Split-weaning before altrenogest synchronization of multiparous sows alters follicular development and reduces embryo survival

This study used split-weaning (SW) to induce differences in follicle size at weaning and study its consequences for follicle development during and after post-weaning altrenogest feeding and for reproductive performance. Multiparous sows (n=47) were assigned to SW (n=23; litter size reduced to the six smallest piglets 3 days before weaning) or control (C; n=24; normal weaning). Altrenogest (20 mg/day) was fed to all 47 sows from Day -1 till Day 5 (complete weaning = Day 0). Follicle size on Day 1, 2 and 8 was smaller in C than in SW (p ≤ 0.05). Ovulation rate was similar, but C sows had higher embryo survival rate (ESR) than SW sows (83 ± 19 and 58 ± 31%, respectively; p=0.001). SW sows with low ESR (<63%; n=10) had a greater follicle size on days 3-6 than SW sows with high ESR (>63%; n=10; p ≤ 0.04). A decrease in follicle size between Day 5 and 6 of altrenogest feeding was associated with increased ESR in both treatments (p=0.002). Follicle pool analyses (assessment of all follicles >2 mm) revealed that on Day 3, sows with low ESR had a higher % of follicles >5 mm compared with sows with high ESR (30% vs 10%; p=0.04). Thus, sows in which follicle growth was less suppressed during altrenogest feeding had a lower ESR. These effects on follicle development and ESR were more pronounced in split-weaned sows.     

Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes

The effects of progesterone (P₄) on follicular growth and fertility in ewes were examined. In Experiment 1, 22 ewes received either one or three packets of P₄ (5 g/packed) or an empty packet subcutaneously (sc) from Days 5 to 15 of the estrous cycle (estrus = Day 0). On Day 6, P₄-treated ewes received 12.5 mg of prostaglandin F₂α. Follicles ⩾3 mm in diameter were observed via transrectal ultrasonography daily from Day 4 through estrus, corpora lutea (CL) were observed 5 to 7 d after estrus. Ewes with low (LOW; ⩽1 ng/ml; n = 5), intermediate (MED; > 1 and <2 ng/ml; n = 10), or normal (NOR; ⩾2 ng/ml; n = 7) P₄ in jugular plasma on Days 7 through 15 differed in follicular development. The largest follicle at estrus was larger in ewes with LOW vs. MED and NOR P₄ (7.8 ± 0.3 vs. 6.9 ± 0.2 mm; P < 0.05). Treatments differed in proportions of multiple-ovulating ewes, in which the oldest ovulatory follicle was first observed before Day 10 (LOW: 3 of 3, MED: 6 of 10, NOR: 0 of 5, respectively; P < 0.05). Estradiol was higher early in the treatment period in LOW ewes than in MED and NOR ewes (day × treatment; P < 0.05). In Experiment 2, ewes received 5 mg of P₄ in corn oil (low progesterone [LP]; n = 51) or 2 ml of corn oil (CON; n = 49) sc every 12 hr on Days 6 through 14 of the estrous cycle before mating. LP ewes received 15 mg of prostaglandin F₂α on Day 6. Mean serum P₄ on Days 7 through 15 was 0.6 ± 0.1 ng/ml in LP and 1.9 ± 0.1 ng/ml in CON ewes. Eleven LP and 12 CON ewes were scanned daily from Day 4 through mating, and in all ewes (n = 93), CL were counted 10 d after mating and embryos were counted at 25, 40, and 60 d of gestation. In multiple-ovulating ewes, day of cycle of appearance was earlier for the oldest (Day 6.1 ± 0.8 vs. 10.4 ± 0.8) but not second oldest (Day 11.7 ± 1.0 vs. 12.2 ± 0.9) ovulatory follicles in LP compared with CON ewes. The conception rate was lower in LP (72%) than in CON ewes (98%; P < 0.01). However, numbers of CL 10 d after mating, and in pregnant ewes, numbers of embryos 25 d after mating and lambs born, did not differ with treatment. In summary, low P₄ increased the size of the largest follicles and the age of the oldest ovulatory follicles. Embryos resulting from the ovulation of older and younger follicles in the same ewe did not differ in their ability to survive.     

Dynamics of ovarian follicular development in cattle during the estrous cycle, early pregnancy and in response to PMSG

Ovarian follicular dynamics of cattle were examined during the estrous cycle, early pregnancy and in response to PMSG. Number and size of follicles were monitored by ultrasonographic examinations. During the estrous cycle, distinct periods of follicular dominance (measured by the increase in difference in size between the largest and second largest follicle) occurred in both the luteal (Days 6-8) and proestrus (18-22) phases of the estrous cycle (two follicular waves). Associated with the well timed development of the first dominant follicle was a change in distribution of follicle numbers in small (less than 5 mm; increased on Days 2-4), medium (6-8 mm; increased on Days 3-5) and large (greater than or equal to 9 mm; increased on Days 6-9) follicular size classes. Follicular development was greater on the ovary bearing the CL for the period that the CL was present. The dominant follicle formed during the first follicular wave was capable of ovulating (6 of 8 heifers) following an injection of a synthetic analogue of prostaglandin F-2 alpha on Day 9 of the estrous cycle. During early pregnancy (Days 6-34), follicular development (size of largest follicle, number of follicles and total accumulated size of all follicles) on the ovary bearing the CL was suppressed between Days 24 and 34 of pregnancy. This was a local effect in that follicular development was sustained on the contralateral ovary. Therefore, the CL or conceptus may be regulating follicular development in a manner to help prevent luteolysis. Associated with the injection of PMSG was an initial increase in the number of small follicles followed by their recruitment into medium and large size classes leading to ovulation. Number of follicles greater than 5 mm on the Day of estrus was related (r = .97) to the number of subsequent embryos and oocytes collected. Ultrasonography is a valuable technique to monitor ovarian follicular dynamics in cattle, and can thereby be used to infer changes in physiological and endocrine states.     

Alarelin active immunization influences expression levels of GnRHR,FSHR and LHR proteins in the ovary and enhances follicular development in ewes

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle‐stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty‐two pre‐pubertal ewes were assigned to experimental groups 1 to 5 (EG‐I to EG‐V) and control group (CG). Ewes in EG‐I, EG‐II and EG‐III were subcutaneously injected with 200, 300 or 400 μg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG‐IV and EG‐V were subcutaneously injected with 200 μg and 300 μg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P < 0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG‐IV and EG‐V were higher than that in EG‐I, EG‐II and EG‐III from days 35 to 45. Expressions of GnRHR protein in EG‐IV and EG‐V were lower than that in CG (P < 0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG‐IV and EG‐V (P < 0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG‐III and EG‐V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin‐immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P < 0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.     

Follicular persistence induced by adrenocorticotropic hormone administration in goats

The objective of the present study was to examine the effect of adrenocorticotropic hormone (ACTH) administration on the induction of persistent cystic follicle in the goat in order to establish a method to experimentally induce cystic follicle. Four cross-bred goats were intramuscularly administered ACTH at 0.78 and 6.25 μg/10 kg twice a day from Days 15 to 21 (Day 0 was defined as the day of last estrus). Follicular status in the ovary was monitored by ultrasound examination. The plasma concentrations of estradiol, progesterone and cortisol were measured. Treatment with ACTH at the 0.78 and 6.25 μg/10 kg levels caused persistent follicles (> 10 days delay from the expected ovulation date) in 50% of the goats in both treatment groups. In those animals, ovulation occurred 17 and 27 days and 11 and 12 days after the expected days in the 0.78 and 6.25 μg/10 kg groups, respectively. The maximum follicle diameters were 10 and 9 mm in the 0.78 and 6.25 μg/10 kg ACTH groups, respectively. In the control group, the estradiol concentration increased on Day 18 and remained at a high level for a few days. However, such an increase was not seen in both ACTH groups. The estradiol concentration increased gradually from Days 21 to 27 in the 6.25 μg/10 kg ACTH group. These results suggest the possibility that ACTH induces persistent follicles in goats, which may be related to the delay of the onset of estradiol secretion followed by its maintenance at a high concentration.     

Patterns of corpora lutea growth and progesterone secretion in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig)

The current study aimed to compare luteal function, as measured by corpora lutea dynamics and progesterone secretion, in 10 sows with obesity/leptin resistance genotype (Iberian pig) and 10 females of lean commercial crosses (Large White × Landrace). In all the animals, the oestrous cycle was synchronized with progestagens, and ovulation was induced by exogenous gonadotrophins. Thereafter, number and size of follicles and plasma oestradiol concentration were determined at oestrus detection, and number and size of corpora lutea and progesterone concentration were evaluated from Day 4 to 12 of the cycle. There were no differences between genotypes in follicle population and oestradiol concentration, and ovulation rate (15.2±1.3 in Iberian vs 12.7±1.8 in LWxL sows); however, there was a higher percentage of Iberian than control sows showing luteal cysts (66.7% vs 30%, respectively; p<0.05). In both breeds, both total luteal area and plasma progesterone concentration grew linearly from Day 4 to 8 (p<0.01) and remained more stable between Days 8 and 12, without significant differences between genotypes. In conclusion, current study supports that ovulatory processes and luteal functionality are not the main limiting factors for prolificacy in a pig model of leptin resistance and obesity.     

Utilization of substrates for testosterone and estradiol-17β production by small follicles of the chicken ovary

A study was conducted to characterize steroidogenesis in small ovarian follicles (1–10 mm in diameter) of the hen. The aims of our study were: 1) to determine basal estradiol-17β (E₂) production by different sizes of small follicles; 2) to determine the ability of intact small follicles to utilize exogenous substrates for testosterone (T) and E₂ production; and 3) to investigate the preferred steroidogenic pathway in small follicles. Small follicles which had not entered the hierarchy were isolated from ovaries obtained 2 hr after oviposition and divided into three groups: small white follicles (SWF; 1–2 mm in diameter), large white follicles (LWF; 2–4 mm in diameter), and small yellow follicles (SYF; 5–10 mm in diameter). Yolk and granulosa cells were removed from LWFs and SYFs and the remaining theca layer was called a follicle shell. Intact follicles or follicle shells (4/4 ml/tube) were incubated in avian Ringer's buffer supplemented with 10 mM HEPES and 0.1% BSA at 37°C for 3 hr with various treatments. Testosterone and E₂ were measured in the medium. The SYFs and their corresponding follicle shells produced the greatest amount of E₂ when E₂ production was expressed per follicle. Addition of 2 mM 8-Br-cAMP to the incubation medium stimulated E₂ production by all sizes of follicles and follicle shells. However, follicle shells produced lower basal- and 8-Br-cAMP-stimulated E₂ secretion compared to corresponding intact follicles. There was no significant difference in E₂ production in response to various concentrations of 25-hydroxycholesterol (25-OH-CHOL; 0–100 μM) by intact follicles and follicle shells. On the contrary, intact follicles and follicle shells produced T and E₂ in a dose-dependent manner in response to increasing concentrations (0–100 μM) of pregnenolone (P₅). Intact follicles also used progesterone (P₄) and dehydroepiandrosterone (DHEA) as substrates for T and E₂ production. DHEA was the preferred substrate for steroid production compared to P₄. In summary, we found that: 1) steroidogenesis in small follicles is regulated by a cAMP-dependent protein kinase A second messenger system; 2) adequate amounts of endogenous cholesterol are available for steroidogenesis; and 3) both Δ⁵ and Δ⁴ pathways are functional in small follicles and the Δ⁵ pathway may be the preferred steroidogenic pathway. pathway.     

Follicle Dynamics and its Relation with Plasma Concentrations of Progesterone, Luteinizing Hormone and Estradiol during the Egg-Laying Cycle in Ostriches

The aims of this study were (i) to describe the changes in the volume of large ovarian follicles (diameter >3 cm) during the 48 h egg laying cycle in farmed ostriches, and (ii) to quantify factors affecting the volume of the largest measured follicle and the plasma concentrations of progesterone (P₄) and estradiol‐17β (E₂β). In eight egg‐producing birds, which all ovulated during the study period, transcutaneous ultrasound scanning and blood sampling was performed at 3 h intervals. The average volume of the total number of visualized large follicles (V_total), the largest measured follicle (V_F1), the second largest follicle (V_F2) and of all follicles smaller than F2 (V_F3–Fn) were each higher before than after oviposition. V_total, V_F2 and V_F3–Fn nearly doubled in the 24‐h period before oviposition, while V_F1 remained at an equal, rather high level until oviposition. Immediately after oviposition V_total, as well as the volume of the other follicle categories, decreased within 6 h, i.e. around the moment of ovulation. By performing statistical analysis on the basis of linear mixed‐effects modelling, we quantified that: (i) V_F1 was 13.2% higher before than after oviposition and increased with 6.5% when LH increased with 1 ng/ml; (ii) P₄ levels were 93.2% higher before than after oviposition and increased with 43.1% for every 3 h closer to oviposition; when LH and E₂β levels and V_F1 increased with 1 ng/ml, 10 pg/ml and 10 ml, respectively, P₄ increased with 116.6%, 50% and 6.1%; and (iii) E₂β levels were 35.6% higher before than after oviposition, increased with 2.7% for every 3 h closer to oviposition and increased with 14.6% when LH increased with 1 ng/ml. It is concluded that during the egg‐laying cycle in ostriches: (i) follicular mass, as estimated by the volume of visualized follicles larger than 3 cm, increases before and decreases after ovulation, and (ii) follicular dynamics and its accompanying endocrine plasma hormone profiles during the egg‐laying cycle in ostriches follow a pattern similar to that in chickens.     

Effect of the dominant follicle aspiration before or after luteinizing hormone surge on the corpus luteum formation in the cow

Luteinizing hormone (LH) surge and follicle rupture act as trigger to start corpus luteum (CL) formation. Thus, we aimed to investigate whether a dominant follicle that has not been exposed to an LH surge can become a functional CL. For this purpose, follicular fluid from the dominant follicles (DF) of cows was aspirated before or after a GnRH-induced LH surge, and subsequent CL formation was observed. Holstein cows were divided into four groups as follows: Luteal phase, a DF was aspirated 7 days after GnRH injection; Pre-LH surge, a DF was aspirated 42 h after PGF(2alpha) injection during the mid luteal phase; Post-LH surge, a DF was aspirated 24 h after GnRH injection following PGF(2alpha); and Intact follicle, ovulation was induced by GnRH injection after PGF(2alpha). Observation of morphological changes in the aspirated follicle using color Doppler ultrasonography and blood sampling was performed on Days 0, 3, 6, and 9 (Day 0 = follicle aspiration). CL formation following DF aspiration was observed only in the Post-LH surge group. In both the Luteal phase and Pre-LH surge groups, however, none of the cows showed local blood flow at the aspirated site or CL formation. Luteal blood flow area, CL volume, and plasma progesterone concentration in the Post-LH surge group were no different from those in the Intact follicle group. The present results clearly demonstrate that rather than follicle rupture, it is the LH surge that is essential for CL formation in cows.     

Endocrine Profiles and Embryo Quality in Japanese Black Cattle Superovulated with Human Menopausal Gonadotrophin and Porcine Follicle Stimulating Hormone

Induction of superovulation using human menopausal gonadotriphin (hMG) in Japanese Black cattle can result in the recovery of a higher percentage of high quality embryos compared with that using porcine follicle stimulating hormone (FSH). In order to clarify the endocrinological mechanism involved in this discrepancy, 30 superovulation sessions of 17 Japanese Black cattle were studied. Fifteen cattle were super‐stimulated with hMG (total 600 IU), and the remaining 15 cattle were given FSH (total 20 mg). The plasma profiles of LH, estradiol‐17β (E₂) and progesterone (P₄) were correlated, and the embryo quality was investigated. The total number of ova recovered and the number of transferable embryos tended to be larger in the hMG‐treated group than in the FSH‐treated group. The percentage of excellent embryos tended to be higher in the hMG‐treated group than in the FSH‐treated group (54.3 and 28.7%, respectively, p < 0.10). The E₂ level increased during the first 3 days after the initial administration of either hMG or FSH and was higher in the hMG‐treated group than in the FSH‐treated group (p < 0.05). During this period, the E₂ level could be categorized into one of the following three types according to whether it increased or decreased and according to the degree of increase or decrease: (1) increase by a factor of 1.2 or more (quick increase type) (2) slight increase by a factor less than 1.2 (slow increase type), and (3) no increase (unstable increase type). In the group treated with hMG, 66.7% of the animals (10 of 15 cattle) showed a quick increase in the E₂ level. However, in the FSH‐treated group, 40% (six of 15) of the animals showed a slow increase in the E₂ level. The plasma LH level increased dramatically 8 h prior to the peak level in both the hMG‐ and FSH‐treated groups, and then it returned to the basal level 12 h later. After the administration of prostaglandin (PG)F_2α, the LH peak level was attained within 44 h in 80% of the animals in the hMG‐treated group, whereas in the FSH‐treated group, the LH peak level tended to be reached later. The P₄ level did not increase during the period of hMG or FSH treatment and decreased drastically following administration of PGF_2α. After the onset of oestrus, the P₄ level was higher in the hMG group than in the FSH group, and 5 to 7 days after oestrus, the level remained higher in the hMG group than in the FSH group (p < 0.05). After the first 3 days of hMG administration, the E₂/P₄ ratio was higher than that after FSH administration. Furthermore, on the day following PGF_2α administration, the ratio was significantly higher in the hMG group than in the FSH group (p < 0.05). These results indicate that superovulation in cattle given hMG results in a significant increase in plasma E₂ during the first 3 days and that the increase in the plasma P₄ level is larger a few days after oestrus and thereafter compared with FSH‐induced superovulation. Therefore, such plasma level profiles may be related to the increased recovery rate of high quality embryos.     

Advances in synchronizing estrus and ovulations in the mare: A mini review

Synchronization of estrus (SE) in mares has been achieved, but not of ovulation (SO). Progestins followed by PGF_2a are useful for SE only. In the two studies reviewed here, SE and SO were attempted by using CIDR-B, an intravaginal (itv) progesterone (1.9 g) releasing device, alone (study 1) or accompanied by estradiol (10 mg) given also itv (study 2). In both studies, Ovuplant™ (OT), an implant containing 2.1 mg of the GnRH analog deslorelin was used for the control of ovulation. Eighty cycling Hanoverian mares, 40 each in studies 1 and 2, received CIDR-B itv for 12 days, with PGF_2a given once at CIDR-B removal. In study 1, 15 mares each received OT when the lead follicle had reached 40 mm (A) or on Day 3 of estrus (B); 10 controls received no OT (C). In study 2, E2 was used in addition on Day 0 (CIDR-B insertion) (10 mares; group II), or on Days 0 and 7 (10; group III) or not (20; groups I and IV). Mares in groups I to III received OT as in study 1 (A); group IV (10) remained untreated. Ovaries were examined and blood samples were taken in studies 1 and 2 from all mares in 1, 2 or 4-day intervals, respectively, and concentrations of FSH, LH, progesterone and estradiol were determined by RIA. In study 1, CIDR-B treatment achieved SE, but not SO as shown by a wide spread of days on which follicles were reaching 40 mm; OT treatment assured ovulations in 48 hours in 93.3% of treated mares vs. 44.4% in controls (P<0.05. In study 2, SE was achieved and SO, but only when estradiol was given once (itv) on Day 0 (group II) but not twice on Days 0 and 7 (group III). In both studies, CIDR-B prevented estrus but stimulated follicle growth: 8 mares in study 1 ovulated with CIDR-Bs in place and 2 in trial 2, respectively. Only when estradiol was used together with CIDR-B, follicle growth was retarded (group II) or suppressed (group III: P<0.05 vs. groups I and IV). The pregnancy rate in study 2 from a single breeding at the first estrus was 52.8% with no significant differences between groups. FSH rose until Day 4 or 8 and had dropped sharply at Day 12; after CIDR-B removal FSH rose most quickly in group II, study 2. LH declined slightly until Day 12 and rose thereafter, reaching peak levels by Day 18 or 20, respectively. In both studies, estradiol had dropped slightly by Day 4 but increase steadily thereafter until ovulation had occurred. Preovulatory rise and postovulatory drop was seen earlier in group II, study 2. Values for progesterone had risen uniformly by Day 4, had declined slowly by Day 12 and precipitously in response to PGF_2a by Day 14. Treatment of cyclic mares with CIDR-B for 12 days, followed by PGF_2a at the day of CIDR-B removal and by Ovuplant™ a deslorelin implant when a follicle had reached 40 mm, resulted in synchronization of estrus. Adding to this scheme a single dose of estradiol (10 mg, intravaginal) on Day 0 resulted also in synchronization of ovulation.     

Comparison of plasma concentrations of estradiol-17β and progesterone, and conception in dairy cows with cystic ovarian diseases between Ovsynch and Ovsynch plus CIDR timed AI protocols

The objectives of this study were 1) to determine the effects of adding a CIDR to the Ovsynch protocol on plasma concentrations of estradiol-17β and progesterone and conception in dairy cows with cystic ovarian diseases and 2) to examine associations among the estradiol-17β and progesterone concentrations and conception. Cows were diagnosed as having cystic ovarian diseases if they were found to have a cystic follicle (diameter ≥25 mm) without a corpus luteum by two palpations per rectum with an interval for 7 to 14 days. They were treated with either the Ovsynch (GnRH on Day 0, PGF(2α) on Day 7 and GnRH on Day 9, with AI on Day 10; n=15) or Ovsynch+CIDR protocol (Ovsynch protocol plus a CIDR from Day 0 to Day 7; n=23). Plasma estradiol-17β concentrations were determined on Days 0, 7 and 9, and plasma progesterone concentrations were determined on Days 0, 7, 9 and 17. The plasma estradiol-17β and progesterone concentrations at all of the days examined and conception rates did not differ significantly between the two timed AI protocols. The progesterone concentrations on Day 17 and conception rates were lower (P<0.05) for cows with low concentrations of estradiol-17β (<2 pg/ml) on Day 9 than for cows with high concentrations of estradiol-17β (≥2 pg/ml). The present study suggests that, in dairy cows with cystic ovarian diseases, addition of a CIDR to the Ovsynch protocol had no remarkable effects on plasma estradiol-17β and progesterone concentrations during and after the treatments or on conception after timed AI. This study indicates that the low plasma estradiol-17β concentration at the second administration of GnRH in the protocols can be a predictor for impaired luteal formation and lower likelihood of pregnancy in dairy cows with cystic ovarian diseases.     

Quantitative analyses of insulin-like peptide 3 and sex steroid hormones in dominant follicles and corpora lutea during the estrus cycle and in follicular cysts in beef heifers

Insulin-like peptide 3 (INSL3) and sex steroids were measured in bovine dominant follicles and corpora lutea during the estrus cycle and in follicular cysts. Paired ovaries from beef heifers (n = 47) were classified, by their morphological features, either into four stages of the estrus cycle (Day 1 = day of ovulation, Day 20 = day of estrus) as Stage I (Days 1–4; n = 8), Stage II (Days 5–10; n = 10), Stage III (Days 11–17; n = 8), and Stage IV (Days 18–20; n = 11) or follicular cystic (n = 10). Cysts (n = 15) were subdivided into estrogen-active (n = 7) and estrogen-inactive (n = 8) cysts. INSL3, testosterone, and estradiol-17β concentrations in the dominant follicular fluid of Stage IV were higher than those in Stages II and III (P < 0.05). INSL3 concentrations in the cystic fluid were similar to those in dominant follicles at Stage IV, whereas testosterone and estradiol-17β concentrations were lower in cysts (P < 0.05). INSL3 content per estrogen-inactive cyst was higher than that of Stage IV (P < 0.05). INSL3 and progesterone concentrations in luteal tissue and contents per corpus luteum were higher in Stages II and III (P < 0.05). In conclusion, INSL3 secretion in bovine dominant follicles increased with maturation. Follicular cysts may retain the production of INSL3 during their formation but tend to lose the capacity for testosterone secretion. Estrogen-inactive cysts subjected to advanced atresia may accumulate more INSL3. INSL3 production in bovine corpora lutea is enhanced during maturation.     

Factors affecting the success of resynchronization protocols with or without progesterone supplementation in dairy cows

The objective of this study was to investigate factors that influence the success of resynchronization protocols for bovines with and without progesterone supplementation. Cow synchronized and not found pregnant were randomly assigned to two resynchronization protocols: ovsynch without progesterone (P4) supplementation (n = 66) or with exogenous P4 administered from Days 0 to 7 (n = 67). Progesterone levels were measured on Days 0 and 7 of these protocols as well as 4 and 5 days post-insemination. Progesterone supplementation raised the P4 levels on Day 7 (p < 0.05), but had no overall effect on resynchronization rates (RRs) or pregnancy per artificial insemination (P/AI). However, cows with Body Condition Score (BCS) > 3.5 had increased P/AI values while cows with BCS < 2.75 had decreased P/AI rates after P4 supplementation. Primiparous cows had higher P4 values on Day 7 than pluriparous animals (p = 0.04) and tended to have higher RRs (p = 0.06). Results of this study indicate that progesterone supplementation in resynchronization protocols has minimal effects on outcomes. Parity had an effect on the levels of circulating progesterone at initiation of the protocol, which in turn influenced the RR.     

Physiological roles of inhibin in regulation of FSH secretion and follicular development during early pregnancy in goats

The aim of the current study was to clarify the physiological role of inhibin in controlling FSH secretion and follicular development during the early pregnancy in goats. Eight goats investigated sonographically on Days 19-21 (Day 0=day of mating) for pregnancy were assigned into control (n=3) and treated (n=5) groups. The ovaries of all animals were daily scanned with ultrasound for follicles 2mm or more in diameter from 1 day before to 5 days after treatment. On Day 25 postbreeding; animals received either 10 ml, of normal goat serum or antiserum against [Tyr (30)]-inhibin alpha (1-30). Jugular blood samples were collected every 6 h starting 24 h before and until 120 h after treatment. The plasma concentration of FSH increased at 6 h and remained at significantly high levels until 120 h in treated vs. control group. The plasma concentrations of estradiol showed a marked increased at 66 h, with peak levels at 120 h after treatment of antiserum. The basal concentrations of LH and the pattern of plasma concentrations of progesterone were not significantly different between the two groups. The number of medium size (3.5-5.0 mm) follicles increased considerably from Day 2, whereas small (3.5 mm or less) and large (5 mm or more) follicles increased noticeably from Day 3, as compared with pre-treatment and controls. These results clearly indicated that inhibin is a key hormone in regulation of follicular development through regulation of endogenous FSH secretion during early pregnancy in goats.     

Ovarian follicular dynamics in heifers: test of two-wave hypothesis by ultrasonically monitoring individual follicles

The two-wave hypothesis for follicular development during the bovine estrous cycle was tested by ultrasonically monitoring individual follicles in 10 heifers during an interovulatory interval. A dominant follicle was defined as one that reached a diameter of at least 11 mm. Subordinate follicles were defined as those that appeared to originate from the same follicular pool as a dominant follicle. A dominant follicle and its cohorts were defined as a wave. Two waves during an interovulatory interval were identified in 9 of 10 heifers. The first wave was first identified, retrospectively, on a mean of Day 0.2 +/- 0.1 (ovulation = Day 0) and gave origin to a dominant anovulatory follicle and a mean of 1.4 +/- 0.3 identified subordinates. The dominant follicle reached maximum diameter (mean, 15.8 +/- 0.8 mm) on an average of Day 7 and then decreased (P less than .04) by Day 11. The subordinate follicles increased in diameter for a few days and then regressed. The second wave was first identified on a mean of Day 10.0 +/- 0.4 and gave origin to the ovulatory follicle and a mean of 0.9 +/- 0.3 subordinates. One of the 10 heifers had 3 waves of follicular activity characterized by an anovulatory wave emerging on Day 0, another anovulatory wave emerging on Day 10, and an ovulatory wave emerging on Day 16. Results strongly supported the two-wave hypothesis but also indicated that a minority of interovulatory intervals in this heifer population may have 3 waves of follicular activity.     

Postpartum gonadotropin secretion in Holstein-Friesian dairy cows differing genetically in liveweight

AIM: To determine whether there are differences in postpartum gonadotrophic activity between strains of Holstein-Friesian dairy cows genetically selected on mature liveweight that might explain differences between the strains in fertility, and the interval between calving and the resumption of ovarian follicular activity. METHODS: Mixed-age Holstein-Friesian cows fed generous allowances of ryegrass/white clover pasture, and genetically selected for heavy (H) or light (L) mature liveweight, were given 10 microg buserelin on Days 21, 28, 35 and 42 (Experiment 1a; n=8/group), or Days 7, 14, 21 and 28 (Experiment 1b; n=8/group) postpartum. The same dose of buserelin was also given to first-calved heifers from each strain (Experiment 1c; n=6/group) on Days 7, 14, 21, and 28 postpartum. Luteinising hormone (LH)concentrations were measured in serial blood samples that were taken for up to 240 min after administration of buserelin. In Experiment 2, serial blood samples were taken at 15-min intervals from H and L cows (n=7/group) over 8 h on Days 14, 21, 28 and 35 postpartum, to examine the endogenous secretion patterns of LH and follicle stimulating hormone (FSH).The time-course of the restoration of positive feedback between oestradiol and LH was examined by giving 1 mg oestradiol benzoate(ODB) 48 h after administration of 500 microg cloprostenol to mixed-age cows from each strain on Days 7 and 21 (n=8/group), or 14 and 28 (n=8/group) after calving (Experiment 3). Relationships between nutrition and the restoration of positive feedback were studied by giving 0.75 mg ODB/500 kg liveweight on Day 17 or 18 after calving to pure-bred Holstein (OSH) and New Zealand Friesian (NZF) cows that were fed either pasture (n=12 OSH, 12 NZF) or a total mixed ration (TMR; n=13 OSH,12 NZF) (Experiment 4). Plasma LH and FSH concentrations were measured in samples collected for 42 h (Experiment 3) or 48 h (Experiment 4) after treatment with ODB. Milk progesterone concentrations were measured 3x weekly to define the reproductive status of animals in each experiment. Conception rates were recorded for animals in all of the experiments. RESULTS: First-service conception rates were lower (p<0.05) in H than L cows (46% vs 59%). In Experiments 1b and 1c, LH response to buserelin increased between Days 7 and 28 postpartum (both p<0.001), but did not differ between strains (p=0.77 and p=0.19, respectively). In Experiment 1a, LH responses to buserelin did not change between Days 21 and 42 postpartum, but overall mean peak concentrations were significantly(p<0.001) greater in L than H cows. In Experiment 2, anoestrous H cows had higher mean (p=0.004) and episodic (p=0.001) concentrations of LH than did L cows, but in cows that had active corpora lutea there were no such differences. There were no differences in FSH concentrations between strains. LH secretion in response to exogenous oestradiol (Experiment 3) increased between Days 7 and 28 postpartum (p<0.001), but there were no differences between strains. Responses were also similar in OSH and NZF cows on Day 17 or 18 postpartum, although there was a significant effect of ration upon the proportion of cows that exhibited an LH surge (20/24 cows on grass vs 12/25 on a TMR; p=0.005). CONCLUSION: These results confirm that H cows have poorer first-service conception rates than L cows, but do not support an hypothesis that there are major differences between these strains of Holstein-Friesian dairy cows in the rate of restoration in the hypothalamo-pituitary axis. However, in anoestrous cows, differences between strains in the endogenous release of LH maybe related to an earlier onset of oestrous cycles in H animals.     

Relationship of the conception rate and the side (left or right) of preovulatory follicle location at artificial insemination in dairy heifers

In this study, we examined the locational effect (left or right ovary) of the preovulatory follicle (PF) on fertility in dairy heifers. In total, 1,111 artificial inseminations (AI) were analyzed. At AI, PF locations were examined using rectal palpation, and heifers were divided into two groups on their PF locations: (i) the PF located in the left ovary (L‐PF); and (ii) the PF located in the right ovary (R‐PF). Pregnancy was diagnosed by rectal palpation 60 days after AI. The conception rate was 50.7% in all heifers. Conception rate was significantly higher in the L‐PF (60.1%) than in the R‐PF (46.2%). The conception rate was significantly lower by sexed semen (48.6%) than conventional semen (59.1%). Conception rates divided by the semen type (sexed: n = 896, conventional: n = 215) were significantly higher in the L‐PF than in the R‐PF for both semen types (sexed; L‐PF vs. R‐PF: 57.3% vs. 44.4%, conventional; L‐PF vs. R‐PF: 72.3% vs. 53.3%). In addition, season, age, AI number, and the number of re‐inseminations at the same estrus did not affect conception rates. In summary, PF development in the left ovary was associated with increased conception rates in dairy heifers.     

Androgens promote the acquisition of maturation competence in bovine oocytes

Recent studies in mice suggest that androgens are important for normal follicle development. However, there have been few reports concerning the action of androgens in the growth of oocytes from large animals. The purpose of this study was to determine the roles of androgens in bovine oocyte growth in vitro. Oocyte-granulosa cell complexes (OGCs) collected from 0.4−0.7 mm early antral follicles were cultured for 14 days with 17β-estradiol (E₂) and a non-aromatizable androgen, dihydrotestosterone (DHT). We also examined the ability of an androgen receptor (AR) inhibitor, hydroxyflutamide, to antagonize the effect of androgens on the oocytes. During growth culture, the OGC structures collapsed in the medium with DHT alone, while in the presence of E₂, the OGC structures were maintained. In the medium with both androgens and E₂, the mean diameter of oocytes was increased from 95 μm to around 120 μm, larger than those grown with E₂ alone (115 μm). Also in the maturation culture, oocytes grown with androgens (A₄ or DHT) and E₂ showed higher percentages of metaphase II oocytes (63% or 69%, respectively) than those grown with E₂ alone (32%). Moreover, these maturation rates were decreased by hydroxyflutamide in a dose-dependent manner. Immunostaining showed that ARs were expressed in oocytes and granulosa cells in early antral follicles, and the nuclei of granulosa cells showed intense AR expression. In conclusion, although E₂ supports the OGC structure, additional androgens promote oocyte growth and their acquisition of meiotic competence via AR during in vitro growth culture.     

Follicular turnover and hormonal association in postpartum goats during early and late lactation

To clarify the effect of lactation period on ovarian follicular activity and associated hormonal levels in goats, six goats were monitored daily by ultrasonographic examination with blood sampling during early (Days 5 to 25; Day 0 was the day of kidding) and late (Days 40 to 60) lactation. While the presence of a corpus luteum of pregnancy retarded follicular growth in the ipsilateral ovary until Days 11-13 postpartum, the total follicular number (TFN) and area (TFA) increased during late lactation due to the significant increase in the number of medium- and large-sized follicles and decrease in the number of small follicles. Four goats showed a similar pattern of follicular development during the period studied characterized by the emergence of five and six waves during the early and late lactation, respectively. The largest follicle diameter of the first three waves monitored during early lactation was significantly smaller as compared with the diameter of those existing during late lactation. TFN showed a positive correlation with FSH but showed a negative correlation with immunoreactive (ir-) inhibin and estradiol during the postpartum period. TFA was positively correlated with ir-inhibin, estradiol and PRL and negatively correlated with FSH during the monitored periods. The plasma levels of ir-inhibin and progesterone were significantly higher during late lactation compared with the levels recorded during early lactation. Ir-inhibin levels showed a significant positive correlation with LH and estradiol during early and late lactation but showed a negative correlation with FSH during the whole lactation period. LH was positively correlated with estradiol and PRL during early and late lactation, respectively. These results suggest that the lactation period has a detrimental effect on ovarian activity during the early postpartum period in goats.