Effect of breeding season and age on follicular dynamics and hemodynamics in embryo donor mares subjected to luteolysis after embryo flushing.

Background: Mares are the only companion animals simulating women in the large diameter of their follicles. Horses start reproduction at the age of three years, and some of them live for >30 years, so aging influences their reproductive capacity. Mares are sensitive to summer heat stress as they can sweat like humans. Aim: The current work aimed to study the effects of age (young versus senile), season (cold versus hot), and the hormonal treatments during embryo collection on the dominant and subordinate follicular dynamics and hemodynamics and circulating ovarian hormones in embryo donor mares ovulated twice spontaneously before inducing ovulation for flushing embryos. Methods: Spontaneous oestrous cycles were studied for young mares (<10 years; N = 6) or senile (>20 years; N = 5) during months of the cold season (November to April) and hot season (May to August). In young embryo donor mares, oestrous cycles after inducing ovulation and luteolysis were studied using Doppler ultrasound. Estradiol (E2), progesterone (P4), nitric oxide (NO), total cholesterol, and lactate dehydrogenase (LDH) were measured in blood serum. Results: A decrease in the dominant follicle antrum diameter (p > 0.05) and LDH (p = 0.016) was observed after inducing luteolysis in young embryo donor mares. Both human chorionic gonadotropin (hCG) and PGF2α treatments increased dominant follicle area (p = 0.0001), antrum area (p = 0.001), perimeter (p = 0.001), granulosa area (p = 0.0001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001). The dominant follicle area, antrum area, perimeter, color area, granulosa area, LDH, cholesterol, NO, and E2 increased (p = 0.0001) during the oestrous cycles of the hot season, but the circulatory % (p = 0.0001) declined. Senile mares had lower dominant follicle area (p = 0.002), antrum area (p = 0.0001), granulosa area (p > 0.05), LDH (p = 0.001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001) but higher circulatory % (p = 0.0001) and color area % (p = 0.023). The dominant follicle possesses the largest diameter, area, perimeter, granulosa area, and color area but the lowest circulatory % during spontaneous oestrous cycles, after inducing ovulation, or luteolysis with significant effects of the day of the spontaneous oestrous cycles on their dynamics and hemodynamics. Conclusion: During hot months, mares treated with hCG ovulated 24 hours later and prostaglandin-induced luteolysis was followed by new ovulation five days later. Follicles ovulated during the hot months were larger than those ovulated during the cold months and both had nearly the same color area %. Senile mares ovulated follicles with a lower area and antrum area but a higher color area %, so senile mares can be used as embryo or oocyte donors during the hot season.

Effects of an ω3 fatty acid-biased diet on luteolysis, parturition, and uterine prostanoid synthesis in pregnant mice.

The ω3 polyunsaturated fatty acids (PUFAs) are known to have beneficial effects on health and diseases, and hence their intake is encouraged. However, it remains unknown as to how ω3 PUFAs affect female reproduction processes, in which ω6 PUFA-derived prostaglandin (PG) E2 and PGF2α play crucial roles. We therefore compared female reproductive performance between ω3 PUFA-biased linseed oil diet-fed (Lin) mice and ω6 PUFA-biased soybean oil diet-fed (Soy) mice. In Lin mice, the uterine levels of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were 0.42 fold and 16 fold of those in Soy mice, respectively, with the EPA/AA ratio being 0.7 (vs 0.02 in Soy mice). Lin mice showed no alterations in any of the fertility indexes, including luteolysis and parturition. The uterine PG synthesis profiles of Lin mice were similar to those of Soy mice, but the levels of PGF2α and PGE2 were 50% of those in Soy mice, as a result of the increased EPA/AA ratio. PGF3α and PGE3 were undetectable in the uterine tissues of Soy and Lin mice. Interestingly, in Lin mice, 'luteolytic' PGF2α synthesis was considerably maintained even in the ω6 PUFA-reduced condition. These results suggest the existence of an elaborate mechanism securing PGF2α synthesis to a level that is sufficient for triggering luteolysis and parturition, even under ω6 PUFA-reduced conditions.

Clinical prospects proposing an increase in the luteolytic dose of prostaglandin F2α in dairy cattle.

Over the past few decades, the luteolytic dose of prostaglandin F2α (PGF2α) and its analogs, used to synchronize estrus for fixed-time insemination in dairy cattle, have remained unchanged. Given the beneficial effects of PGF2α on a young corpus luteum and on multiple ovulations in a fixed-time insemination protocol, and its therapeutic abortive effects on multiple ovulations in pregnant cows, we propose the use of a double PGF2α dose or two PGF2α treatments 24 hours apart. Ultrasonography procedures serve to identify luteal structures and may therefore help to determine the best PGF2α dose to improve the fertility of high-producing dairy cows.

The role of vaspin in porcine corpus luteum.

Vaspin, visceral adipose tissue-derived serine protease inhibitor, plays important roles in inflammation, obesity, and glucose metabolism. Our recent research has shown the expression and role of vaspin in the function of ovarian follicles. However, whether vaspin regulates steroidogenesis and luteolysis in the corpus luteum (CL) is still unknown. The aim of this study was first to determine the expression of vaspin and its receptor GRP78 in porcine CL at the early, middle, and late stages of the luteal phase. Next, we investigated the hormonal regulation of vaspin levels in luteal cells in response to luteinizing hormone (LH), progesterone (P4), and prostaglandin PGE2 and PGF2α. Finally, we determined vaspin's direct impact on luteal cells steroidogenesis, luteolysis and kinases phosphorylation. Our results are the first to show higher vaspin/GRP78 expression in middle and late vs early stages; immunohistochemistry showed cytoplasmic vaspin/GRP78 localization in small and large luteal cells. In vitro, we found that LH, P4, PGE2, and PGF2α significantly decreased vaspin levels. Furthermore, vaspin stimulated steroidogenesis by the activation of the GRP78 receptor and protein kinase A (PKA). Also, vaspin increased the ratio of luteotropic PGE2 to luteolytic PGF2α secretion via GRP78 and mitogen-activated kinase (MAP3/1). Moreover, vaspin, in a dose-dependent manner, decreased GRP78 expression, while it, in a time-dependent manner, increased kinases PKA and MAPK3/1 phosphorylation. Taken together, we found that vaspin/GRP78 expression depends on the luteal phase stage and vaspin affects luteal cells endocrinology, indicating that vaspin is a new regulator of luteal cells steroidogenesis and CL formation.

Early resynchronization protocols for goats: Progestogens can be used prior to an early pregnancy diagnosis without affecting corpus luteum functionality.

This study aimed to evaluate the exogenous progesterone (P4) effect on the luteal function from Day 16 to Day 21 of the oestrous cycle in inseminated goats with unknown pregnancy status. A total of 54 does passed through a short progestin-based synchronization protocol and, on Day 16 of the following oestrous cycle, 27 does received a new P4 device which was retained until Day 21. Blood samples were collected daily from all does during this period, as well as on Day 24. Pregnancy diagnoses were performed on Day 30. Serum P4 values from 26 animals (GNPSP : Group of non-pregnant does with second sponge: n = 8; GNPNSP : Group of non-pregnant does without second sponge: n = 6; GPSP : Group of pregnant does with second sponge: n = 5; GPNSP : Group of pregnant does without second sponge: n = 7) were determined by radioimmunoassay commercial kits. No P4 differences were found between groups (GNPSP : 3.1 ± 2.8; 1.7 ± 1.8; 0.4 ± 1.0; and 0.0 ± 0.0 vs. GNPNSP : 4.4 ± 1.8; 3.0 ± 2.2; 0.8 ± 0.8; and 0.0 ± 0.0 or GPSP : 4.2 ± 1.0; 3.4 ± 0.6; 3.3 ± 1.6; 3.2 ± 0.9; 3.6 ± 1.2; 3.5 ± 1.3; 2.7 ± 1.3 vs. GPNSP : 4.4 ± 1.6; 3.6 ± 1.5; 3.7 ± 1.5; 3.8 ± 1.4; 3.2 ± 1.2; 3.1 ± 1.2; 3.6 ± 1.1; D16, D17, D18, D19, D20, D21, D24, respectively) or for the interaction of group and time. In conclusion, a second progestogen device had no effect on luteolysis or early pregnancy in the following oestrous cycle.

Responses to intra-luteal administration of cloprostenol in dairy cows.

The aim of the study was to determine the luteolytic dose of cloprostenol administered directly into the corpus luteum (CL; intra-luteal treatment, ILT) in dairy cattle. Cows of two control groups were treated with 500 µg of cloprostenol (Estrumate®) intramuscularly (IM-500) or via ILT with 0.2 mL of physiological solution (ILT-0). Cows of four experimental groups were treated by ILT with cloprostenol in doses 5, 25, 50 and 100 µg (ILT-5, -25, -50 and -100 groups). Progesterone concentrations (P4) and size of CL were evaluated to assess luteolysis at 0, 0.5, 1, 2, 4, 8, 24 and 48 h or at 0, 24 and 48 h after ILT, respectively. Cows in the ILT-0 and -5 groups were unaffected by ILT. The P4 concentrations were less in cows of the IM-500, as well as ILT-25, -50 and -100 groups at 48 h subsequent to ILT. The size of the CL was less in cows of IM-500, as well as ILT-25, -50 and -100 groups at 48 h after ILT. There were P4 concentrations of about 1 ng/mL 48 h after ILT in cows of the IM-500, as well as ILT-50 and -100 groups. In conclusion, the cloprostenol dose of 50 µg administered intra-luteally is a luteolytic dose in cows.

Short communication: Heat stress does not affect induced luteolysis in Holstein cows.

Heat stress (HS) has deleterious effects on bovine reproduction, including prolongation of the luteal phase in Holstein cows, perhaps due to compromised luteolysis. The objective was to characterize effects of HS on luteolytic responses of nonlactating Holstein cows given 25 or 12.5 mg of PGF2α on d 7 of the estrous cycle. Cows were randomly distributed into 2 environments: thermoneutral (n = 12; 25°C) or HS (n = 12; 36°C). In each environment, cows were treated with 2 mL of saline, 25 or 12.5 mg of PGF2α (n = 4 cows per group). The HS environment induced a significant increase in rectal temperature and respiratory rate compared with the thermoneutral environment. Heat stress did not have significant effects on luteolytic responses or circulating progesterone concentrations. Rapid and complete luteolysis occurred in all cows given 25 mg of PGF2α and in 4 of 8 cows given 12.5 mg; the other 4 cows given 12.5 mg had partial luteolysis, with circulating progesterone concentrations initially suppressed, but subsequently rebounding. Therefore, we conclude that HS does not change corpus luteum sensitivity to PGF2α.

Intravaginal instillation of prostaglandin F2α was as effective as intramuscular injection for induction of luteal regression in lactating dairy cows.

Our objectives were to test the efficacy of intravaginal (IVG) administration of PGF2α to induce corpus luteum (CL) regression, compare circulating progesterone (P4) profiles in cows receiving IVG versus intramuscular (IM) treatment with PGF2α, and evaluate reproductive outcomes. Lactating Holstein cows were synchronized using a Double-Ovsynch protocol [GnRH, 7 d later PGF2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF2α, 1 d later PGF2α, 32 h later GnRH, 16 to 20 h timed artificial insemination (TAI)] to receive TAI at 67 ± 3 d in milk. Seven days after the first GnRH treatment (time 0), cows with at least 1 visible CL ≥15 mm were blocked by parity and randomly assigned to a treatment that consisted of IM injection (IM-PGF; n = 201) or IVG instillation (IVG-PGF; n = 201) of PGF2α. Cows in IM-PGF received a single 25-mg dose of PGF2α (dinoprost tromethamine) intramuscularly. Cows in IVG-PGF received two 25-mg doses of PGF2α 12 h apart delivered through a catheter in the cranial portion of the vagina. Blood samples were collected at 0, 12, 48, and 72 h after treatment. Ovulation to the first GnRH of Double-Ovsynch was determined through transrectal ultrasonography. Only cows with P4 ≥1 ng/mL (functional CL) at time 0 (IM-PGF = 169; IVG-PGF = 179) were included in the analyses. Binary and quantitative data were analyzed by logistic regression and ANOVA with repeated measures, respectively. Results are presented as least squares means. Concentrations of P4 and the proportion of cows with a new CL at time 0 did not differ. Overall, the proportion of cows with CL regression using 1 ng of P4/mL (IM-PGF = 89.0%; IVG-PGF = 86.7%) or 0.5 ng of P4/mL (IM-PGF = 82.2%; IVG-PGF = 82.1%) as the cutoff did not differ. Concentrations of P4 were affected by treatment, time, and treatment × time interaction. Cows in IVG-PGF had greater mean P4 at 12 h than cows in IM-PGF. Mean P4 did not differ at 48 or 72 h after treatment. The proportion of cows with estrus recorded within 3 d of treatment (IM-PGF = 45.4%; IVG-PGF = 48.9%), ovulation risk after treatment (IM-PGF = 88.5%; IVG-PGF = 85.1%), and pregnancies per artificial insemination after TAI (IM-PGF = 51.5%; IVG-PGF = 57.8%) did not differ. We concluded that 2 IVG doses of 25 mg of PGF2α 12 h apart were as effective as a single 25-mg IM dose of PGF2α for inducing luteal regression in lactating dairy cattle.

Association of pregnancy per artificial insemination with gonadotropin-releasing hormone and human chorionic gonadotropin administered during the luteal phase after artificial insemination in dairy cows: A meta-analysis.

One strategy for improving fertility in cattle is administration of GnRH or human chorionic gonadotropin (hCG) during the luteal phase, which increases progesterone (P4) secretion and delays luteolysis. To provide an overview of how GnRH or hCG treatment between 4 and 15 d after artificial insemination (AI) improves pregnancy per AI (P/AI) in cows, a meta-analysis was performed on 107 different trials from 52 publications. Data from 18,082 treated cows and 18,385 untreated controls were meta-analyzed. The meta-analysis explained the relative risk for P/AI with GnRH or hCG treatment under various circumstances. The results did not show any difference in P/AI between cows treated with hCG and cows treated with GnRH. Compared with no treatment, treatment with GnRH or hCG improved the chances of P/AI in cows with very poor (<30%) and poor (30.1 to 45%) fertility, whereas treatment did not benefit cows with very good fertility (>60.1%). Moreover, treatment with GnRH and hCG improved the chances of P/AI in primiparous cows. The improvement was much better in primiparous cows with very low fertility. Treatment with buserelin at a dose above 10 µg and with hCG at a dose above 2,500 IU was associated with increased chances of P/AI compared with lower doses. Treatment with GnRH 10 d after AI was also associated with increased chances of P/AI compared with earlier treatment. The present meta-analysis showed that the use of GnRH and hCG after AI should be focused on cows expected to have low or moderate fertility. Day and dose of treatment have to be considered as well.

Effects of follicular ablation and induced luteolysis on LH and follicular fluid factors during the periovulatory period in mares.

Haemorrhagic anovulatory follicles (HAFs) are the most common pathological anovulatory condition in the mare. To enhance understanding of the physiopathology of HAFs, the aim of the present study was to determine the effects of an induced-follicular wave on LH concentrations and follicular fluid factors relevant to the ovulatory process. Mares were allocated to treatment or control groups (n = 7/group) in a crossed over design during 14 oestrous cycles with a period of one cycle occurring when there were no treatments between the times when treatments were administered. In the treatment group, all antral follicles ≥8 mm were ablated on Day 10 after ovulation followed by administration of a luteolytic dose of PGF2α. All mares of both groups were treated with 1500 IU of hCG when a follicle ≥32 mm was detected (Hour 0), and follicular fluid was aspirated 35 h later. Blood samples were collected every 48 h from Day 10 until Hour 0 from all mares. Follicular fluid was assayed for PGE2, estradiol and progesterone. Plasma was assayed for LH concentrations. A follicular wave followed follicle ablation in the treated mares. Concentrations of LH were greater (P = 0.05) in mares ot the treatment compared with control group. Concentrations of PGE2, estradiol and progesterone in follicular fluid did not differ between groups (P > 0.05). Treatment resulted in an earlier increase in circulating LH, however, there was no effect on concentrations of intra-follicular PGE2, estradiol or progesterone in hCG-stimulated preovulatory follicles.

Effects of prostaglandin F2α on angiogenic and steroidogenic pathways in the bovine corpus luteum may depend on its route of administration.

Prostaglandin (PG) F2α and its analogs (aPGF2α) are used to induce regression of the corpus luteum (CL); their administration during the middle stage of the estrous cycle causes luteolysis in cattle. However, the bovine CL is resistant to the luteolytic actions of aPGF2α in the early stage of the estrous cycle. The mechanisms underlying this differential luteal sensitivity, as well as acquisition of luteolytic sensitivity by the CL, are still not fully understood. Therefore, to characterize possible differences in response to aPGF2α administration, we aimed to determine changes in expression of genes related to (1) angiogenesis-fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2); and (2) steroidogenesis-steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), and hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 1 (HSD3B) in early- and middle-stage CL that accompany local (intra-CL) versus systemic (i.m.) aPGF2α injection. Cows at d 4 (early stage) or d 10 (middle stage) of the estrous cycle were treated as follows: (1) systemic saline injection, (2) systemic aPGF2α injection (25 mg), (3) local saline injection, and (4) local aPGF2α injection (2.5 mg). Progesterone (P4) concentration was measured in jugular vein blood samples during the entire set of experiments. After 4 h of treatment, CL were collected by ovariectomy, and mRNA and protein expression levels were determined by reverse transcription quantitative-PCR and Western blotting, respectively. Local and systemic aPGF2α injections upregulated FGF2 expression but decreased expression of VEGFA in both CL stages. Both aPGF2α injections increased the expression of STAR in early-stage CL, but downregulated it in middle-stage CL. In the early-stage CL, local administration of aPGF2α upregulated HSD3B, whereas systemic injection decreased its mRNA expression in early- and middle-stage CL. Moreover, we observed a decrease in the P4 level earlier after local aPGF2α injection than after systemic administration. These results indicate that aPGF2α acting locally may play a luteotrophic role in early-stage CL. The systemic effect of aPGF2α on the mRNA expression of genes participating in steroidogenesis seems to be more substantial than its local effect in middle-stage CL.

Ganglionic and ovarian action of acetylcholine during diestrous II in rats. Neuroendocrine control of the luteal regression.

Our aim was to investigate if acetylcholine (Ach), added to the celiac ganglion-superior ovarian nerve-ovary system (CG-SON-ovary) or in ovary incubations, modifies the release of progesterone (P4), androstenedione (A2), dopamine (DA), norepinephrine (NE), gonadotropin-releasing hormone (GnRH), and alters the expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 20α-hydroxysteroid dehydrogenase (20α-HSD), and apoptotic genes in ovarian tissue during the diestrous II (DII) in rats. The CG-SON-ovary system or the ovary alone were removed and placed into separate cuvettes both containing Krebs-Ringer solution (control groups). In experimental groups, 10-6 M Ach was added into the ganglion compartment or into the ovary compartment. P4, A2 and GnRH were measured by RIA, mRNA expression by RT-PCR, and catecholamines by HPLC. In addition, a routine histological technique was applied. In ex-vivo system, 10-6 M Ach into the ganglion compartment decreased P4 and NE release, altered 3ß-HSD and 20α-HSD expression, and decreased bax/bcl-2 ratio, while increasing the release of A2 and DA, and bcl-2 expression. In ovary incubations, 10-6 M Ach decreased P4 and GnRH release, decreased 3ß-HSD and bcl-2 expression, increased A2 release, increased 20α-HSD and bax expression, and the bax/bcl-2 ratio, and induced disorganization of the corpus luteum structure. The peripheral nervous system protected the ovary from the apoptotic mechanisms while in the ovary incubation the effect was reversed. Our results indicate that Ach in DII regulates steroidogenesis and apoptosis in the ovary, by modulating the concentration of neurotransmitters. In vivo, an alteration in the extrinsic cholinergic innervation of the ovary could disrupt the endocrine control of the reproductive function.

Spatial relationships of ovarian follicles and luteal structures in dairy cows subjected to ovulation synchronization: Progesterone and risks for luteolysis, ovulation, and pregnancy.

Objectives were to determine relative ovary location of follicles, GnRH-induced corpora lutea (CL), and older CL present in ovaries as part of ovulation synchronization and their associations with progesterone concentration and risk for luteolysis, ovulation, and pregnancy. Cows were exposed to a timed artificial insemination (AI) program [GnRH-1-7 d-PGF2α (1 dose or 2 doses 24 h apart)-56 h after first or only dose of PGF2α-GnRH-2-16 h-timed AI at 72 ± 3 d in milk]. Blood was collected to assess progesterone when ovarian structures were mapped in 694 cows before GnRH-1 and before and 48 h after PGF2α and, in a subset of cows, size of CL (n = 599) and progesterone (n = 380) at 6 d after AI. Dominant follicles and CL in single-ovulating cows were detected more often in right than left ovaries (follicles before GnRH-1: 60.6% right and GnRH-2: 61.2% right; and CL before GnRH-1: 58.6% right and GnRH-2: 66.4% right). Dominant follicles in single-ovulating cows before GnRH-1 tended to be ipsilateral to the CL more often than contralateral (54.8 vs. 45.2%) with co-dominant follicles identified in both ovaries (19.3%). In response to GnRH-1 or GnRH-2, more left-ovary follicles ovulated contralateral to CL (left to right, 54.7%; right to left, 34.7%) than right-ovary follicles, but fewer left-ovary follicles ovulated ipsilateral to CL (left to left: 45.3%) than right-ovary follicles ovulated ipsilateral (right to right: 65.3%). Preovulatory follicles in single-ovulating cows before PGF2α tended to be detected more often ipsilateral than contralateral to CL induced by GnRH-1 (younger CL; 56.5 vs. 43.6%), but were of equal frequency ipsilateral or contralateral to older CL present before GnRH-1. Luteolytic risk was less in cows bearing co-dominant follicles in both ovaries compared with those in either right or left ovaries. Luteolytic risk in single-ovulating cows did not differ between ovaries. Luteolytic risk was greater for cows bearing older CL (86.5%) than for cows bearing younger GnRH-1-induced CL (65.3%) or both (79.6%). Pregnancy risk at 60 d after AI was or tended to be greater in cows having both CL types compared with either younger or older CL, respectively, partly because of greater embryonic loss in the latter 2 cases. More female calves tended to be carried in right horns when conception occurred after first service, whereas the opposite greater female frequency occurred in left horns after repeat services. Right-ovary dominance is evident before and after GnRH treatment.

Endogenous and exogenous effects of PGF2α during luteolysis in mares.

An inhibitor of PGF2α biosynthesis (flunixin meglumine, FM) was used to study the role of endogenous PGF2α on the luteolytic effect of exogenous PGF2α in mares. A 2-h infusion of PGF2α at a constant rate (total dose, 0.1 mg) on Day 10 (ovulation = Day 0) was used to mimic the maximal concentrations of a spontaneous pulse of a PGF2α metabolite (PGFM). Treatment with FM (1.7 mg/kg) was done 1 h before and 5 h after the start of PGF2α infusion. In hourly blood samples beginning 1 h before the start of PGF2α infusion, progesterone decreased (P < 0.05) similarly by 5 h in each of the PGF2α and PGF2α+FM groups but not in the controls (n = 5). In a study of spontaneous luteolysis, the same FM dose was given every 6 h from Day 13 until Day 17 or earlier if CL regression was indicated by an 80% decrease in luteal blood-flow signals. Blood was sampled for progesterone assay each day and 8 h of hourly blood sampling was done each day to characterize PGFM concentrations and pulses. Progesterone (P4) was lower (P < 0.05) in controls than in an FM group (n = 7) by Day 15. Luteolysis (P4 < 1 ng/mL) ended on Days 14-19 in individual controls. In contrast, luteolysis did not end until after Day 20 in 4 of 7 FM-treated mares. In the three mares with completion of luteolysis before Day 20 in the FM group, the interval from beginning to end of luteolysis was longer (P < 0.02) (4.5 ± 0.6 days) than in the controls (3.0 ± 0.4 days). During 8-h sessions of hourly blood sampling on Day 14, concentration of PGFM was significantly lower in the FM group for the minimal, mean, and maximal per session. Pulses of PGFM were identified by a CV methodology on each day in 7 of 7 and 3 of 7 mares in the controls and FM group, respectively. The four FM-treated mares without a CV-identified pulse were the four mares in which luteolysis did not occur before Day 20. In mares with detected pulses, PGFM was lower at each nadir and at the peak (86% lower) in the FM group than in controls, but the interval between nadirs or base of a pulse was not different between groups. Hypothesis 1 that endogenous PGF plays a role in the luteolytic effect of exogenous PGF2α was not supported. Hypothesis 2 that an inhibitor of PGF2α biosynthesis prevented or minimized the prominence of PGFM pulses and increased the frequency of persistent CL was supported.

Supplementation with long-acting progesterone in early diestrus in beef cattle: II. Relationships between follicle growth dynamics and luteolysis.

The aims were to characterize follicular dynamics in response to supplemental progesterone (P4) and to investigate the relationships between follicular growth and onset of luteolysis in P4-treated cows, submitted or not to artificial insemination (AI). Nonsuckled beef cows detected in estrus were assigned to receive AI or to remain non-AI. Three days after ovulation (ie, D3), AI cows were injected with 150 mg of long-acting P4 (AI + injectable P4 formulation [iP4]; n = 22), and the non-AI cows were assigned to receive 150 mg iP4 (n = 19) or saline (control, n = 19). Between D3 and D21, growth dynamics of the dominant follicles (DFs) was monitored by ultrasonography. Plasma P4 concentrations were measured every other day from D9 to D19. Pregnancy status (ie, P: pregnant and NP: nonpregnant) was examined by ultrasound on D28 to D32. Injectable P4 formulation supplementation decreased average maximum diameter of first-wave DF (DF1). Neither day of emergence of DF2 or DF3 nor the proportion of two- or three-wave cycles were altered by supplemental P4. Daily mean diameter of DF2 and DF3 was also similar between control and iP4 groups. Consistently, daily mean diameter of DF1 in iP4-treated cows was smaller for cows that underwent luteolysis by D15 compared to a later onset. Progesterone concentrations between D9 and D19 decreased earliest in the iP4 group, latest in the control group and was intermediate for the NP-AI + iP4 group. In addition, three-wave cycles presented a delayed decrease on plasma P4 concentrations than two-wave cycles. Further analysis revealed that on two-wave cycles, P4 concentrations on D15 were lowest in the iP4 and NP-AI + iP4 animals compared to the control and P-AI + iP4 groups. Conversely, for three-wave cycles, on D15, P-AI + iP4, NP-AI + iP4, and controls had greater P4 concentrations than the iP4 group. In summary, our data indicate that impairment of first follicular growth was associated with P4-induced shortened luteal lifespan (D14-D15) and that three-wave cycles after AI can be more supportive for pregnancy maintenance in P4-treated cows. We speculate that such conditions play a critical role in the embryonic ability to inhibit iP4-induced early luteolysis reported in part I of this series.

Serum and tissue pregnanes and pregnenes after dexamethasone treatment of cows in late gestation.

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3ßHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3ßHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3ßHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Intra-ovarian dynamic blood flow in pseudopregnant rabbits during prostaglandin F2α-induced luteolysis.

In the present study, we evaluated the dynamic changes of intra-ovarian blood flow, by real-time colour-coded and pulsed Doppler ultrasonography, as well as the immunopresence of prostaglandin F2α (PGF2α) receptor (FP) and peripheral plasma progesterone concentrations in pseudopregnant rabbit after PGF2α treatments at either early- (4 days) and mid-luteal (9 days) stages. During the pre-treatment observation interval of one hour, the ovarian blood flows showed a fluctuating pattern. Independently of luteal stage, PGF2α administration caused a fourfold decline in the blood flow within 40 min that was followed 50 min later by a reactive hyperaemia that lasted several hours, while the resistive index showed an opposite trend. Twenty-four hour later, the blood flow was one half that measured before PGF2α injection. At day 4 of pseudopregnancy, PGF2α did not affect peripheral plasma progesterone concentrations, but at day 9, it caused functional luteolysis as progesterone levels declined 6 hr later to reach basal values after 24 hr. The changes in the ovarian blood flows of pseudopregnant rabbits receiving PGF2α were accompanied by simultaneous changes in the resistance index. This biphasic response in the blood flow and vascular resistances likely reflects reactive hyperaemia following vasoconstriction. By immunohistochemistry, strong positive immune reaction for FP was detected in the cytoplasm of endothelial cells of ovarian arteries, veins and capillaries. In conclusion, these results suggest that PGF2α could acutely regulate the ovarian blood flow of pseudopregnant rabbits, even if there is no evidence of a blood flow reduction anticipating luteolysis.

Comparison of luteolysis and timed artificial insemination pregnancy rates after administration of PGF2α in the muscle or the ischiorectal fossa in cattle.

Prostaglandin F2α (PGF2α) is commonly injected intramuscularly (IM) in female cattle in synchronization protocols. A novel site for administration of PGF2α that improves beef quality assurance is the ischiorectal fossa (IRF). The objective of this study was to determine whether administration of PGF2α in the IRF results in a similar physiological response to an intramuscular injection. Yearling angus-cross heifers (n = 112) were blocked by weight and randomly assigned within blocks to be injected with 5 mL PGF2α either IM in the neck or in the IRF. Blood samples taken at 0, 8, 16, 24, 36, and 48 h post-injection were analyzed for serum progesterone concentration using a radioimmunoassay. Progesterone concentration curves for each heifer were plotted to determine luteolysis. The median times to luteolysis for neck and IRF injections were 18.1 h and 20.0 h, respectively (p = 0.06). Angus cross commercial beef cows (n = 1471) at least 30 days post-partum were blocked by age and randomly assigned within blocks to be injected with 5 mL PGF2α either IM in the neck muscle or in IRF as part of a 7-Day CO-Synch + CIDR synchronization protocol. Pregnancy diagnosis was performed via ultrasound at 60 days post insemination. Results were analyzed with Proc Glimmix (SAS). Pregnancy rates for neck and IRF injections were 52.6% and 57.2%, respectively (p = 0.06). In summary, injection of PGF2α in the IRF for synchronization of estrus and luteolysis did not differ from IM injection. Utilizing the ischiorectal fossa as an injection site for PGF2α may serve as an alternative that more closely aligns with beef quality assurance.

Level of circulating concentrations of progesterone during ovulatory follicle development affects timing of pregnancy loss in lactating dairy cows.

The objective of this experiment was to determine the effect of high versus low progesterone (P4) during the pre-dominance or dominance phase (or both) of ovulatory follicle development on follicular dynamics and fertility of lactating dairy cows. Progesterone (P4) was manipulated to reach high (H) or low (L) serum concentrations during the pre-dominance phase (d 0 to 4 of the wave) and dominance phase (d 5 to 7 of the wave) of a second follicular wave ovulatory follicle, creating 4 treatments: H/H, H/L, L/H, and L/L. Luteolysis was induced with PGF2α on d 7 of the wave and ovulation was induced with GnRH 56 h after PGF2α. Cows (n = 558) received artificial insemination (AI) 16 h following GnRH. Pregnancy was determined at 6 intervals during gestation and at calving to quantify pregnancy loss beginning at d 23 post-AI utilizing pregnancy-specific protein B (PSPB) in novel within-cow comparisons. Cows with single ovulations assigned to the L/L treatment had greater pre-ovulatory follicle diameter compared with cows assigned to the L/H or H/L treatments. Cows with single ovulations had greater pre-ovulatory follicle diameter compared with cows with double ovulations. Low P4 in H/L, L/H, and L/L increased double ovulation rate compared with H/H. Cows with double ovulations had greater pregnancies per AI (P/AI) on d 23 post-AI compared with cows with single ovulations but had greater losses if ovulations were unilateral. Cows with low P4 during the entire period of the ovulatory follicle development also had greater P/AI on d 23 post-AI compared with cows with high P4 during both phases. However, full-term P/AI was not different between treatments. This was a result of the greater incidence of pregnancy losses between d 35 and 56 of gestation for cows with unilateral double ovulations compared with bilateral double ovulations and single ovulatory cows. Cows with single ovulation and low circulating P4 during the dominance period of follicle development had increased pregnancy losses between d 35 and 56 of gestation compared with cows with single ovulations and high P4. The PSPB measurements on d 16 and 23 post-AI were highly accurate in the prediction of pregnancy at d 28. The PSPB differed on d 23 and 28 between cows that had versus cows that did not have pregnancy losses between d 28 and 35 of gestation. In summary, circulating concentrations of P4 during ovulatory follicle development affected numbers of follicles ovulated and timing of subsequent pregnancy losses.

Effects of altering the ratio of dietary n-6 to n-3 fatty acids on spontaneous luteolysis in lactating dairy cows.

Objectives were to evaluate the effects of altering the dietary ratio of omega-6 (n-6) to omega-3 (n-3) fatty acids on the profile of fatty acids and expression of genes related to the prostaglandin biosynthesis on endometrial tissue, uterine secretion of PGF2α, and timing of spontaneous luteolysis in dairy cows. Multiparous lactating Holstein cows (n = 45) were blocked based on milk yield and, within each block, assigned randomly to 1 of 3 dietary treatments at 14 d postpartum for 90 d. Diets were supplemented with a mixture of Ca salts of fish, safflower, and palm oils to create 3 different ratios of n-6 to n-3 fatty acids, namely R4, R5, and R6, which resulted in 3.9, 4.9, and 5.9 parts of n-6 to 1 part of n-3 fatty acids, respectively. Blood was sampled every 2 h from d 16 to 23 of the estrous cycle and assayed for concentrations of progesterone and the PGF2α metabolite 13,14-dihydro-15-keto-PGF2α (PGFM). In a subsequent estrous cycle, endometrial tissue was collected for biopsy on d 8 and endometrial fatty acids profile and gene expression were quantified. The proportion of arachidonic acid of the endometrial fatty acids increased as the dietary ratio n-6 to n-3 fatty acids increased (R4 = 9.05, R5 = 11.64, and R6 = 13.41%). On the other hand, proportions of eicosapentaenoic (R4 = 2.85, R5 = 2.14, and R6 = 2.02%) and docosahexaenoic (R4 = 3.30, R5 = 1.57, and R6 = 1.08%) decreased as the ratio of n-6 to n-3 fatty acids in the diet increased. Increasing the ratio of dietary n-6 to n-3 fatty acids increased mRNA expression of estrogen receptor 1, oxytocin receptor, cyclooxygenase 2, prostaglandin E and F synthases, and steroidogenic acute regulatory protein in endometrium, but decreased expression of peroxisome proliferator-activated receptor gamma and insulin-like growth factor-1. The changes in endometrium gene expression caused by dietary treatments were associated with changes in the ratio of n-6 to n-3 fatty acids in the endometrium. As the ratio increased from R4 to R6, the number of PGFM pulses (R4 = 5.6, R5 = 4.3, and R6 = 3.8 ± 0.6 pulses; least squares means ± standard error of the means) decreased, but the amplitude of the greatest PGFM pulse increased (R4 = 226, R5 = 267, and R6 = 369 ± 38 pg/mL). Luteolysis by d 23 of the estrous cycle was observed in 79.6% of the cows (R4 = 11/14; R5 = 13/15; and R6 = 11/15) and day of spontaneous luteolysis did not differ among treatments (R4 = 20.8; R5 = 21.1; and R6 = 21.0 ± 0.4). Three pulses of PGFM was the best predictor of luteolysis in dairy cows. Collectively, supplying the same quantity of fatty acids in the diet of lactating dairy cows, but altering the ratio of n-6 to n-3 fatty acids, influenced the endometrial fatty acids profile and gene expression and altered the pattern of prostaglandin synthesis; however, the changes were not sufficient to alter the length of the estrous cycle.