Circulating P4 Concentrations Research Articles

204 PERIPHERAL PROGESTERONE CONCENTRATION AFFECTS BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

The developmental competence of cumulus–oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175–176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation = Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5 mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1 ng mL–1), follicle phase 1 (Fp; P4 <1 ng mL–1), and induced CL phase (iCL; P4: ≥1 ng mL–1). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4 ± 3.1 days v. 23.8 ± 1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9 ± 2.3 ng mL–1 v. 3.0 ± 1.6 ng mL–1). For mRNA analysis, denuded COC were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

109 EFFECT OF SHORT TERM PROGESTERONE SUPPLEMENTATION ON CIRCULATING PROGESTERONE CONCENTRATION, CORPUS LUTEUM SIZE, AND EARLY EMBRYO DEVELOPMENT IN CATTLE

The aim of this study was to investigate the effect of short term progesterone (P4) supplementation on circulating P4 concentrations, corpus luteum (CL) size, and conceptus development in cattle. The oestrous cycles of crossbred beef heifers were synchronised using a 7-day PRID® Delta (1.55 g P4) treatment with administration of a PGF2α analog (Enzaprost®) the day before PRID® Delta removal. Only those recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to 1 of 5 groups: (1) control: no treatment, (2) placebo: insertion of a blank device (no P4) from Day 3 to 7, (3) insertion of a PRID® Delta from Day 3 to 7, (4) insertion of a PRID® Delta from Day 3 to 5, or (v5) insertion of a PRID® Delta from Day 5 to 7. In vitro produced blastocysts were transferred to each heifer on Day 7 (10 blastocysts per heifer) and conceptuses were recovered at slaughter on Day 14. In Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to 1 of 3 treatment groups (1) placebo, (2) PRID® Delta from Day 3 to 5, or (3) PRID® Delta from Day 3 to 7. All heifers were slaughtered on Day 16, and recovered conceptuses were incubated in synthetic oviduct fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-tau (IFNT). In both experiments, daily blood samples were taken to measure serum P4 concentration. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA). Insertion of a PRID® Delta resulted in an increase (P < 0.05) in serum P4, which declined following removal. In Experiment 1, serum P4 concentration was significantly lower from Day 9 to 14 (P < 0.05) and Day 14 CL weight was lower in the PRID® Delta Day 3 to 7 group than the placebo or control groups. P4 supplementation from Day 3 to 5 (17.0 ± 1.4 mm) or Day 3 to 7 (11.3 ± 2.3 mm) increased conceptus length compared to the placebo (2.1 ± 1.8 mm). In Experiment 2, serum P4 was significantly lower in the two supplemented groups following PRID® Delta removal compared with the placebo (P < 0.05) and was associated with a lower CL weight in the Day 3 to 7 group. Supplementation from Day 3 to 5 (94.0 ± 18.8 mm) or Day 3 to 7 (143.6 ± 20.6 mm) increased conceptus length on Day 16 compared to the placebo (50.3 ± 17.4 mm). Conceptus length was strongly correlated with the concentration of IFNT in the uterine flush (r = 0.58; P = 0.011) and spent culture medium (r = 0.68; P < 0.002). These findings highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrus period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan. Supported by CEVA Sante Animale and Science Foundation Ireland (07/SRC/B1156).

Immunolocalization of Progesterone Receptors in the Canine Oviduct around Ovulation

In the bitch, oocyte maturation, sperm storage, fertilization and early embryo development take place within the oviducts under high and increasing circulating progesterone concentrations. To investigate the potential effects of progesterone on the canine oviduct, nuclear progesterone receptors (PR) were localized. Oviducts were collected by ovariectomy from adult Beagle bitches during anestrus, after the onset of proestrus but prior to the Luteinizing Hormone (LH) peak (Pre-LH), after the LH peak but before ovulation (Pre-ov) and on Days 1, 4 and 7 after ovulation (n = 3 bitches per stage). The cellular distribution of PR was studied by immunohistochemistry (IHC) in the ampulla, isthmus and tubal part of the utero-tubal junction (UTJ). Plasma progesterone and 17β-oestradiol were assayed on the day of surgery. PR were specifically expressed in the nuclei of epithelial, stromal and muscular cells in the ampulla, isthmus and UTJ. The IHC scores did not vary from one oviductal region to another. However, the epithelium displayed higher scores than the stroma at anestrus, Pre-ov, Days 4 and 7, and also higher scores than muscle at Days 4 and 7 (p < 0.05). Immunohistochemistry scores in the stroma and muscle decreased at Days 4 and 7 compared with previous stages (p < 0.05). Furthermore, muscular IHC scores were positively correlated with circulating 17β-oestradiol concentrations and negatively correlated with circulating progesterone concentrations (p < 0.05). In conclusion, PR were identified in the canine oviduct, with differences in expression between tissues and times around ovulation, suggesting that progesterone may regulate tubal functions and reproductive events in this species.

Effects of treatment with human chorionic gonadotrophin or intravaginal progesterone-releasing device after AI on circulating progesterone concentrations in lactating dairy cows

Adequate circulating progesterone (P4) is important for pregnancy. Lactating dairy cattle have lower circulating P4, particularly when smaller follicles are ovulated during timed AI protocols. The aim of the present study was to determine the supplementation strategy that resulted in P4 concentrations in lactating dairy cattle similar to those in heifers. Lactating Holstein cows (n=61) were synchronised using the Double-Ovsynch method and, on Day 5, were randomly assigned to receive no treatment (control), controlled internal drug release (CIDR), human chorionic gonadotrophin (hCG; 3300 IU) or CIDR+hCG. Heifers after normal oestrus were followed as controls (n=10). Profiles of circulating P4 concentrations were compared using repeated-measures ANOVA. Heifers had greater P4 concentrations than control cows at all times after Day 5 (P<0.0001). Cows receiving CIDR had lower P4 concentrations than heifers (P=0.0037) on Days 8-16. Treatment with hCG generally caused ovulation and resulted in circulating P4 concentrations greater than those in control lactating cows by 3 days after treatment (Day 8 after AI), but the treatment×time interaction (P=0.01) showed that cows treated with hCG generally had lower P4 concentrations than heifers. Supplementation with CIDR+hCG resulted in P4 concentration profiles similar to those in heifers. Thus, the use of CIDR and the production of an accessory corpus luteum with hCG elevates P4 concentrations in lactating cows to those seen in heifers. This information may be useful for designing future trials into P4 supplementation and fertility.

Effect of progesterone on magnitude of the luteinizing hormone surge induced by two different doses of gonadotropin-releasing hormone in lactating dairy cows

Ovulation to the first GnRH injection of Ovsynch-type protocols is lower in cows with high progesterone (P4) concentrations compared with cows with low P4 concentrations, suggesting that P4 may suppress the release of LH from the anterior pituitary after GnRH treatment. The objectives of this study were to determine the effect of 1) circulating P4 concentrations at the time of GnRH treatment on GnRH-induced LH secretion in lactating dairy cows and 2) increasing the dose of GnRH from 100 to 200μg on LH secretion in a high- and low-P4 environment. A Double-Ovsynch (Pre-Ovsynch: GnRH, PGF2α 7d later, GnRH 3d later, and Breeding-Ovsynch 7d later: GnRH, PGF2α 7d later, and GnRH 48h later) synchronization protocol was used to create the high- and low-P4 environments. At the first GnRH injection of Breeding-Ovsynch (high P4), all cows with a corpus luteum ≥20mm were randomly assigned to receive 100 or 200μg of GnRH. At the second GnRH injection of Breeding-Ovsynch (low P4) cows were again randomized to receive 100 or 200μg of GnRH. Blood samples were collected every 15min from −15 to 180min after GnRH treatment, and then hourly until 6h after GnRH treatment. As expected, mean P4 concentrations were greater for cows in the high- than the low-P4 environment. For cows receiving 100μg of GnRH, the LH peak and area under the curve (AUC) were greater in the low- than in the high-P4 environment. Similarly, for cows receiving 200μg of GnRH, the LH peak and AUC were greater in the low- than the high-P4 environment. Cows receiving 100 or 200μg of GnRH had greater mean LH concentration in the low- than the high-P4 environment from 1 to 6h after GnRH treatment. On the other hand, when comparing the effect of the 2 GnRH doses in the high- and low-P4 environments, cows receiving 200μg of GnRH had a greater LH peak and AUC than cows treated with 100μg of GnRH both in the high- and low-P4 environments. For the high-P4 environment, mean LH was greater from 1.5 to 5h after GnRH treatment for cows receiving 200μg of GnRH than for those receiving 100μg of GnRH. In the low-P4 environment, mean LH was greater for cows receiving 200μg of GnRH than for those receiving 100μg of GnRH from 1 to 2.5h after GnRH treatment. We conclude that the P4 environment at GnRH treatment dramatically affects GnRH-induced LH secretion, and that a 200-μg dose of GnRH can increase LH secretion in either a high- or a low-P4 environment.

85 EFFECT OF FOLLICULAR ASPIRATION JUST PRIOR TO OVULATION ON CORPUS LUTEUM CHARACTERISTICS, CIRCULATING PROGESTERONE CONCENTRATIONS AND UTERINE RECEPTIVITY IN SINGLE-OVULATING BEEF HEIFERS

Progesterone (P4) has a crucial impact on the transcriptome of the uterine endometrium and the preparation of the uterus to support implantation. The aim of this study was to investigate the effect of follicle aspiration just before ovulation on corpus luteum (CL) development, circulating P4 concentrations and the ability of the uterus to support embryo development and conceptus elongation. We tested the hypothesis that the unavoidable loss of follicular fluid and some granulosa cells during aspiration of the preovulatory follicle would compromise the development and function of the developing CL and that this would be associated with reduced P4 and a poorer uterine environment. Oestrous cycles of crossbred beef heifers were synchronized using an 8-day CIDR treatment with administration of a prostaglandin F2α analogue on the day before CIDR removal to ensure CL regression. Heifers were checked for signs of oestrus 4 times per day commencing 30 h after CIDR withdrawal and only those recorded in standing oestrus (Day 0, n = 20) were used. All heifers received a gonadotropin-releasing hormone agonist (0.01 mg buserelin) 48 h after CIDR removal to induce an LH surge. Half of the animals underwent follicle aspiration 20 h later, while the remainder underwent ovulation. Daily transrectal ultrasonography was carried out from Day 3 to 13 to record CL development. Daily blood samples were collected from Day 0 to 14 for circulating P4 concentrations. To test the ability of the uterus to support embryo development and conceptus elongation, Day 7 in vitro-produced blastocysts were transferred to the uteri of synchronised recipients (7 to 10 blastocysts per recipient). All recipients were slaughtered on Day 14 to assess embryo survival and conceptus size. CL diameter and CL area were significantly reduced in the follicle aspirated group compared with controls from Day 6 onwards (P ≤ 0.05). Similarly, at slaughter on Day 14, CL weight (4.17 ± 0.48 vs 7.05 ± 1.65 mm), diameter (19.89 ± 1.35 vs 24.64 ± 2.07 mm) and area (321.94 ± 45.01 vs 510.18 ± 69.41 mm2) were lower in aspirated heifers (P ≤ 0.05). Circulating P4 concentrations were lower at all time points from Day 3 to Day 14 but were only significantly lower from Day 12 onwards (P ≤ 0.05). Conceptus length (2.08 ± 0.29, n = 56 vs 4.55 ± 0.78 mm, n = 45) and area (2.52 ± 0.39 vs 5.61 ± 1.12 mm2) were lower (P ≤ 0.05) in heifers undergoing follicular aspiration compared with those undergoing ovulation. In conclusion, aspiration of the preovulatory dominant follicle just before expected ovulation was associated with a compromised CL in terms of size and P4 output and this, in turn, was associated with a reduced capacity of the uterus to support the initiation of conceptus elongation. Supported by Science Foundation Ireland (07/SRC/B1156).

126 INFLUENCE OF PERIPHERAL PROGESTERONE CONCENTRATION ON MORPHOLOGICAL OOCYTE QUALITY IN REPEATED OVUM PICKUP SESSIONS

Today, ovum pickup (OPU) followed by in vitro production (IVP) of bovine embryos is an integral part of many breeding programs. The quality of the obtained cumulus–oocyte complexes (COC) limits the success of embryo production. The developmental competence of the COC is dependent on several factors, including the stage of the oestrous cycle, the stage of follicular development and the follicular diameter. The aim of the present study was to determine the influence of circulating progesterone (P4) concentrations during repeated OPU sessions on the morphological quality of bovine COC. Cumulus–oocyte complexes were aspirated twice weekly for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle (ovulation = Day 0). During each session, number and diameter of the punctuated follicles and diameter, consistency and cavities in the corpus luteum (CL) were recorded. Follicles were assigned to 3 groups according to their diameter (3 to 5 mm = small follicles; 6 to 8 mm = intermediate follicles; &gt;8 mm = large follicles). Additionally, blood samples were taken at the time of each OPU session and blood P4 concentration was determined using a radioimmunoassay. The COC were categorised as IVP-suitable (round, ≥3 layers of cumulus cells, homo- or heterogeneous ooplasm) or unsuitable according to their morphological quality. All animals showed signs of oestrus accompanied by the presence of large follicles during the course of the OPU sessions. Statistical analysis was performed by an ANOVA followed by a Tukey test. A P-value of &lt;0.05 was considered significant. The mean (±s.e.m.) cycle lengths for all heifers were 23.8 ± 4.6 days. Following the aspiration of a large follicle, a CL-like structure could be detected (referred to as “induced CL”). According to the P4 concentrations, the cycle was divided into 4 phases: natural CL phase (nCL; P4 ≥1 ng mL–1), follicle phase 1 (Fp1; P4 &lt;1 ng mL–1), induced CL phase (iCL; P4 ≥1 ng mL–1), or follicle phase 2 (Fp2; P4 &lt;1 ng mL–1). During the nCL phase, blood P4 concentrations were significantly higher than during the iCL phase (4.9 ± 2.3 ng mL–1, n = 12 vs 3.0 ± 1.6 ng mL–1, n = 10). There were no differences in follicle numbers, the diameter distribution of follicles, recovery rates, or number of retrieved IVP-suitable COC (nCL: 3.1 ± 3.4; Fp1: 3.3 ± 3.7; iCL: 2.7 ± 3.0; Fp2: 3.7 ± 3.7; Table 1). In summary, circulating P4 concentrations had no effect on follicle number, diameter, recovery rate, or IVP suitability of recovered COC. Table 1.Number of follicles, retrieved oocytes, follicle sizes and recovery rates of the ovum pickup (OPU) sessions

Improving fertility to timed artificial insemination by manipulation of circulating progesterone concentrations in lactating dairy cattle

This manuscript reviews the effect of progesterone (P4) during timed AI protocols in lactating dairy cows. Circulating P4 is determined by a balance between P4 production, primarily by the corpus luteum (CL), and P4 metabolism, primarily by the liver. In dairy cattle, the volume of luteal tissue is a primary determinant of P4 production; however, inadequate circulating P4 is generally due to high P4 metabolism resulting from extremely elevated liver blood flow. Three sections in this manuscript summarise the role of P4 concentrations before breeding, near the time of breeding and after breeding. During timed AI protocols, elevations in P4 are generally achieved by ovulation, resulting in an accessory CL, or by supplementation with exogenous P4. Elevating P4 before timed AI has been found to decrease double ovulation and increase fertility to the timed AI. Slight elevations in circulating P4 can dramatically reduce fertility, with inadequate luteolysis to the prostaglandin F2α treatment before timed AI being the underlying cause of this problem. After AI, circulating P4 is critical for embryo growth, and for establishment and maintenance of pregnancy. Many studies have attempted to improve fertility by elevating P4 after timed AI with marginal elevations in fertility. Thus, previous research has provided substantial insights into mechanisms regulating circulating P4 concentrations and actions. Understanding this prior research can focus future research on P4 manipulation to improve timed AI protocols.

The effect of progesterone replacement on gene expression in the corpus luteum during induced regression and late luteal phase in the bonnet monkey (Macaca radiata)

BackgroundIn higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles.MethodsTo identify differentially expressed genes encoding PR, PR binding factors, cofactors and PR downstream signaling target genes, the genome-wide analysis data generated in CL of monkeys after LH/P4 depletion and LH replacement were mined and validated by real-time RT-PCR analysis. Initially, expression of these P4 related genes were determined in CL during different stages of luteal phase. The recently reported model system of induced luteolysis, yet capable of responsive to tropic support, afforded an ideal situation to examine direct effects of P4 on structure and function of CL. For this purpose, P4 was infused via ALZET pumps into monkeys 24 h after LH/P4 depletion to maintain mid luteal phase circulating P4 concentration (P4 replacement). In another experiment, exogenous P4 was supplemented during late luteal phase to mimic early pregnancy.ResultsBased on the published microarray data, 45 genes were identified to be commonly regulated by LH and P4. From these 19 genes belonging to PR signaling were selected to determine their expression in LH/P4 depletion and P4 replacement experiments. These 19 genes when analyzed revealed 8 genes to be directly responsive to P4, whereas the other genes to be regulated by both LH and P4. Progesterone supplementation for 24 h during the late luteal phase also showed changes in expression of 17 out of 19 genes examined.ConclusionThese results taken together suggest that P4 regulates, directly or indirectly, expression of a number of genes involved in the CL structure and function.

Changes in the Endometrial Transcriptome During the Bovine Estrous Cycle: Effect of Low Circulating Progesterone and Consequences for Conceptus Elongation

In cattle, elevated concentrations of circulating progesterone (P4) in the immediate postconception period are associated with advanced conceptus development, while low P4 is implicated as a causative factor in low pregnancy rates observed in dairy cows. This study aimed to: 1) describe the transcriptional changes that occur in the bovine endometrium during the estrous cycle, 2) determine how elevated P4 affects these changes, 3) identify if low P4 alters the expression of these genes, and 4) assess the impact that low P4 has on conceptus development. Relatively few differences occurred in endometrial gene expression during the early luteal phase of the estrous cycle (Day 5 vs. 7), but comparison of endometria from more distant stages of the luteal phase (Day 7 vs. 13) revealed large transcriptional changes, which were significantly altered by exogenous supplementation of P4. Induction of low circulating P4 altered the normal temporal changes in gene expression, and these changes were coordinate with a delay in the down-regulation of the PGR from the LE and GE. Altered endometrial gene expression induced by low P4 was associated with a reduced capacity of the uterus to support conceptus development after embryo transfer on Day 7. In conclusion, the present study provides clear evidence that the temporal changes in the transcriptome of the endometrium of cyclic heifers are sensitive to circulating P4 concentrations in the first few days after estrus. Under low P4 conditions, a suboptimal uterine environment with reduced ability to support conceptus elongation is observed.

Effects of rumen-protected polyunsaturated fatty acid supplementation on reproductive performance of Bos indicus beef cows1

Five experiments evaluated the effects of rumen-protected PUFA supplementation on reproductive function of Bos indicus beef cows. In Exp. 1, 910 lactating primiparous Nelore cows were randomly assigned to receive 0.4 kg/d of a protein-mineral mix in addition to 0.1 kg/d of a rumen-inert PUFA source (PF) or 0.1 kg/d of kaolin (rumen-inert indigestible substance; control), from the beginning of estrus synchronization protocol (d -11) until 28 d after fixed-time AI (TAI; d 28). Cows supplemented with PF had greater (P = 0.04) pregnancy rates compared with control cows (51.2 vs. 39.6%). In Exp. 2, 818 lactating primiparous Nelore cows were assigned to the same TAI schedule from Exp. 1 and randomly allocated to receive 1) control from d -11 to 28, 2) PF from d -11 to 16 and control from d 17 to 28, or 3) PF from d -11 to 28. Cows receiving PF until d 28 had greater (P = 0.02) pregnancy rates compared with control cows and tended to have greater (P = 0.10) pregnancy rates compared with cows receiving PF until d 16 (42.9, 31.3, and 35.8%, respectively). In Exp. 3, 435 nulliparous and multiparous lactating B. indicus-crossbred cows were randomly assigned to receive control or PF from the end of synchronization protocol (d 0) until 21 d after fixed-time embryo transfer (d 28). Cows supplemented with PF had greater (P = 0.07) pregnancy rates compared with control cows (49.6 vs. 37.7%). In Exp. 4, 504 lactating multiparous Nelore cows were randomly assigned to receive PF or a similar supplement containing a rumen-protected SFA source (SF) for 28 d beginning after TAI. Cows supplemented with PF had greater (P = 0.02) pregnancy rates compared with SF cows (47.9 vs. 35.5%). In Exp. 5, 9 nonlactating, nonpregnant, ovariectomized Gir x Holstein cows inserted with an intravaginal progesterone (P4)-releasing device were stratified by BW and BCS and divided into 3 squares. Squares were randomly assigned to receive control, PF, or a protein-mineral mix containing 0.2 kg/d of rumen-inert PUFA source (PF2), in a Latin square 3 x 3 design containing 3 periods of 14 d each. Cows supplemented with PF had greater (P = 0.03) mean serum P4 concentrations compared with control and PF2 cows (1.81, 1.66, and 1.68 ng/mL, respectively). These results indicate that supplementing 0.1 kg/d of rumen-inert PUFA to beef cows, particularly after breeding, may be a method of enhancing their reproductive performance, perhaps by increasing circulating P4 concentrations.

Luteal function in jennies following PGF2α treatment 3 days after ovulation

Native PGF(2alpha) and its analogs have been used in the horse mare to manipulate ovarian activity, primarily as luteolytic agents to induce estrus. Despite numerous studies on the effects of these luteolysins in the mare, to date only a single investigation has been conducted in the jenny. The aim of this study was to evaluate the response of the corpus luteum (CL) to a single dose of PGF(2alpha) given 3 days (72h) after ovulation and to establish the plasma progesterone (P4) profile from pre-treatment to post-treatment ovulation in the Martina Franca donkey. Twenty-two jennies were ultrasonographically monitored and treated 72h after the detection of ovulation with 0.075 mg i.m. of R-cloprostenol. From the day of ovulation until ovulation post-treatment, blood was collected daily for P4 determination by enhanced luminescence immunoassay. All the jennies except one, exhibited behavioral signs of PGF(2alpha)-induced estrus within 4 days of treatment lasting 5.4+/-1.16 days. Post-treatment ovulation was also hastened, reducing the interovulatory interval (9.6 days). In response to treatment, plasma P4 concentrations fell to estrus levels and then remained constant until the next ovulation in all but the non-responding animal. Our findings indicate that PGF(2alpha) treatment on Day 3 post-ovulation causes the functional regression of the CL in the jenny, reflected both by the rapid induction of estrus and ovulation and by an abrupt drop in circulating P4 concentrations.

238 THE EFFECT OF TRANSVAGINAL FOLLICULAR ASPIRATION AND THE ROLE OF PROGESTERONE IN FOLLICULAR CYSTS OF DAIRY CATTLE

Ovarian follicular cyst (CYS) is a common condition affecting 5-25% of postpartum dairy cows, increasing the number of days open, calving intervals, cow costs, and culling rates, and with an effect on fertility rates. Current hormonal treatment has variable responses. Debate exists regarding progesterone (P4) influence on CYS formation and the effect of the aspirated CYS in resumption of cyclicity. The objectives of this descriptive study were to investigate the effect of ultrasound-guided transvaginal follicular aspiration on the resumption of follicular dynamics, to determine the fate of the aspirated CYS, and to find the P4 relationship. Resumption of ovarian cyclicity was determined by ovulation, formation of a corpus luteum (CL), and a P4 rise over 3.2 nmol/L. Ten (10) postpartum (35-60 days in milk (DIM)) lactating Holstein cows with CYS were selected. They were monitored every other day by rectal palpation, ultrasonography, and P4 concentration for 30 days post-aspiration. Change in size of the aspirated CYS and emergence of a new follicular wave were recorded and images were saved electronically. Results were analyzed using Pearson correlations, linear regression, and one-way ANOVA. Of these cows, 5/10, 3/10, and 1/10 resumed cyclicity after the 1st, 2nd, and 3rd aspirations, respectively. Two (2/10) cows did not resume cyclicity during the observation period. Size of aspirated CYS was 34.6 � 0.2 mm (mean � SEM). Seven (7/10) cows had intermediate (0.98 � 0.3 nmol/L) and 3/10 cows had high (8.6 � 3.6 nmol/L) P4 concentration at aspiration. Mean times from aspiration to emergence of a new follicle, to ovulation, and to detection of a new CL were 3.6 � 1.4, 10.5 � 2.0, and 15.2 � 3.0 days, respectively. New cysts grew in 4/10 cows after the first aspiration and one aspirated CYS refilled twice. Dominant follicles present at first aspiration (DFPF) had the tendency to undergo cyst turnover (4/9) or to ovulate (2/9) at a larger size (25.7 � 3.2 mm). The CYS size was correlated (r = 0.58; P = 0.05) with circulating P4 concentration. These data, together with a significant (P = 0.05) drop in P4 concentration following aspiration (from 3.26 � 1.5 to 2.16 � 0.1 nmol/L), suggest that the CYS may be the source of P4. Persistence of the aspirated cyst was associated (r = 0.87) with increased days to ovulation. The significant association (r = 0.96; P = 0.01) between P4 at ovulation (2.29 � 0.6 nmol/L) and the size of the ovulatory follicle (18.5 � 2.0 mm) may explain the tendency of existing follicles to persist (cyst turnover) or to ovulate at a larger size. In summary, follicular aspiration was successful in inducing resumption of cyclicity and shortening days to ovulation in half of the cases with one aspiration. Persistence of the aspirated CYS increased days to ovulation. P4 may have a follicular origin and an influence on the size of the cyst and the ovulatory follicle. DFPF tend to undergo cyst turnover or to ovulate at a larger size. However, resumption of normal cyclicity may also be influenced by other factors, such as stress, disease, and energy balance.