Establishment of a vascular system within the corpus luteum (CL) is critical for progesterone (P₄) secretion. Measurement of luteal blood flow (LBF) may be a feasible way to determine luteolysis induced with cloprostenol sodium (CLO). Our overall objective was to establish timelines to assess luteolysis via Doppler ultrasonography. Estrous cycles were synchronized in 11- to 12-mo-old Holstein heifers (n = 37). Heifers were injected CLO at a random stage of the estrous cycle 12 d from treatment. Gonadotropin-releasing hormone (GnRH) was administered 2 (d -10) and 8 d (d -4) after the initial CLO. This satisfied the study objective of inducing simultaneous presence of CL at d 4 of development (D4 CL) and CL at d 10 of development (D10 CL) on the day of treatment with different CLO doses (hereafter referred to as d 0). Heifers were randomly assigned to 1 of 5 treatments on d 0: negative control (NC) consisting of no treatment with CLO (n = 8); a quarter dose of CLO (0.125 mg; n = 8); half dose of CLO (0.25 mg; n = 8); full dose of CLO (0.5 mg; n = 8); or positive control (PC) consisting of 4 doses of 0.5 mg of CLO at 24-h intervals starting at d 0 (n = 5). Data collection was performed at d 0 (before and 1 h after treatment) and 2, 4, 6, and 8 d following treatment, to determine luteal volume (LV), LBF, and circulating concentrations of P₄. Both NC and PC were efficient in mimicking physiological scenarios that occur during normal luteal development and luteolysis. Heifers that received PC had complete LBF disappearance of both D4 and D10 CL between d 2 and 4 after the first of 4 CLO treatments given 24 h apart (average 4.0 ± 0.0 and 3.2 ± 0.7 d, respectively). Complete LBF disappearance was used as a luteolysis marker. Treatment with different doses of CLO did not impair luteal development of the D4 CL. However, concurrent complete LBF disappearance for D10 CL in heifers treated with half (5/8 heifers) and full doses of CLO (8/8 heifers) resulted in less LBF in the half dose, and less LV and LBF in the full-dose treatment, in D4 CL at d 8 post-treatment, compared with NC. Treatment with various doses of CLO induced an acute increase in LBF 1 h after treatment, regardless of dose in D10 but not in D4 CL. We found a lack of dose response in LV reduction of D4 and D10 CL. Interestingly, LV of the D10 CL decreased in untreated NC between d 0 and 8 after treatment (d 10-18 of luteal development). Assessment with color Doppler ultrasound was sensitive enough to identify dose-response patterns in Holstein heifers (absence, partial, or complete luteolysis) following various doses of CLO. Variability in time to complete LBF disappearance of mature D10 CL following a full dose of CLO limits the use of Doppler ultrasonography to detect luteolysis at a single time point following treatment.

Keywords: cloprostenol sodium; color Doppler; luteal blood flow; luteolysis.

The present study was aimed to investigate the effects of double-dose gonadotropin-releasing hormone (GnRH) injection on the induction of oestrus and some reproductive performance parameters in Awassi ewes during the non-breeding season. In the study, 100 ewes were treated with a vaginal sponge containing 60 mg medroxyprogesterone acetate for 7 days in the anoestrus (day 0). PMSG 500 IU and 250 μg cloprostenol sodium were injected on the day of removal of the sponge (day 7). Ewes in Group 1 (n = 31) were not subjected to any hormonal treatment. Ewes in Group 2 (n = 31) were given 50 μg GnRH 48th hour after removal of the sponge. Ewes in Group 3 (n = 33) were given 50 μg GnRH 48th hour after the removal of the sponge and 50 μg GnRH 12th day after post-mating. The results obtained in the study showed that there were no statistical differences between the Groups 1, 2 and 3 in terms of oestrus rates (82.8%, 68.9%, 72.7%), conception rates (66.7%, 55.0%, 54.2%), multiple pregnancy rates (28.5%, 50.0%, 30.7%) and litter sizes (1.28, 1.50, 1.31). No significant increases in P4 concentration were observed in Group 3 treated with GnRH at the 12th day after post-mating; however, a numerically lower (p > 0.05) late embryonic-early fetal mortality rate was observed in Group 3 (0%), when compared with the values obtained in Group 1 (12.5%) and Group 2 (9.1%). In conclusion, after short-term progestagen administration during the non-breeding season, double-dose GnRH injections did not increase P4 concentration and had no significant differences on reproductive performance parameters among groups.

Keywords: Awassi ewes; Embryonic mortality; GnRH; Litter size; Progesterone.

Aims: To assess the effect on conception rate to first service, 3- and 6-week in-calf rates, final pregnancy rate and the hazard of pregnancy, of addition of a second prostaglandin (PG) injection 24 hours after the first, in a synchrony programme combining Ovsynch and progesterone in pasture-based dairy cows that had not been detected in oestrus before the start of the seasonal mating period.

Methods: This prospective, negative-controlled study was conducted using anoestrous cows (n = 1,411) from nine spring-calving dairy herds in the Waikato region of New Zealand. Ovaries of cows not detected in oestrus were examined by transrectal ultrasonography and the presence of a corpus luteum determined. All cows were then treated with an intravaginal progesterone-releasing device and injected I/M with gonadorelin (Day -9). Cows were injected I/M with cloprostenol at device removal (Day -2) and on Day -1 were injected with either water (single PG; n = 720), or cloprostenol (double PG; n = 692). On Day 0 cows were injected again with gonadorelin, unless previously detected in oestrus, and were inseminated on Day 1. Based on dated pregnancy diagnosis by transrectal ultrasonography, cows were categorised as conceiving to first insemination, becoming pregnant in the first 3 or 6 weeks, or becoming pregnant by the end of the mating period.

Results: Cows in the double PG group had increased conception rate to first service (45.3 (95% CI = 45.1-45.4)% vs. 41.1 (95% CI = 41.0-41.3)%; p < 0.001), 3-week in-calf rate (53.4 (95% CI = 53.6-54.0)% vs. 49.2 (95% CI = 49.0-49.4)%; p < 0.001), and 6-week in-calf rate (67.2 (95% CI = 65.7-68.8)% vs. 63.5 (95% CI = 62.0-65.0)%; p = 0.014) compared to cows in the single PG group. Final pregnancy rate and hazard of pregnancy did not differ between treatment groups (p >0.2).

Conclusions: Addition of a second PG injection 24 hours after the first, increased conception rate to first service and the proportion of cows pregnant by 3 and 6 weeks after commencement of the mating period.

Clinical relevance: Improved outcomes can be achieved when treating those cows not detected in oestrus by the start of the seasonal mating period by addition of a second PG injection to the currently recommended treatment protocol for these animals.

Keywords: Anoestrus; conception rate; dairy cows; dose; prostaglandin; treatment.

The objective of this study was to test the effect of low circulating concentrations of progesterone (P4) on pre-ovulatory follicle development in heifers as part of an overarching objective to develop a model to understand this phenomenon in dairy cattle without the confounding factors of lactation. Holstein heifers between 12 and 13 mo of age were pre-synchronized to ensure all heifers were on d 6 of the estrous cycle at the start of the Ovsynch program. Only heifers with CL regression and ovulation to the following pre-treatment strategy were used in the study: 0.5 mg cloprostenol (PGF_2α), 2 d later, 0.1 mg GnRH, 6 d later, GnRH (G1; 1st GnRH of Ovsynch). Heifers (n = 159) responding to pre-treatment were randomly assigned to 4 groups and completed the Ovsynch program: high P4 control (HPC), low P4 control (LPC; PGF_2α 24 h after G1), PG2 (PGF_2α 24 and 48 h after G1) and PG3 (PGF_2α 24, 48, and 96 h after G1). Only heifers that had ovulation to G1 remained in the study. Blood samples were collected in all heifers on d 7 (n = 157) and in a subset of heifers on d 1, 2, 3, 4 (n = 82) after G1 to measure serum P4. Pre-ovulatory follicle size at G1 (13.0 ± 0.1 mm; P = 0.53) and mean serum P4 24 h after G1 (d 1; 3.62 ± 0.11 ng/mL; P = 0.46) did not differ among treatments. HPC heifers had greater (P < 0.001) mean serum P4 compared to LPC, PG2 and PG3 on d 2, 3, 4, and 7. On d 2, 3 and 4, mean serum P4 of LPC, PG2 and PG3 heifers did not differ (P > 0.10). On d 7, LPC heifers had greater (P < 0.001) serum P4 compared to PG2 and PG3 heifers. Mean ± SEM serum P4 on d 7 after G1 was 8.43 ± 0.39, 2.55 ± 0.36, 1.58 ± 0.20, and 1.21 ± 0.15 ng/mL for HPC, LPC, PG2, and PG3, respectively. Percentage of heifers with P4 < 0.50 ng/mL on d 7 was greater (P < 0.05) for LPC, PG2 and PG3 (27, 32 and 26%, respectively) compared to HPC (0%). A greater (P < 0.05) proportion of heifers ovulated before G2 in the LPC, PG2 and PG3 than in the HPC. For heifers that ovulated after G2, low serum concentrations of P4 in LPC, PG2 and PG3 induced double ovulations in 6/97 heifers after the final GnRH of Ovsynch compared to 0/33 in HPC. In summary, PGF_2α treatments during early CL development reduced circulating P4 concentrations 7 d after G1 compared with both HPC and LPC. However, it did not effectively control CL and follicle function to be utilized as a model to test high vs. low serum P4 on fertility parameters in Holstein heifers.

Keywords: Cloprostenol; Corpus luteum; Dairy heifer; Progesterone; Prostaglandin F2α.

The effect of hCG administration on accessory corpus luteum (ACL) formation, CL area, and plasma progesterone (P4) concentration (ng/mL) seven days after breeding was studied in nulliparous Santa Inês sheep. Intravaginal 60 mg MAP sponges were inserted into ewes for six days and 300 IU eCG i.m. and 30 µg d-cloprostenol latero-vulvar were administered 24 h before sponge removal. Ewes were naturally bred and, seven days after first mating (Day 0; D0), were treated with either 250 IU hCG (hCG group; n = 7) or 1 mL saline solution (control group; n = 7). Blood was collected to determine plasma P4 concentrations and sonograms were performed on Days 7, 10, 13, 16, 19, and 22. Number of CL on D7 was similar (P > 0.05) between hCG (1.3 ± 0.5) and control (1.3 ± 0.5) groups; however, on D13, it was greater (P < 0.05) in the hCG group (2.3 ± 0.5) than in the control group (1.3 ± 0.5). A greater (P < 0.05) luteal tissue area was detected in hCG-treated ewes (n = 4) on Days 16 to 22 than in the animals in the control group (n = 7). Plasma P4 concentration on D13 to D22 was higher (P < 0.05) in hCG-treated animals than in control ewes. Administration of hCG seven days after estrus onset efficiently induced accessory CL formation in ewes, increasing luteal tissue area and plasma P4 concentration.

Keywords: CL; luteotropic effect; ovulation induction; progesterone; sheep; ultrasound.

Induction of farrowing with prostaglandins is a way of increasing farrowing supervision and to provide adequate care for piglets in the first hours of life. However, some studies observed negative effects associated with induction, including decreased piglet viability, reduced birth weight and decreased colostrum yield. Furthermore, the farrowing response of sows to prostaglandins treatment varies among studies, largely influenced by the induction protocol applied. Thus, a systematic review and meta-analysis was carried out to evaluate the effects of farrowing induction with prostaglandins on stillbirth rate, birth weight, pre-weaning mortality, weaning weight, farrowing duration and colostrum and milk characteristics as well as the farrowing response to prostaglandin treatment. The interval from farrowing induction to onset of farrowing (IFIOF) was 31 h, and a twice application of prostaglandin increased by 37% the proportion of sows farrowing during the next working day. Prostaglandins had no effect on farrowing duration (P > 0.05). Piglet birth weight and weaning weight were only decreased (P < 0.05) when farrowing was induced ≥3 days before the expected farrowing date (based on herd average or in gestational length of the control group). Induction three or two days before the expected farrowing date had no effect on stillbirth rate; conversely, stillbirth rate was reduced by 28% (P < 0.05) when induction was performed one day before the expected farrowing date. Farrowing induction had no influence on pre-weaning mortality. The present study strengthened the observations that farrowing induction with prostaglandins is a valuable tool to reduce gestational length variation and to synchronize farrowing during the working day, allowing better assistance to sows and piglets. To obtain the maximum benefit of farrowing induction, it is recommended that induction should be performed one or two days before the expected farrowing date.

Keywords: Birth weight; Cloprostenol; Dinoprost; Sow; Stillbirth; Swine.

The aim was to study reproductive outcomes of Nelore (Bos indicus) cows submitted to a 7-d estradiol (E2)/progesterone (P4)-based timed artificial insemination (TAI) protocol, receiving various combinations of doses and hormones. Primiparous (n = 962) and multiparous (n = 1935) cows were submitted to synchronization (n = 2012) and resynchronization (n = 885 non-pregnant cows at pregnancy diagnosis 30 d after TAI) protocols, following a 2 × 2 × 2 factorial arrangement of eight treatments. At the initiation of the TAI protocol (Day -9), all cows received a 1.0 g intravaginal P4 insert, 2.0 mg E2 benzoate and received (PGF1) or not (PGF0) 0.5 mg cloprostenol sodium (PGF). On Day -2, the P4 insert was removed, all cows received 0.5 mg PGF, 300 IU equine chorionic gonadotropin (eCG) and 0.5 (EC0.5) or 1.0 mg estradiol cypionate (EC1.0). On Day 0, cows were treated (G1) with 8.4 μg buserelin acetate (GnRH) or not (G0), concurrently with TAI. The eight treatments were generated: 1) PGF0-EC0.5-G0 (n = 364), 2) PGF0-EC0.5-G1 (n = 363), 3) PGF1-EC0.5-G0 (n = 363), 4) PGF1-EC0.5-G1 (n = 360), 5) PGF0-EC-1.0-G0 (n = 360), 6) PGF0-EC1.0-G1 (n = 363), 7) PGF1-EC1.0-G0 (n = 361), and 8) PGF1-EC1.0-G1 (n = 363). Pregnancy per AI (P/AI) was greater at first AI compared with resynchronization (58.9 [n = 2012] vs. 54.9% [n = 885]). Presence of CL on Day -9 resulted in more cows expressing estrus (81.3 [n = 680] vs. 67.1% [n = 2033]) and greater P/AI (66.0 [n = 692] vs. 54.9% [n = 2106]). There was no difference in P/AI between cows that received or not PGF on Day -9 (58.7 [n = 1447] vs. 56.6% [n = 1450]). In contrast, PGF tended to increase P/AI of cows with CL on Day -9 (with PGF = 69.1 [n = 375] vs. without PGF = 62.5% [n = 317]). Cows that received 1.0 mg EC expressed more estrus than those treated with 0.5 mg (73.8 [n = 1414] vs. 67.9% [n = 1398]) and had greater P/AI (60.2 [n = 1447] vs. 55.1% [n = 1450]). P/AI was greater in cows treated with GnRH at TAI (59.8 [n = 1449] vs. 55.5% [n = 1448]), particularly in cows that did not show estrus (52.7 [n = 393] vs. 38.1% [n = 420]). Moreover, GnRH on Day 0 increased P/AI in cows with BCS < 3.0 (57.1 [n = 723] vs. 48.6% [n = 698]), in primiparous (50.1 [n = 465] vs. 41.9% [n = 497]) and in cows that received 0.5 mg EC (58.9 [n = 723] vs. 51.3% [n = 727]). In conclusion, 1.0 mg of EC on Day -2 and GnRH at TAI improved P/AI, but the combination of a higher dose of EC and GnRH treatment at AI did not enhance this effect. Furthermore, GnRH improved P/AI especially in Bos indicus cows with lower expression of estrus, such as primiparous, thinner cows, and cows treated with 0.5 mg of EC.

Keywords: Beef cattle; Estrus; Fertility; GnRH; Synchronization; TAI.

Cloprostenol (CLO) is an important chiral drug widely used in veterinary practice. In high-performance liquid chromatography (HPLC), the acetonitrile/water system is more effective in the chiral resolution of CLO. The change in the ratio of acetonitrile and water has a great influence on the chiral selectivity of CLO. The enantioselective adsorption and nonselective adsorption of CLO on the polysaccharide stationary phase with three groups of mobile phases (Acetonitrile / 0.1 TFA; 95 Acetonitrile/5 H₂O/0.1 TFA; 45 Acetonitrile/55 H₂O/0.1 TFA) were determined and fitted with the subtraction fitting method (SFM). Adding a small amount of water to the mobile phase mainly reduced the enantioselective equilibrium adsorption constants K_s of (-)-CLO and thus changed the selectivity. Among them, the true separation factor (α_true) of the 95 Acetonitrile/5 H₂O mobile phase was as high as 92.86. Chiral preparation was performed on the basis of this mobile phase. The preparation performance in kkd (kilograms per kilogram of stationary phase purified compound per day) was 0.25 kg racemate/kg CSP/day.

Keywords: Adsorption model; Chiral preparation; Cloprostenol; Enantioselectivity; Polysaccharide stationary phase.

This study assessed the outcomes of nonsurgical embryo recovery (NSER) after superovulation (SOV) in five locally adapted Brazilian breeds of sheep and goats. The objective was to evaluate the feasibility and efficiency of using SOV combined with a less-invasive embryo collection technique for supplying the Brazilian animal gene bank with germplasm from specific genotypes of interest. Morada Nova (n = 20), Santa Inês (n = 20), and Somalis (n = 20) ewes received an intravaginal progesterone (330 mg) device for 9 days, while Canindé (n = 15) and Moxotó (n = 15) goats received an intravaginal medroxyprogesterone acetate (60 mg) device for 6 days. All females received 133 mg of porcine follicle-stimulating hormone (pFSH) administrated in six decreasing doses 12 hours apart, starting 60 hours before device removal, plus 37.5 μg of d-cloprostenol at the fifth and sixth pFSH dose. Donors in estrus were mated with fertile males. The corpora lutea (CL) number was assessed by ultrasonography 1 day before NSER. On day 6.5 or 7 after estrus, NSER was performed following hormonally induced cervical relaxation. A total of 97% of sheep and 90% of goats responded with estrus, and among those, 91% of sheep and 85% of goats presented a CL. In ewes, the numbers of CL were greater (p < 0.05) in the Santa Inês breed, while similar (p > 0.05) CL numbers were found among the goat breeds. All viable embryos were freezable (excellent and good quality) and the number per donor was 7.8 for sheep and 4.9 for goats. All parameters of NSER efficiency, embryo yield, and fertility post-NSER did not differ (p > 0.05) between breeds among each species. The SOV-NSER procedures applied for an embryo biobank supply of locally adapted Brazilian breeds of small ruminants were efficient regarding production of cryopreservable embryos, and preservation of donor fertility. Therefore, SOV followed by NSER is recommended for embryo biobank assembly in sheep and goats.

Keywords: caprine; cryopreservation; genetic resources conservation; ovine; superovulation; transcervical.

Information on the follicular population and oocyte quality of cows in the final period of reproductive life is scarce. The present study aimed to compare the antral follicle count (AFC), oocyte production and embryonic developmental competence of young versus long-lived and senescent Bos indicus beef cows. Nellore cows (Bos indicus) were classified into three groups according to age: young (4-9 years, n = 10), long-lived (14-17 years, n = 10) and senescent (17-23 years, n = 10). At a random time in the estrus cycle, the cows received cloprostenol sodium salt (0.5 mg, IM), estradiol benzoate (1 mg, IM) and an intravaginal P4 device (1.4 g). Five days later, the P4 devise was removed and oocyte collection (OPU1) was performed. A second OPU (OPU2) was performed 5 days after the first in order to aspirate only growing follicles. During each OPU, AFC and the number and quality of cumulus-oocyte complexes (COCs) were evaluated. Then, the COCs were placed in standard maturation medium (IVM), fertilized and incubated for 9 days. The data were subjected to ANOVA and Multinomial Logistic Regression. The AFC was smaller in long-lived and senescent cows in both OPU1 and OPU2 when compared to younger cows. There was no difference in AFC between OPU1 (19.9 ± 1.8) and OPU2 (17.6 ± 1.9) in young cows, however, more follicles were punctured in long-lived and senescent cows in OPU1 (12.0 ± 2.6 and 19.3 ± 4.6) than in OPU2 (9.2 ± 1.9 and 10.3 ± 2.3), respectively (P < 0.01). The numbers of COCs recovered from young cows (OPU1 = 14.2 ± 1.8; OPU2 = 8.4 ± 0.9) were higher than those obtained from long-lived cows (OPU1 = 5.9 ± 2.3; OPU2 = 4.3 ± 1.0) and senescent cows (OPU1 = 7.2 ± 3.0; OPU2 = 4.1 ± 1.7), respectively (P < 0.05). The cleavage rate did not differ between groups. However, the rate of blastocyst formation was higher for young (64.8%) and long-lived (65.0%) compared to senescent (16.5%) cows (P < 0.01). In conclusion our results indicate that the AFC is lower in long-lived and senescent cows compared with young cows. However, unlike in senescent cows, the embryonic development of long-lived cows is similar to that of young cows. This suggests that Nellore cows aged >17 years begin to have reduced embryonic development capacity due to ovarian aging.

Keywords: AFC; Ageing; Bos indicus cows; Embryo; OPU.

Development of efficient and stereoselective synthesis of prostaglandins (PGs) is of utmost importance, owing to their valuable medicinal applications and unique chemical structures. We report here a unified synthesis of PGs <em>cloprostenol</em>, bimatoprost, PGF_2α, fluprostenol, and travoprost from the readily available dichloro-containing bicyclic ketone <b>6a</b> guided by biocatalytic retrosynthesis, in 11-12 steps with 3.8-8.4% overall yields. An unprecedented Baeyer-Villiger monooxygenase (BVMO)-catalyzed stereoselective oxidation of <b>6a</b> (99% ee), and a ketoreductase (KRED)-catalyzed diastereoselective reduction of enones <b>12</b> (87 : 13 to 99 : 1 dr) were utilized in combination for the first time to set the critical stereochemical configurations under mild conditions. Another key transformation was the copper(ii)-catalyzed regioselective <i>p</i>-phenylbenzoylation of the secondary alcohol of diol <b>10</b> (9.3 : 1 rr). This study not only provides an alternative route to the highly stereoselective synthesis of PGs, but also showcases the usefulness and great potential of biocatalysis in construction of complex molecules.

This study aimed to evaluate the progesterone (P4) release profile provided by four commercially available intravaginal P4 devices, as well as the effect of circulating P4 concentrations exclusively from these devices on the development of the dominant follicle (DF) in Nelore (Bos indicus) cows. Therefore, non-lactating multiparous Nelore cows were enrolled in an experimental design, over three replicates, starting on Day -9 with the insertion of a reused P4 device (2 g - original P4 load) for 7 d, followed by two treatments of cloprostenol sodium (PGF; 0.482 mg), 24 h apart, on Days -3 and -2. Just before device removal, on Day -2, a norgestomet ear implant was inserted and, 2 d later (Day 0), at the time of norgestomet withdrawal, cows were randomly assigned to receive one of the intravaginal devices: Primer (0.5 g); Prociclar (0.75 g); Sincrogest (1 g); or CIDR (1.9 g), and 2 mg of estradiol benzoate (EB) im. Blood samples were collected immediately before P4 device insertion, 12 h later and daily over 15 d (1 d after P4 device removal). Ultrasound examinations were performed on Days 0, 7, 8, 9, 10, 12, and 14 to evaluate ovarian dynamics. Results are presented as mean ± SEM and differences were considered when P ≤ 0.05. Overall, the devices resulted in distinct circulating P4 concentrations over 10 d, varying according to their initial P4 load and P4 impregnated surface area. Primer provided the lowest circulating P4 concentrations over time, whereas, CIDR had the greatest concentration. Sincrogest and Prociclar were similar, producing intermediary circulating P4. There was no effect of treatment on the DF diameter on any specific day, nor on follicular growth rate from Day 7-10. However, the Primer device resulted in a greater mean DF diameter over time. Additionally, greater circulating P4 concentrations, mainly during the first 3 d of device insertion, were associated with smaller DF diameters regardless of the treatment. In conclusion, results from this study provided a better understanding of the P4 profile of intravaginal P4 devices, as well as, their effect on DF development in Bos indicus cows. These data contribute to optimize the use of P4 devices in the reproductive management of beef cattle.

Keywords: Beef cattle; Bos indicus; Dominant follicle; P4 insert; Steroid hormone.

Two experiments evaluated the effects of gonadotropin releasing hormone (GnRH) treatment on fertility of suckled Nelore beef cows treated with an estradiol/progesterone (P4)-based protocol for timed artificial insemination (TAI). Experiment 1 was designed to determine the effect of GnRH administered 34 h after P4 insert removal (GnRH34) on time of ovulation. Suckled cows (n = 34) were treated with 2 mg estradiol benzoate (EB) and an intravaginal insert containing 1.9 g of P4. Eight days later, P4 inserts were removed, and all cows received 150 μg of d-cloprostenol (prostaglandin F2 alpha analogue), 300 IU of eCG, and 1 mg of estradiol cypionate (ECP). On Day 9 (05:00 p.m.), cows were randomly distributed, according to the diameter of the pre-ovulatory follicle, in two treatments: 1) GnRH (n = 17) cows that received 10.5 μg of buserelin acetate, or 2) no further treatment (control, n = 17). Cows treated with GnRH 34 h after P4 insert removal ovulated earlier (P = 0.02) than control cows (66 ± 0.0 and 77.2 ± 4.3 h). Experiment 2 was designed to determine the effect of GnRH34 on the fertility of suckled beef cows. Nelore cows (n = 506) were treated as Experiment 1. On Day 8, cows were painted in the sacrocaudal region to identify cows that displayed estrus. On Day 9 (05:00 p.m.), cows were randomized to receive same treatment as Experiment 1, control (n = 252), or GnRH (n = 254). All cows were TAI 48 h after P4 insert removal. At TAI, estrus was evaluated, and deemed to have occurred in cows without a tail-head chalk mark (>75% paint loss). Cows treated with GnRH 34 h after P4 insert removal had greater (P = 0.01) pregnancy per AI (P/AI) than cows that only received ECP (63.0% and 50.4%). No difference (P = 0.5) was observed in the proportion of cows that displayed estrus between treatments. Furthermore, cows that displayed estrus had greater (P < 0.01) P/AI than cows that did not. Treatment with GnRH, given at 34 h after P4 insert removal, increased (P < 0.05) P/AI in cows that did not show estrus at TAI. In summary, treatment with GnRH 34 h after P4 insert removal was associated with earlier ovulation and resulted in greater P/AI in suckled Nelore cows treated with an estradiol/P4-based protocol for TAI.

Keywords: Beef cattle; Fertility; Ovulation; Pregnancy.

Two experiments were conducted in acyclic Alpine (A) and Saanen (S) goats that received intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 6 days, as well as 200 IU of eCG and 30 μg d-cloprostenol i.m. 24 h before sponge removal. On day 7 (day 0 = onset of synchronized estrus), all goats were randomly divided into two groups: animals treated with 300 IU of hCG i.m. (hCG; Exp.1: n = 8A; Exp.2: n = 75A + S) and untreated controls (Control; Exp.1: n = 8A; Exp. 2: n = 70A + S). In Exp.2, all goats were artificially inseminated. Transrectal ovarian ultrasonography and blood collection were done on days 7, 10, 13, 17, and 21 (Exp.1), and pregnancy detection on day 60 (Exp.2). Estrus and ovulations occurred in five hCG and seven Control animals. Accessory CL (aCL) were detected in all hCG does. The total luteal area of ovulatory corpora lutea (oCL) increased (P < 0.05) on day 10 in hCG does and remained greater (P < 0.05) than in Control until day 21. Total and high-velocity color Doppler area were greater (P < 0.05) for oCL of hCG does on days 13 and 17. Progesterone concentrations were greater (P < 0.05) in hCG does from days 13 to 21 and related directly to the total luteal and oCL area for the duration of the study in all does. The pregnancy rate was higher (P < 0.05) in hCG than in Control by 22.5 %. Human chorionic gonadotropin given on day 7 of the synchronized estrous cycle positively affected CL function and pregnancy rates in seasonally anovular dairy goats.

Keywords: Corpus luteum; Human chorionic gonadotropin; Ovarian ultrasonography; Pregnancy; Progesterone.

Due to the limited literature available evaluating doses of Prostaglandin F2α in donkeys, doses for horses have been extrapolated and used as guidelines. This study aimed to assess the efficacy and side effects of four different cloprostenol sodium and dinoprost tromethamine doses to induce luteolysis in jennies. Sixty-three cycles of seven Jennies (nine cycles per jenny) were used in this study. Seven days after ovulation, jennies randomly received one of the treatments in a crossover design as follows: Control, no treatment was administered; C1, 250 µg of cloprostenol sodium (CS, Estrumate , Merck Animal Health, USA); C2, 125 µg of CS; C3, 65.5 µg of CS, C4, 37.5 µg of CS; DT1, 5 mg of dinoprost tromethamine (DT, Lutalyse, Zoetis, USA); DT2, 2.5 mg of DT; DT3, 1.25 mg of DT; DT4, 0.625 mg of DT. Jennies were monitored for 30 minutes following treatment, and adverse effects were recorded. The measurement of the corpus luteum (CL) and the length of the estrous cycle were recorded. All DT and CS treatment doses were effective (P < .0001) in reducing the estrous cycle length compared to jenny's Control cycle. The CL volume was decreased in all treated groups one day after treatment (P < .05). The adverse effects were reduced as the dose of both Prostaglandin F2α analogs were reduced. In conclusion, a single low dose of dinoprost tromethamine (0.625 mg) or cloprostenol sodium (37.5 µg) can induce luteolysis and shorten the estrous length in jennies producing fewer adverse effects.

Keywords: Corpus luteum; Diestrus; Estrous cycle; Jenny; Prostaglandin.

Inadequate luteolysis in fertility programs is a problem for lactating dairy cows treated with a single dose of PGF_2α. The proportion of cows with complete luteolysis can be increased by administering 2 doses of PGF_2α 24 h apart. This study hypothesized that a double dose of cloprostenol sodium (1.0 mg) could take the place of 2 doses 24 h apart due to its enhanced half-life. Cows were allocated to receive 1 of 3 treatments: negative controls: 0.5 mg of cloprostenol sodium (single; n = 337); positive controls: two 0.5-mg doses of cloprostenol sodium 24 h apart (two/24; n = 313); and treated: 1.0 mg of cloprostenol sodium (double; n = 298) at the final PGF_2α of Double-Ovsynch. Cows received artificial insemination (AI) 16 h after final GnRH of Double-Ovsynch. Pregnancy diagnosis was determined at 24, 34, 62, and 184 d post-AI. Pregnancy loss was categorized in the following periods: between 24 to 34, 34 to 62, and 62 to 184 d post-AI. Ultrasonography (B-mode and color Doppler) was used to assess luteal function pre- and posttreatment with various doses of cloprostenol sodium. Luteal volume and luteal blood flow (LBF) from d 7 and 14 corpora lutea were determined before treatment (d -1), and 2 and 4 d after treatment. No evidence was observed of an effect of treatment on pregnancy rates per AI at 24, 34, or 62 d post-AI. No effect was observed of treatment on pregnancy losses occurring between d 24 and 34, 34, and 62, and between 62 and 184 d post-AI. However, third-plus parity cows treated with the single treatment had greater pregnancy loss compared with two/24 and double between d 24 and 34 post-AI. Third-plus parity cows that received the double treatment had lower LBF 2 and 4 d after treatment compared with cows treated with single. Amount of LBF present 4 d after treatment was not a predictor of pregnancy or pregnancy loss. A double dose (1.0 mg) of cloprostenol sodium may be a feasible alternative for fertility programs based on nondifferent outcomes to the two/24 treatment, lower pregnancy losses, and reduced LBF disappearance following treatment in third-plus parity cows in comparison with the single treatment.

Keywords: cloprostenol sodium dose; dairy cow; luteal blood flow; pregnancy.

The aim of the study was to conduct an ultrasound analysis of quantitative parameters of the corpus luteum (CL) in recipient heifers on days 6-8 after oestrus, and to compare reproduction potential of both types of CL in those females. Analyses were performed on 300 heifers, synchronized with two injections of cloprostenol. Clinical and ultrasound examinations of ovaries were performed and measurements of the CL were recorded. The blood samples were taken to determine progesterone level. Pregnancy examination was conducted after 6-8 weeks from the ET. Cavitary CL was found in 32.7% heifers In 48.0% of the cavitary CL, its luteal tissue area was reduced by 14.3% compared to the compact CL, while 16.3% of the CL had luteal tissue reduced by more than 33.8%. Progesterone level in blood serum was higher in heifers with the cavitary CL (p < 0.001). Pregnancy rate was higher for females with a cavitary CL (52%) than those with compact ones (33%, p < 0.05). The ultrasound assessment of luteal tissue should be included in the evaluation of the functional status of the CL in ET-recipient heifers. The cavitary CL presence may indicate a higher potential of the recipient in maintaining the pregnancy.

Keywords: cavitary corpus luteum; compact corpus luteum; corpus luteum; embryo transfer; heifers; ultrasound examination.

This study evaluated the relationship between proestrus length and follicular size, estrous behavior, and pregnancy rate in Bos taurus beef heifers subjected to fixed-time artificial insemination (FTAI). A total of 911 heifers received a synchronization treatment protocol for FTAI (J-Synch) consisting of an intravaginal progesterone device for 6 d, estradiol benzoate at the time of device insertion cloprostenol sodium and eCG at device removal and GnRH at the time of FTAI. The presence or absence of a corpus luteum (CL) was determined by ultrasonography at device insertion and all heifers were tail painted at device removal for estrus detection at the time of FTAI. For the establishment of different periods of proestrus length (i.e., interval from device removal to FTAI), GnRH was administered i.m. at 48 h (n = 308), 60 h (n = 290) or 72 h (n = 313) after device removal. The diameter of the largest follicle at the time of GnRH administration was determined by ultrasonography, expression of estrous was determined by percentage of tail paint removal, and FTAI was performed at the time of GnRH administration in all heifers. The diameter of the largest follicle was greater when GnRH/FTAI was performed at 72 or 60 h (12.9 ± 0.2 mm and 12.8 ± 0.1 mm, respectively) than at 48 h (12.2 ± 0.1 mm, P < 0.05). The proportion of heifers in estrus tended to be greater when GnRH/FTAI was performed at 72 h (77.0%, 137/178) than at 48 h (68.2%, 122/179; P = 0.06), and intermediate at 60 h (71.4%, 120/168). Pregnancy rate tended to be greater in heifers with the longest (72 h: 70.0%, 219/313) than the shortest (48 h: 63.6%, 196/308; P < 0.1) proestrus length, while 60 h proestrus length was intermediate (63.1%, 183/290; P= NS). Pregnancy rate was affected by the presence of a CL at device insertion (71.3%, 352/494 in heifers with a CL, vs. 59.0%, 246/417 for those without a CL; P < 0.01). For those heifers bearing a CL, pregnancy rate was greater in heifers with a 72 h proestrus length (77.0%, 134/174) than with 48 or 60 h proestrus length (67.7%, 107/158 and 68.5%, 111/162; respectively; P < 0.05). In heifers without a CL, proestrus length did not affect pregnancy rate. In summary, extending proestrus length by delaying the interval from device removal to GnRH/FTAI from 48 to 72 h, was associated with a greater diameter of the preovulatory follicle, greater proportion of heifers expressing estrus at the time of FTAI, and greater pregnancy rate in cycling beef heifers.

Keywords: Follicle; Ovulation; Timed artificial insemination; Ultrasonography.

The aims of the present study were to analyze the effect of different hormonal protocols using an implant containing Norgestomet, at the ovulation synchronization in buffaloes and to verify the effect of the stress caused by manipulation of the herd during the experiment. Twenty-four female Murrah breed buffaloes, lactating, multiparous, aged from three to eight years, with a body condition score of 3.5 or higher (0 to 5) and with more than 45 days post partum, were used. These females were randomly distributed into one of the three groups (group 1, group 2 and group 3) with eight subjects in each. On day 0 (day 0), all animals received an ear progesterone implant with 3.0 mg of Norgestomet and an intramuscular (IM) injection with 2.0 mg of estradiol benzoate (EB). The implants were removed after nine days (day 9) and one single dose of PGF2α (0.15 mg d-cloprostenol, IM) was administered to all animals. On the same day, the group´1 and group 3 buffaloes were treated with 500 UI of eCG (IM). Two days later (day 11), 1000UI of hCG (IM) were administered to the group 1 and group 2 buffaloes. After the implant had been removed, an ultrasound evaluation was performed every 12 h, in order to access the ovarian follicular dynamics, using an Aloka 500 equipment with a 5 MHz transrectal probe. Blood samples were also taken on days 0, 9 and 11 to determine the plasmatic concentrations of cortisol and progesterone. No difference was observed in the ovulation rate (group 1: 62.5%, group 2: 50%, group 3: 75%). However, the size of the preovulatory follicles and the plasmatic concentration of cortisol had (P < 0.05). G3 was the most efficient group in promoting the ovulation synchronization, which suggests that this protocol can be used in Fixed Timed Artificial Insemination programs (FTAI) among postpartum, lactating Murrah breed buffaloes'.

Keywords: FTAI; cortisol; eCG; hCG; progesterone.

Aim: Both human and rat myometrium express stromal interaction molecule (STIM) and Orai/ transient receptor potential canonical (TRPC) proteins, which are components of plasma membrane Ca²⁺ store-operated channels. There are reports that these proteins mediate agonist-induced Ca²⁺ influx in cultured myometrial cells. In this study, we aimed to determine the effects of Pyr6, an Orai channel blocker, on different agonist-induced contractions in isolated segments of rat uterus.

Main findings: In Ca²⁺ -free Tyrode's solution, Pyr6 (3 μM) promoted a reduction in both the magnitude and frequency of Ca²⁺ (1 mM)-induced uterine contractions after the addition of carbachol (CCh, 100 μM), but not after the addition of oxytocin (OT, 150 nM). In Ca²⁺ (0.18 mM)-Tyrode's solution, Pyr6 completely relaxed uterine contractions induced by both CCh and cloprostenol (300 nM), but not those induced by either KCI (40-80 mM) or OT. The addition of Pyr6 abolished the oscillatory uterine contractions induced by Ca²⁺ after the addition of cyclopiazonic acid (CPA, 10 μM). When pre-incubated (5 min), Pyr6 reduced the magnitude of both CCh-induced phasic and tonic contractions. The addition of Pyr2 (3 μM), an Orai and TRPC channel blocker, abolished uterine contractions induced by CCh or OT.

Conclusion: Considering Pyr6 as an Orai channel blocker and its inhibitory effect on uterine contractions induced by CCh, CPA, and cloprostenol, we suggest that Orai channels are required for the maintenance of contractions induced by these agonists in rat uterus.

Keywords: Orai channels; Pyr6; TRPC channels; rat uterus; uterine contractions.

Multiple Ovulation and Embryo Transfer (MOET) technology is a potential technique to upgrade livestock species' genetics. The varied response to super-stimulatory treatments remains one of the limiting factors to this technology's widespread use. The present study was aimed to improve the superovulation response and in-vivo embryo production by using controlled internal drug release (CIDR)-GnRH or CIDR-EB (Estradiol Benzoate) along with conventional superovulation protocol in Holstein Frisian (HF): Bos taurus; n = 42) and Crossbred (XB: Cholistani (Bos indicus) × HF; n = 28) cows. In the CIDR-GnRH/CIDR-EB treatment, CIDR was implanted in the cows after confirming the presence of a corpus luteum (CL) on the 8th day after estrus. 2 ml GnRH (Lecirelin acetate 0.0262 mg/ml) or 2 mg EB was also administered in CIDR-GnRH/CIDR-EB groups, respectively. Both groups were given super-stimulatory treatment from the 11th day after estrus (FSH in tapering doses twice a day for four consecutive days). On day 13, two doses of 2 ml prostaglandin (75 µg/ml of dextrorotatory cloprostenol) were administered (am: pm), and CIDR was removed the following day. Two artificial inseminations (AI) of the cows were performed (12 h apart) on the 15th day. No CIDR and GnRH/E.B were given in the control group, but the remaining superovulation protocol was the same. Later on, seven days after the first AI, non-surgical embryo flushing was done. The transferable embryos produced from three different superovulation protocols were then transferred into the recipient cows (n = 90) for determining their fertility. Statistical analysis revealed that the number of super-estrus follicles (SEF), multiple corpora lutea (MCL), ovulation/fertilization percentage, fertilized structures recovered (FSR), and transferable embryos (TEs) remained significantly higher (p < 0.05), and days taken for return to estrus (RTE) after embryo collection remained significantly lower (p < 0.05) in CIDR-GnRH (n = 18) and CIDR-EB (n = 15) groups as compared to the control (n = 37). The comparison between XB and HF cows revealed that the TEs production in CIDR-GnRH (XB = 5 vs HF = 13) and CIDR-EB (XB = 6 vs HF = 9) based superovulation protocols were 11.60 ± 4.08 vs 04.31 ± 0.98 and 09.33 ± 1.78 vs 05.22 ± 1.36, respectively. TEs production in XB cows (n = 5) of the CIDR-GnRH group was significantly higher (11.60 ± 4.08) than other groups. On the other hand, the days taken for RTE after embryo collection remained significantly lower (p < 0.05) in HF cows of treatment groups. However, the fertility of TEs was neither affected significantly (p > 0.05) by the superovulation protocol used nor by breed differences among donor cows. In conclusion, using CIDR-GnRH or CIDR-EB along with conventional superovulation protocol may enhance the efficiency of MOET programs in cattle. Furthermore, XB donor cows demonstrated a better performance than HF donor cows under subtropical conditions.

Keywords: Bovine embryo transfer; ETT in Pakistan; Improved in-vivo embryo production; Synchronization of the follicular wave; Wave emergence based superovulation.

We aimed to compare the effect of three estradiol benzoate (EB) doses on follicular wave emergence (FWE) and dominant follicle growth of suckled Nelore cows submitted to TAI (D0). On a random day of estrous cycle (D-10), multiparous (MULT; n=36) and primiparous (PRIM; n=20) suckled Nelore cows received an intravaginal progesterone (P4) device and were assigned in three groups. Cows in the EB-1 (n=20), EB-1.5 (n=15) or EB-2 (n=21) groups received, respectively, an im treatment with 1, 1.5 or 2 mg EB. A subgroup (n=10-13 cows/group) were subject to daily ovarian evaluations from D-10 to D0. On D-2, P4 devices were removed, and all cows received the same treatment: 1 mg estradiol cypionate, 0.53 mg sodium cloprostenol, and 300 IU eCG. Statistical analyses were performed considering only the main effects of treatment group and parity order. The proportion of cows with a synchronized FWE and the moment of the FWE did not differ (p>0.05) among the treatment groups (overall: 80% [28/35] and 4.1 ± 0.4 days); however, the FWE occurred earlier (p=0.007) in MULT (3.8 ± 0.2 days) than PRIM (5.1 ± 0.4) cows. The proportion of animals detected in estrus was greater (86% [31/36] vs. 70% [14/20]; p=0.02) and the dominant follicle was larger on D-2 (9.7 ± 0.3 mm vs. 7.8 ± 0.7 mm; p=0.006) and D0 (11.9 ± 0.4 mm vs. 10 ± 0.5 mm; p=0.008) in MULT than PRIM cows. In conclusion, the three EB doses presented similar efficiency to synchronize the FWE in suckled Nelore cows. Moreover, a delayed FWE and smaller dominant follicle is observed in PRIM cows, contributing to the reduced reproductive performance in this parity category when using similar TAI protocols of MULT cows.

Keywords: Bos indicus; cattle; estradiol benzoate; follicular dynamics; multiparous; primiparous.

Objective: To investigate if modification of a gonadotrophin-releasing hormone (GnRH) based protocol to synchronise oestrus in Bos indicus cattle will improve response rates to the first administration of GnRH and improve pregnancy rates to artificial insemination (AI).

Design: Randomised control study.

Methods: Bos indicus heifers, nonlactating and lactating cows allocated to a GnRH-18 treatment (n = 237) were treated with an intravaginal device (IVD) and cloprostenol (0.5 mg IM) on day -11 and on day 0 remaining animals in the GnRH-7 treatment (n = 222) were administered an IVD and every animal was treated with GnRH (100 μg IM). On day 7, equine chorionic gonadotrophin (400 IU IM) and cloprostenol were administered and IVD's were removed. Animals detected in oestrus on day 9 were artificially inseminated while those not detected in oestrus were administered GnRH (100 μg IM) at 1700 hours and inseminated on day 10. Bulls were inserted 2 weeks after completion of AI and remained until day 65.

Results: The GnRH-18 protocol increased the diameter of the largest follicle in the ovary on day 0, increased the percentage of new CL's induced after day 0 (46.3% vs 36.1%, for GnRH-18 and GnRH-7; P = 0.022), decreased circulating concentrations of progesterone on day 7 and increased odds of pregnancy to AI in heifers but not in nonlactating and lactating cows.

Conclusion: Treatment with the GnRH-18 compared to the GnRH-7 protocol increased pregnancy rates to AI in heifers but not in nonlactating or lactating cows.

Keywords: AI; Bos indicus; GnRH; oestrous synchronisation.

Effects of conservative treatment on uterine blood flow and morphometric findings are still unknown in bitches. Thus, this study aimed to compare uterine changes of pyometra bitches subjected to distinct modes of treatment. Pyometra bitches were assigned to: OHE (ovariohysterectomy immediately after diagnosis), Aglepristone (days 1, 2 and 8) and Associative (aglepristone treatment coupled with cloprostenol for 7 days) groups. After 9 days, bitches were ovariohysterectomized. Before surgery, uterine area was measured ultrasonographically and the uterine artery Doppler velocimetry analyzed blood flow velocity and indexes. Uterine horns were classified according to resistance index (RI) as more compromised and less compromised. Endometrial vasculature was quantitatively evaluated by color flow Doppler. Blood samples were collected to determine nitric oxide (NO) concentrations. Histological uterine structures were quantified by stereology and VEGF-A (vascular endothelial growth factor) and eNOS were (endothelial nitric oxide synthase) immunohistochemically analyzed. Aglepristone and Associative groups had lower uterine area and vascularization, and higher blood flow velocity and indexes compared to OHE group. Less compromised horn of Associative group had higher blood flow velocity compared to OHE group. Aglepristone group presented lower inflammatory infiltrate and larger uterine stroma. Associative group had lower volume density and absolute surface of endometrial cysts and lower VEGF-A expression for glandular epithelium and stromal cells. Blood NO and e-NOS immunostaining were not different among groups. In conclusion, association between aglepristone and prostaglandin is more effective in decreasing uterine vascularization and modulating uterine blood flow. Moreover, associative therapy promotes marked morphological changes.

Lay summary: This research compared two medical protocols of treatment for uterine infection (pyometra) in bitches, using a hormone blocker (anti-progesterone aglepristone) solely or in association with a uterine contraction inducer (prostaglandin; associative therapy). After treatment, bitches were gonadectomized and a microscopic analysis of uterine blood vessel formation and uterine tissue elements were performed as well as uterine blood flow evaluation through Doppler ultrasonography. According to vascular resistance, uterine horns were additionally classified as more compromised and less compromised. Both treatment protocols led to reduction of uterine dimensions and vascularization, and higher blood flow compared to untreated bitches. Less compromised uterine horn of the associative treatment had higher blood flow compared to untreated bitches. The hormone blocker treatment had lower inflammatory cells and larger uterine histological structure, while associative treatment had less uterine pathological cysts and lower blood vessel formation. The associative therapy is effective in decreasing uterine vascularization and modulating uterine blood flow as well as reestablishing endometrium structure in bitches with uterine infection.

Keywords: Doppler; aglepristone; angiogenesis; nitric oxide; uterus.