that early induction of ovulation with GnRH can cause shortened luteal function in cattle and, ultimately, reduced fertility.

The influence of the corpus luteum (CL) at the time of buserelin treatment and of buserelin-induced ovulation on the dynamics of ovarian follicular development was determined in 18 postpartum cyclic beef cows injected i.m. with 8 micrograms of buserelin on d 0 (d of treatment) and with 500 micrograms of cloprostenol (PGF) 6 d later. From d 0 to onset of estrus, ovaries were examined ultrasonographically, and blood samples were collected daily. Number of medium (5 to 10 mm) follicles and diameters of the two largest (F1 and F2) follicles in each cow were recorded. Cows were grouped according to the presence (CL+) or absence (CL-) of an active CL before buserelin injection and the presence (OV+) or absence (OV-) of a buserelin-induced ovulation (OV). Three groups were formed: CL-OV+ (n = 8), CL+OV+ (n = 6), and CL+OV- (n = 4). Buserelin induced an ovulation in all CL- cows (progesterone [P4] < .3 ng/mL) and in CL+ cows that had P4 < 4 ng/mL but did not in CL+ cows that had P4>> 8 ng/mL. Within CL+ cows, buserelin-induced ovulation (OV+) was associated with a smaller (P < .0001) increase in F1 and F1-F2 diameters and a transient increase in the number of medium follicles that was greater (P < .03) and of a longer duration (P < .01) than in OV- cows. After PGF, the increase in F1 and F1-F2 diameters was still greater in OV+ than in OV- cows (day x OV; P < .05). In all cows, the selection of the preovulatory follicle occurred before PGF injection. Results indicate that a buserelin-induced ovulation was dependent on P4 concentrations at the time of treatment. Subsequently, ovarian follicular dynamics were altered by a GnRH-induced ovulation, but emergence and selection of a large growing follicle occurred in all cows within 6 d of treatment. This follicle became the preovulatory follicle following PGF-induced luteolysis.

Reproductive performance in cows following synchronization of estrus with intravaginal progesterone releasing devices (IVD) has varied with the length of treatment, cyclic status and prolonged return to estrus intervals in some cows following first AI. The objective of this study was to compare two methods of synchronizing and resynchronizing estrus on the reproductive performance of lactating dairy cows. Cows were treated with an IVD (Day 0) for 7 days (n = 350) or 8 days (n = 350), cloprostenol (0.5 mg i.m.) at the time of device removal and estradiol benzoate (EB) at the time of device insertion (1.5mg i.m.), and again 9 days later (1.0 mg i.m.). Cows were also resynchronized starting on Days 23 and 46 by reinsertion of IVDs for either 7 or 8 days and treatment with EB (1mg i.m.) at the time of device insertion and again 9 days later. Cows were inseminated on detection of estrus for 4 days after removal of devices at each of the synchronized estrous cycles. No significant differences in reproductive performance were detected between each treatment throughout the study period. Synchrony of estrus was more precise at the first and second estrus after treatment with an IVD for 8 days compared to 7 days. Cows classified as anestrous had lower reproductive performance than cows classified as cycling and had longer intervals to estrus at the second (P < 0.001) and third estrus (P < 0.06), but not at the first estrus (P = 0.09). Mean time to onset of estrus after IVD removal was less in cows treated with an IVD for 8 days compared to 7 days at each synchronized estrus (P < 0.01). More Holstein-Friesian cows were classified as non-pregnant and not detected in estrus than crossbreed cows (15.7%, 54/343 versus 9.0%, 24/266; [P < 0.05). The results of the study suggested that the main effects of the treatments that were used to synchronize and resynchronize estrus were to alter the timing and synchrony of estrus without affecting fertility.

In previous studies we demonstrated that the administration of a luteolytic dose of cloprostenol to dairy cows in luteal phase, followed by hCG plus estradiol benzoate (EB) 12 h later, led to successful timed AI 48 h after the initiation of treatment. This article reports two consecutive studies. In Study 1 we determined the pregnancy rate of dairy heifers in luteal phase (established by palpation per rectum) treated with cloprostenol followed by 250 IU of hCG plus 1 mg of EB 12 h later, and inseminated 48 h after cloprostenol injection. Study 2 was designed to evaluate the efficiency this synchronization protocol, irrespective of the estrus stage of the animals. In Study 1, 1272 Friesian heifers aged 14 to 16 months with a palpable corpus luteum received 500 mcg cloprostenol. Heifers were then synchronized either according to the hCG plus EB protocol (hCG-EB, n=637), or by a second dose of cloprostenol 11 d later (PG, n=636). Animals in this last group served as controls and were inseminated 72 and 96 h after the second cloprostenol injection. The pregnancy rate was significantly higher (P<0.0001) in the hCG-EB group (59.5%, 379/637) than in PG (44.8%, 285/636). In Study 2, 135 contemporary heifers (with no corresponding information on estrus stage) were subjected to the same protocol as those in the hCG-EB group of Study 1. These animals were classified in retrospect according to estrus stage established by plasma progesterone concentration. Pregnancy rates were 66.7% (24/36), 51% (25/49) and 58% (29/50) for animals in the follicular, early/late luteal, and mid-luteal phase, respectively. The total pregnancy rate was 57.8% (78/135). These findings indicate an improved pregnancy rate for heifers subjected to single insemination after cloprostenol/hCG/EB synchronization, compared to double insemination after synchronization by 2 cloprostenol injections 11 d apart. The cloprostenol/hCG/EB protocol did result in acceptable pregnancy rates after timed AI of dairy heifers regardless of their estrus cycle phase.

This study examined the effects of altered serum FSH concentration on subsequent ovarian response to superovulation. Synchronized heifers were assigned randomly on Day 1 of the cycle (estrus = Day 0) to three pretreatment groups that consisted of 6-d of saline (7ml, s.c., b.i.d.; Group I), FSH-P (0.5 mg, i.m., b.i.d.; Group II) or charcoal-extracted bovine follicular fluid (BFF; 7 ml, s.c., b.i.d.; Group III) injections. Superovulation was initiated on Day 7 and consisted of FSH-P in decreasing dosages over 4 d (4,3,2,1 mg; i.m., b.i.d.), with cloprostenol (500 mug) on the morning of the third day. A second replicate with 14 heifers was conducted using the same protocol but twice the pretreatment dosage of FSH-P (1 mg) and BFF (14 ml). Endogenous plasma FSH decreased during BFF and FSH-P pretreatments compared to controls (P < 0.02). Endogenous FSH concentrations in both primed groups (II and III) were similar to control values (Group I) 12 h after the start of superovulation. Basal LH concentrations were not different between pretreatment groups. The interval from cloprostenol treatment to the preovulatory LH surge in Group III was 21.3 and 23.9 h longer (P < 0.0001) than it was in Groups I and II. The postovulation progesterone rise was delayed in Group III. The number of corpora lutea (CL) was lowest in the BFF-primed group (4.2 +/- 0.8) compared with the FSH-primed (7.4 +/- 1.3) and the control (12.0 +/- 1.8; P < 0.003) groups. In the FSH-primed group (0.68 +/- 0.06 cm(3)), CL volumes were larger than in the control group (0.45 +/- 0.03 cm(3)), whereas in the BFF-primed group (0.27 +/- 0.02 cm(3)) CL volumes were smaller compared with the control group (P < 0.0001). Mean FSH concentrations for 48 h preceding superovulation and the number of CL per cow were positively correlated (r = 0.55; P < 0.004; n = 26). We concluded that both FSH-P and BFF pretreatments decreased the superovulatory response of heifers to FSH-P. The mechanism for this would appear to be associated with reduced endogenous FSH prior to the start of superovulation.

This study evaluated the effect of equine chorionic gonadotropin (eCG) on reproductive performance, when incorporated into the first Ovsynch + P4 synchronization following planned start mating (PSM) in pasture-based lactating dairy cows. Two synchrony programs were compared in a randomized controlled trial in Queensland, Australia. Lactating cows from a single dairy herd (n = 782) were randomly allocated to Control and eCG groups. Control cows had their estrous cycles synchronized by treatment with 100 μg gonadotropin releasing hormone (GnRH; im) and insertion of a progesterone (P4) releasing intravaginal device that contained 1.0 g of P4 on Day 0; removal of P4 device and administration of 500 μg of an analogue of PGF2α on Day 7 (cloprostenol; im); 100 μg im of GnRH on Day 9, and fixed-time artificial insemination (FTAI) on Day 10. The eCG group were treated the same as the Control group except for the addition of 400 IU of eCG, im on Day 7 of the first synchronized estrous cycle. Following the first insemination, non-pregnant cows from both groups had their estrous cycles synchronized with the same treatment protocol without using eCG. The effects of eCG on 42d cumulative incidence of pregnancy and pregnancy per AI (P/AI) were determined using logistic regression models. The effect of eCG on time to pregnancy was determined using Kaplan-Meier survival analysis and Cox proportional hazards models. Adjusted 42 d cumulative incidence of pregnancy for eCG and control groups were 47.2 and 39.3% respectively (Odds ratio [OR] = 1.38, 95% CI: 1.01-1.88). Hazard of pregnancy tended to be higher in eCG cows overall (Hazard ratio [HR] = 1.18, 95% CI: 0.99-1.41) and was significantly higher when restricting to the first 42 days after PSM (HR = 1.31, 95% CI: 1.04-1.64). Hazards of pregnancy were not different between groups when restricting to>> Day 42 post PSM (HR = 1.00, 95% CI: 0.77-1.31). P/AI tended to be higher in eCG treated cows at the first AI (44.0 vs 37.7%, OR = 1.30, 95% CI: 0.94-1.78). P/AI for second and third AIs were not significantly different between groups. In this herd, a single treatment of eCG at the first synchronized estrus after PSM improved reproductive performance in the short term, but not at subsequent inseminations.

Dinoprost tromethamine (DIN), a molecule similar to endogenous PGF_2α, has a half-life of approximately 9 min. Cloprostenol sodium (CLO), a synthetic analog of PGF_2α, has a half-life of approximately 3 h. We hypothesized that treatment of Holstein heifers with CLO would improve estrous detection rate, estrous characteristics, service rate, and overall reproductive performance compared with DIN. Currently in the United States, heifers are largely inseminated based on signs of estrus, which is detected visually or with the aid of mounting detection devices (MD). Automated estrous detection devices (AED) are becoming more accessible to producers, but it is not clear whether they present advantages in the reproductive management of heifers. Therefore, we hypothesized that the use of an AED would improve service and pregnancy rates compared with detection of estrus with the aid of a MD. Holstein heifers (n = 1,019) were enrolled in the experiment at 10 to 11 mo of age, when they were fitted with a Heatime HR LD System (SCR Ltd., Netanya, Israel). At 12 mo of age, we paired heifers according to estrous cycle phase and randomly assigned them to treatments in a 2 × 2 design: PGF_2α formulation (CLO vs. DIN) and estrous detection treatment (AED vs. MD). Heifers in the AED treatment were detected in estrus only by the Heatime HR LD System, whereas heifers in the MD treatment were detected in estrus only by the Kamar Heatmount Detector (Kamar Products Inc., Zionsville, IN). Treatments with the same PGF_2α formulations were repeated 14 d after the first treatment if heifers had not been detected in estrus. A sub-group of heifers had blood sampled on the day of PGF_2α treatment and within 24 h of onset of estrus to determine progesterone and estradiol concentrations. Treatment with CLO reduced the progesterone concentration within 24 h of onset of estrus compared with DIN (0.04 ± 0.01 vs. 0.11 ± 0.01 ng/mL). Among heifers in mid diestrus on the day of PGF_2α treatment, CLO reduced the interval to estrus compared with DIN (72.0 ± 2.2 vs. 82.4 ± 2.4 h). Prostaglandin F_2α formulation and estrous detection treatment did not affect pregnancy to the first service. The interval between the first and second services tended to be reduced for the AED treatment compared with the MD treatment (24.4 ± 0.5 vs. 25.7 ± 0.6 d). Prostaglandin F_2α formulation and estrous detection treatment did not affect the hazard of pregnancy. Although CLO treatment may shorten the interval to estrus in heifers at mid diestrus compared with DIN, PGF_2α formulation did not affect reproductive performance. In the current experiment, no advantages in reproductive performance were observed when estrous detection was based on an AED compared with a MD.

A protocol for presynchronization of ovarian status with 2 injections of PGF2α given 14 d apart, with the last PGF2α injection given 12 or 14 d before Ovsynch increases pregnancy per artificial insemination (P/AI) in dairy cows. We determined the efficacy of reducing the interval from the last PGF2α injection (500 μg of cloprostenol) of presynchronization to initiation of Ovsynch on response to treatment and P/AI. Lactating dairy cows were assigned to an Ovsynch protocol, with the initial injection of GnRH given either 9 (PRE-9; n=135) or 12d (PRE-12; n=135) after the second PGF2α injection of presynchronization. The Ovsynch protocol consisted of 2 injections of 100 μg of GnRH given 9 d apart and 1 injection of PGF2α given 7 d after the initial GnRH injection, and cows were subjected to timed artificial insemination (TAI; 70±3.5 DIM) approximately 16 h after the second GnRH injection. Body condition score (1-5 scale) was recorded at TAI. Blood samples were taken for progesterone determination at the PGF2α injection of Ovsynch, at TAI, and at 11 d after TAI. Ultrasonographic examinations were done in all cows at the second PGF2α injection of presynchronization, initial GnRH injection, PGF2α injection of Ovsynch, at TAI, and 24 h after TAI for cyclicity status and ovarian responses to treatments, and at 32 and 60 d after TAI for confirmation of pregnancy. Overall, 29 cows (10.7%) were determined acyclic or cystic and excluded from the study. The percentage of cows responding to initial GnRH injection (62.2 vs. 61.5%) did not differ between PRE-9 and PRE-12 but more cows in the PRE-9 group failed to respond to PGF2α treatment of Ovsynch compared with PRE-12 (22.7 vs. 10.7%). Body condition score at TAI (2.9±0.02) and mean ovulatory follicle diameter (16.4±0.2 mm) were not different between treatments. Overall P/AI at 32 d was reduced in PRE-9 (33.6%) compared with PRE-12 (44.3%) but pregnancy losses (5.0 vs. 3.7%) did not differ between treatments. Primiparous cows in the PRE-12 group had higher mean progesterone concentration 11 d after TAI and greater P/AI 32 after TAI than primiparous cows in the PRE-9 group (6.4±0.5 vs. 4.6±0.5 ng/mL and 55.8 vs. 30.0%, respectively). In conclusion, reducing the interval from the last PGF2α injection of the presynchronization treatment to initiation of Ovsynch (from 12 to 9 d) did not affect ovulatory response to initial GnRH injection but reduced response to PGF2α injection of Ovsynch and P/AI at 32 and 60 d after TAI. The reduction in P/AI was particularly evident in primiparous cows of the PRE-9 group.

An experiment was designed to evaluate treatments to promote ovarian follicular maturity in advance of administration of exogenous gonadotropin-releasing hormone (GnRH; 100 μg gonadorelin) for control of the bovine estrous cycle. We hypothesized prostaglandin F_2α (PGF_2α; 500 μg cloprostenol) followed by an intravaginal progesterone-releasing insert (CIDR; 1.38 g progesterone) would induce greater follicle size and serum estradiol at the time of GnRH administration. Postpartum cows (n = 194) in two locations were assigned to one of five treatments based on age, days postpartum, and body condition score. Cows in Treatment 1 were treated with the standard 7-d CO-Synch + CIDR protocol: administration of GnRH and CIDR insertion on Day -10, and administration of PGF_2α and CIDR removal on Day -3. Treatments 2-5 were designed in a 2 × 2 factorial arrangement, with Treatment 1 included as an additional reference. On Day -17, cows in Treatments 2-5 received a CIDR insert, either with (Treatments 2 and 3) or without (Treatments 4 and 5) administration of PGF_2α at CIDR insertion. On Day -10, all cows were administered GnRH, and CIDR inserts were either removed (Treatments 2 and 4) or remained in place until Day -3 (Treatments 3 and 5). Treatment with PGF_2α and CIDR in advance of GnRH (Treatments 2 and 3) resulted in increased diameter of the largest ovarian follicle (P < 0.001) and increased serum concentrations of estradiol (P < 0.0005) on Day -10. In addition, variation among cows in CL status (no CL vs. a single CL vs. multiple CL) on Day -3 tended to be decreased (P = 0.08), with cows more likely to have a single CL rather than no CL or multiple CL. Lastly, the proportion of cows expressing estrus prior to fixed-time artificial insemination tended (P = 0.08) to be improved. Results support the hypothesis that administration of PGF_2α and treatment with a CIDR for 7 days prior to GnRH promotes follicular maturity in advance of GnRH administration and may provide an approach by which to enhance response of postpartum beef cows to GnRH-based estrus synchronization programs.

Keywords: Estrous cycle; Gonadotropin-releasing hormone; Prostaglandins.

OBJECTIVE

To compare the timing of onset of oestrus and ovulation, characteristics of oestrus, and fertility in Bos indicus heifers synchronised with a progesterone releasing intravaginal insert (IVP4) and administration of oestradiol benzoate (ODB) either at the time of removal of the insert or 24 h later.

METHODS

Cohort study.

METHODS

Bos indicus and Bos indicus cross heifers were treated on two farms (Farm A, n = 273; Farm B, n = 47) with an IVP4 for 8 days with 1.0 mg of ODB administered at the time of device insertion and 250 mg of cloprostenol at the time of device removal. Heifers in the ODB-0 group were administered 0.75 mg of ODB at the time of device removal while heifers in the ODB-24 group were administered the same dose of ODB 24 h after device removal. Heifers were inseminated once daily after detection of oestrus. Heifers not detected in oestrus by 72 h after removal of inserts were inseminated at that time. Oestrus was detected in heifers on Farm A using heatmount detectors while on Farm B oestrus in heifers was monitored using radiotelemetry of mounting pressure. Ovarian follicular development was monitored daily in 30 heifers on Farm B from the time of administration of inserts until ovulation to a maximum of 96 h after removal of inserts, and again 11 days after removal of inserts (Day 19). A blood sample was collected from all heifers on Farm B on Day 19 and analysed for plasma concentration of progesterone. Pregnancy was diagnosed 6 to 8 weeks after insemination.

RESULTS

Administration of ODB at the time of removal of inserts shortened the time interval to oestrus and ovulation (P < 0.001), increased the number of mounts recorded during oestrus (P = 0.04) and reduced the odds of pregnancy (P = 0.03). The proportion of heifers ovulating on Farm B was 67% and was not affected by treatment group (P = 0.61). The mean diameter of the largest follicle measured in ovaries was greater at the time of removal of inserts (9.1 +/- 0.6 vs 10.7 +/- 0.4; P = 0.03) and at the expected time of the LH surge (8.1 +/- 0.4 vs 11.5 +/- 0.3 mm; P < 0.001) in heifers that ovulated compared to heifers that failed to ovulate, respectively. Emergence of a new follicular wave was not detected during the synchronisation treatment in heifers that failed to ovulate. Concentrations of progesterone in plasma on Day 19 were less in non-pregnant heifers (P = 0.05) compared to heifers subsequently diagnosed as pregnant to insemination and were affected by the diameter of the ovulatory follicle (P = 0.01).

CONCLUSIONS

Administration of ODB at the time of removal of inserts can shorten the time interval to oestrus and ovulation and can reduce fertility when insemination is carried out once daily. Further work is needed to determine if prolonged suppression of follicular development, anovulatory oestrus and premature ovulation occuring in some heifers is associated with administration of ODB.

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.

The growth profiles of the future dominant follicle (DF) and subordinate follicle (SF) and the gene expression of the granulosa cells during luteolysis induction in Bos indicus cows were evaluated. Forty cows were synchronized with a progesterone and estradiol based protocol. After synchronization, cows with a corpus luteum (CL) were evaluated by ultrasonography every 12 h, beginning at eight days post ovulation. Cows identified with a follicle of at least 6.0 mm in diameter in the second wave were split into two groups (BD-before follicular deviation and AD-after follicular deviation. In the BD group cows received 500 μg of cloprostenol (a synthetic analogue of prostaglandin F2α) when the DF reached a mean diameter of 7.0 mm (6.5-7.5 mm). In the AD group, cows received 500 μg of cloprostenol when the DF reached a mean diameter of 8.0 mm (7.5-8.5 mm). Cows in both groups were submitted to aspiration of the DF at 96 and 72 h after prostaglandin was given. Follicular aspirations were performed to quantify IGF1R, LHR and PAPPA transcripts in the granulosa cells. The diameter of the DF at the moment of prostaglandin administration (P = 0.001) and the growth rate of the SF (P = 0.05) were greater in the AD group. There was greater abundance of LHR transcripts in BD cows (P = 0.04). The remaining variables tested were similar between the experimental groups (P>> 0.05). In conclusion, the induction of luteolysis before follicular deviation does not interfere with dominant follicle dynamics. However, it causes granulosa cell LHR down regulation.

The present study characterizes the relationship between the levels of eCG, ovarian morphology, resumption of cyclicity, and fertility in postaborted embryo transfer recipient mares. A total of 32 pregnant recipient mares carrying a male fetus were aborted at approximately 65 days of gestation by single transcervical administration of cloprostenol. In addition, 25 gestation age-matched mares were used as nonaborted controls. The concentration of progesterone, but not of eCG, differed significantly between controls and aborted mares 48 hours after abortion. Of treated mares, 84.4% (27 of 32) expelled the fetus within 48 hours of treatment. The eCG concentration and the number of supplementary luteal structures were lower in mares aborted in November (equivalent to May in Northern Hemisphere) than in January. A total of 6.2%, 37.5%, and 56.2% of the mares entered anestrus, ovulated normally, and had 1 to 2 consecutive anovulatory cycles, respectively. The mean interval from abortion to the first ovulation was 28.5 ± 3.3 days (range, 5-65 days). The correlation between the levels of eCG at abortion and the interval to the first ovulation was poor (r = 0.38; P = 0.03). Of aborted mares, 90% (18 of 20) were reused and became pregnant after embryo transfer at a mean of 57.6 ± 4.4 days after abortion (range, 19-103 days) and eCG concentration of 0.9 ± 0.3 IU/mL (range, 0.1-3.6 IU/mL). In conclusion, the levels of eCG at the time of abortion were extremely variable and did not correlate well with the number of luteal structures or the interval from abortion to the first ovulation.

Until now, there have been no reports of foals born through embryo transfer after artificial insemination using frozen semen in Japan. The aims of this study were to develop a riding crossbred horse and evaluate the prospects of embryo transfer technology in multiplying horse population. In both donor and recipient mares, luteolysis was induced by the administration of 0.1 mg Cloprostenol to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU human chorionic gonadotropin (hCG) or 0.75 mg Deslorelin. Frozen semen from an Irish Connemara pony stallion was used to breed a Hokkaido native pony mare by deep-horn artificial insemination (dose, 400 × 10⁶ sperm). A non-surgical technique was used to collect embryos from the donor mare at day 7 post-ovulation and transfer them transcervically into the uterus of recipient mares (n = 4) immediately after collection. Weekly blood samples were collected from the recipients throughout pregnancy. A total of four embryos were recovered from seven collection attempts (57% recovery) from a donor mare in a single breeding season. Three of the four transferred embryos maintained successful pregnancy and delivered a healthy live foal (75% birth). A normal progesterone profile was observed throughout gestation in recipient mares. In conclusion, for the first time, to the best of our knowledge, this study describes the birth of foals through non-surgical transcervical embryo transfer in Japan after artificial insemination using frozen semen. We expect that this new crossbreed (Connemara pony × Hokkaido native pony) will be a good riding breed.

Progesterone Releasing Intravaginal Devices (Prids) were inserted into six post-partum beef cows for nine days and 0.5 mg cloprostenol was injected i m on day eight. Blood samples were taken via jugular venous catheters at frequent intervals for seven days after Prid removal and assayed for LH, FSH and progesterone. The induced pre-ovulatory type LH and FSH surges occurred between 35 and 123h after Prid withdrawal in five of the cows. In four cows which underwent surges during the time of most intensive sampling, LH levels were significantly higher during the 30h period prior to the LH surge than during the 30h period after the surge. FSH values were low for the 30h period preceding and the 14h period following the time of maximum FSH/LH concentrations. 16 - 30h after the FSH and LH surges, FSH values were again significantly raised compared with the period immediately after the surge. Despite the success of this Prid/PG regime in inducing ovulation, the variability in time between progestagen withdrawal and the LH surge and ovulation is such that the use of fixed time artificial insemination may give poor results.

The pattern of pulsatile secretion of inhibin, oestradiol and androstenedione by the ovary at different stages of the oestrous cycle in sheep was studied in five Finn-Merino ewes in which the left ovary had been auto-transplanted to the neck. The ewes had jugular venous blood samples collected at 4-hourly intervals from 42 h before the induction of luteolysis by i.m. injection of cloprostenol (100 micrograms) on day 10 of the oestrous cycle, until day 3 of the following cycle. There were five periods of intensive blood sampling, when both ovarian and jugular venous blood samples were collected, as follows: (a) mid-luteal phase, before the second injection of cloprostenol on day 10 (15-min intervals for 6 h); (b) early follicular phase, 24 h after the second injection of cloprostenol (10-min intervals for 4 h); (c) late follicular phase, 48 h after the second injection of cloprostenol (10-min intervals for 4 h); (d) after the LH surge on day 1 of the cycle, 76 h after the second injection of cloprostenol (10-min intervals for 4 h); (e) early luteal phase on day 3 of the cycle, 120 h after the second injection of cloprostenol (10-min intervals for 3 h). Plasma was collected and the samples assayed for LH, FSH, progesterone, oestradiol, androstenedione and inhibin. The ovarian secretion rates for oestradiol, androstenedione and inhibin were calculated. All ewes responded normally to the luteolytic dose of cloprostenol with the preovulatory surge of LH occurring within 56.4 +/- 1.6 h (mean +/- S.E.M.) followed by the establishment of a normal luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to assess the effect of the duration of progesterone-based estrus induction protocols on preovulatory follicular dynamics, ovulatory response, and embryo yield after non-surgical embryo recovery (NSER) in Lacaune ewes. Females received acetate medroxyprogesterone intravaginal sponges for six (G-6; n = 14) or nine (G-9; n = 14) days plus d-cloprostenol and eCG 24 h before sponge removal (Day 0). Preovulatory follicular dynamics and the luteal characteristics are evaluated by B-mode and Color-Doppler ultrasonography. NSER was performed five to six days after ovulation. The estrous behavior rate was 85.7% for both groups, and the percentage of ewes that ovulated was 92.9% in G-6 and 100% in G-9. The day of wave emergence (relative to Day 0) did not differ (P > 0.05) between G-6 (-3.0 ± 0.5) and G-9 (-4.2 ± 0.5). The number of follicles of size 4.1-5.0 mm was higher (P < 0.05) in G-9 (1.4 ± 0.2) compared to G-6 (0.8 ± 0.2) during the Days -4 to 0. At NSER, the transcervical penetration rate was 95.2% (20/21) and its duration time was lower (P < 0.05) in G-9 (3.4 ± 0.6 min) than in G-6 (7.2 ± 1.3 min). The number of ovulations and viable embryos was higher (P < 0.05) in G-9 (2.9 ± 0.3 and 1.3 ± 0.4, respectively) than in G-6 (1.9 ± 0.3 and 0.4 ± 0.2, respectively). In conclusion, the 9-day protocol promoted higher ovulation rate and embryo yield; moreover, the cervical dilation treatment allowed NSER in a high percentage of Lacaune ewes.

Experiments were conducted to determine the role of estrogens on endogenous PGF2 alpha secretion and luteolysis following injection of cloprostenol in heifers. In Exp. 1, eight luteal-phase heifers were used to evaluate tamoxifen (T) as an estrogen antagonist. Heifers received T (35 mg i.v.) or ethanol:saline vehicle (ES) every 4 h for 44 h. All received cloprostenol (500 micrograms i.m.) immediately after the start of T or ES, and received estradiol-17 beta (500 micrograms i.m.) 12 h later. Each ES heifer had a surge of luteinizing hormone (LH) within 48 h of estradiol injection, whereas T-treated heifers did not. Estrus was observed in three ES-treated heifers, but not in T-treated heifers. In Exp. 2, 10 heifers received T (35 mg i.v.) or ES every 4 h for 64 h beginning on d 15 postestrus. Cloprostenol (500 micrograms i.m.) was injected 16 h after the start of treatment. Concentrations of LH were similar (P greater than .05) in both groups. In ES heifers, concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) increased; in T-treated heifers, PGFM remained at pre-cloprostenol levels. Luteolysis was induced in all heifers. Progesterone (P4) decreased to less than or equal to 1 ng/ml at similar (P greater than .05) rates in ES-treated and T-treated heifers. Mean concentration of P4 288 h post-cloprostenol was greater (P less than .05) in ES-treated than in T-treated heifers. Three ES-treated heifers, but no T-treated heifers, were in standing estrus. We conclude that T effectively antagonizes estrogen in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study aimed to determine the effect of administrating prostaglandin F2α (PGF2α) and GnRH at the time of artificial insemination (AI) on the pregnancy per artificial insemination (P/AI) and the pregnancy survival rate of dairy cows. A number of 830 lactating Holstein cows were randomly divided into four groups. Cows in group 1 (n=200) treated with 150 µg d-cloprostenol. In group 2 (n=212), cows received 10 µg buserelin acetate, and group 3 (n=205) was treated with both 150 µg d-cloprostenol and 10 µg buserelin acetate. In addition, 213 cows were assigned as control group which received normal saline as placebo (group 4). To measure progesterone, milk samples were collected at the insemination day and five days later. Pregnancy diagnosis was performed 28 and 60 days after the insemination, and the size and number of corpus luteum (CL) and twin pregnancies were recorded. Hormone therapies had no effect on the P/AI, pregnancy survival rate, and the size and number of CL. The P/AI ratio in groups 1, 2, 3 and 4 were 38.50%, 42.92%, 41.46% and 40.84%, and the pregnancy survival rates in groups 1, 2, 3 and 4 were 84.42%, 86.81%, 88.23% and 83.91%, respectively. The probability of a twin pregnancy was significantly higher in group 1 (15.58%) than other groups. There was no significant difference between groups in terms of the offspring gender. In conclusion, the administration of d-cloprostenol or buserelin acetate at the time of AI had no effect on P/AI and pregnancy survival rate in dairy cattle under no heat stress condition, while the administration of d-cloprostenol increased the probability of twin pregnancies.

The objective of the present study was to compare the concentrations of 17 beta-estradiol, progesterone, cyclic adenosine monophosphate and cyclic quinosine monophosphate in the largest follicles of cows that persist for seven days after insemination following the preceding synchronization of oestrus and superovulation and in follicles of the luteal phase of cycle (5th-10th days). Animals included in the experiment were selected on the basis of rectal examination. Synchronization of oestrus was achieved in 24 crossbreds of Slovak Pied x Lowland Black Pied breeds (SS x Nc) using two doses of cloprostenol of Czechoslovak provenience Oestrophan Spofa, 500 micrograms in each, within 11 days. Serum gonadotrophin at the amount of 2500 I. U. was administered forty-eight hours before administration of the second dose PGF2 alpha. Experimental animals were inseminated after 72 hours. On the 7th day after mating the cows were killed at a slaughterhouse. Evaluated were only the ovaries of the 14 cows in which the persistent large follicles occurred. Ovaries of the 13 control cows in the luteal phase between the 5th-10th days were obtained at the slaughterhouse by the method after Ireland et al. (1980). Correct determination of the phase of sexual cycle was substantiated by determination of progesterone concentrations in blood serum. Follicular fluid was obtained from the largest follicles by aspiration and centrifuged in a cooled centrifuge at 3000 G. The concentrations of 17 beta-estradiol and progesterone in follicular fluid were determined using kits from URVJT at Kosice, designated RIA-test-ESTRA (SI-125-9) or RIA-test-Prog (SI-125-6).2+ persistent follicles (9.15 +/- 5.47 nmol.l-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Eighteen lactating Holstein cows were randomly divided into three groups of equal size. Six cows were not superovulated; the remaining cows were superovulated using either FSH-P or PMSG beginning on Day 12 of the estrous cycle (day of ovulation = Day 0). Animals treated with FSH-P were injected intramuscularly (i.m.) with 4 mg FSH-P every 12 h for 5 d. PMSG was administered i.m. as a single injection of 2350 IU. Cloprostenol (PG, 500 ug) was injected i.m. 56 and 72 h after commencement of treatment and at the same time in the cycle of controls. All cows were inseminated 56, 68 and 80 h after the first PG injection. Blood samples (5 ml) were collected daily and every 15 min for a period of 9 h on Days -1, 0, 2, 8 and 10, with continuous blood sampling at 15-min intervals during Days 3 to 6. Ovulation rate was 27.7 +/- 8.22 in animals treated with PMSG, and 8.0 +/- 3.2 embryos per donor were recovered. In the FSH group, ovulation rate was 8.3 +/- 1.48 and 3.0 +/- 1.1 embryos per donor were recovered. Progesterone concentrations were similar in all three groups until the onset of the LH surge, when progesterone concentrations were greater (P<0.05) in animals of the PMSG group. After the preovulatory LH surge, concentrations of progesterone started increasing earlier (44 h) in cows treated with PMSG, followed by FSH-treated cows (76 h) and controls (99 h). The LH surge occurred earlier (P<0.05) in PMSG-treated cows (37 h after first PG treatment), than in animals treated with FSH-P (52 h) or controls (82 h). In animals treated with FSH-P, the magnitude of the preovulatory LH surge (24.2 +/- 1.02 ng/ml) was higher (P<0.05) than in the other two groups (PMSG = 17.1 +/- 2.04 ng/ml; control, 16.7 +/- 1.24 ng/ml). Superovulation with FSH-P or PMSG did not affect either mean basal LH concentration, frequency or amplitude of LH pulses during Days -1, 0, 2, 3, presurge periods, or Days 8 and 10 post-treatment. At ovariectomy, 8 d post-estrus, more follicles>> 10 mm diam. were observed in the ovaries after treatment with PMSG (8.5 +/- 5.66) than after treatment with FSH-P (0.7 +/- 0.42) (P<0.05). Maximum concentrations of PMSG were measured 24 h after administration. Following this peak, PMSG levels declined with two slopes, with half-lives of 36 h and 370 h.

Expression of estrus near timed artificial insemination (TAI) is associated with greater fertility, and estrus detection could improve TAI fertility or direct TAI management, although accurate estrus detection can be difficult and time-consuming using traditional methods. The aim of this study is to evaluate influence of estrus on pregnancy (artificial insemination pregnancy rates (P/AI)) and to validate an alternative method to classify estrus/heat expression using tail chalking (HEATSC) in postpartum Bos indicus cows subjected to TAI in progesterone-estrogen-based protocols. In experiment 1 (Exp. 1), cows (5491) were subjected to visual observation of estrus after progesterone device removal, before TAI, and P/AI was evaluated according to estrus and body condition score (BCS). Cows received a progesterone device and 2 mg estradiol benzoate (EB). After 8 days, the device was removed and 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotrophin was given. Later, animals in Exp. 1 received 1 mg EB and TAI 44 to 48 h. In the Exp. 2 - 3830 cows using similar protocol, received different ovulation inducers: 1 mg EB (n=1624) or 1 mg estradiol cypionate (EC; n=2206) on day 8 (D8). Cows were then marked with chalk, and HEATSC evaluated at TAI on D10 (HEATSC1 - no chalk removal=no estrus expression; HEATSC2 - partial chalk removal=low estrus expression; HEATSC3 - near complete/complete chalk removal=high estrus expression). In Exp. 1, cows showing estrus presented greater P/AI (48.4% v. 40.2%, P<0.05). In Exp. 2, P/AI (HEATSC1 - 40.0%; HEATSC2 - 49.7%; HEATSC3 - 60.9%; P<0.001), and larger follicle timed artificial insemination (LFTAI) (<0.001) varied according to HEATSC. There was no difference in P/AI (P=0.41) or LFTAI (P=0.33) according to ovulation inducer. Cows with greater BCS showed greater P/AI in both experiments (P<0.05). Estrus presence and greater HEATSC improved P/AI, and EC v. EB used promoted differential estrus manifestation (cows showing HEATSC2 and HEATSC3: 79.5% with EB v. 69.98% with EC use, P<0.001), however, with similar P/AI. The use of HEATSC in B. indicus cows subjected to TAI is useful to identify cows with greater estrus expression and consequently improved pregnancy rates in TAI, allowing the cows with low HEATSC to be targeted for additional treatments aimed at improving P/AI.

In Experiment 1, an osmotic minipump containing oxytocin was implanted s.c. in ewes for 12 days beginning on Day 10 of the oestrous cycle, producing approximately 100 pg oxytocin/ml in the plasma. Two days after the start of infusion, all ewes were injected with 100 micrograms cloprostenol and placed with a fertile ram. At slaughter 22 days later, 9 (75%) of the 12 control (saline-infused) ewes were pregnant compared with 1 (11%) of the 9 ewes infused with oxytocin. In the control group, midcycle plasma concentrations of oxytocin were significantly higher in nonpregnant than in pregnant ewes. In Experiment 2, an infertile ram was used throughout to avoid any possible effects of pregnancy and oxytocin infusions were given at different stages of the oestrous cycle. Otherwise the protocol was similar to that in Exp. 1. Oxytocin infusion during luteolysis and the early follicular phase had no effect on the subsequent progesterone secretion pattern, but infusions beginning the day before cloprostenol-induced luteolysis and lasting for 7 or 12 days and infusions beginning on the day of oestrus for 4 days all delayed the subsequent rise in plasma progesterone by approximately 3-4 days. In these animals, the cycle tended to be longer. It was concluded that an appropriate oxytocin secretion pattern may be necessary for the establishment of pregnancy in ewes and that a high circulating oxytocin concentration during the early luteal phase delays the development of the young corpus luteum.