Follicle Ablation Research Articles

Effect of use and dosage of p-follicle-stimulating hormone for ovarian superstimulation before ovum pick-up and in vitro embryo production in pregnant Holstein heifers

The benefit of ovarian superstimulation using exogenous FSH before ovum pick-up (OPU) and in vitro embryo production (IVEP) has been the subject of conflicting results. The objective of the present study, therefore, was to evaluate the effect of use and dose of porcine FSH (p-FSH) before OPU/IVEP on ovarian response and embryo production in pregnant heifers. Pregnant Holstein heifers (n = 48) were assigned randomly to receive 0, 160, or 300 mg NIH-FSH-P1 in a crossover design. Ovum pick-up was performed at 49, 63, and 77 d of gestation with a 14 d "washout" between OPU sessions. Follicle ablation was performed on D 0 (p.m.) and p-FSH treatments, consisting of 4 decreasing dose injections administered 12 h apart, were initiated 36 h after follicle ablation (D2 a.m.). Heifers underwent OPU on D 5 (a.m.), 40 h after the last p-FSH treatment, and cumulus oocyte complexes (COC) were subjected to IVEP procedures. Differences between treatment groups were evaluated using generalized linear mixed models. There were quadratic effects of treatment on both number and percentage of small (<6 mm), medium (6-10 mm) and large (>10 mm) follicles. Number and percentage of medium follicles increased with increasing p-FSH dosages, although the magnitude of the change was greater between 0 and 160 mg, than between 160 and 300 mg of p-FSH. Total number of follicles, number of COC recovered and number of viable COC increased linearly with increasing p-FSH dose. Conversely, there was no evidence for an effect of p-FSH dose on COC recovery percentage nor the percentage of viable COC. Cleavage percentage, number of cleaved oocytes, blastocyst percentage, and number of blastocysts increased linearly with increasing p-FSH dose. In conclusion, use of p-FSH before OPU resulted in greater superstimulatory response and oocyte competence which in turn increased IVEP. Furthermore, these effects were dose dependent such that use of a greater dose of p-FSH up to 300 mg progressively increased embryo yield.

Follicular fluid and plasma concentrations of progesterone following hCG administration before ultrasound-guided ablation of dominant follicles ≥25mm in mares

Ultrasound-guided transvaginal follicle ablation of dominant follicles ≥25mm during the follicular phase results in luteinization and an increase in circulating concentrations of progesterone (Ignácio et al., Theriogenology. 2021; 160:128-133). Although LH receptors are present in early antral follicles of 5mm diameter (Goudet et al., Biol Reprod. 1999; 60:1120-7) and increase after deviation (Bastos et al., Theriogenology. 2014; 82:1131-6), the ablation-induced luteinization rate may be less for smaller-sized dominant follicles <30mm and progesterone may not achieve concentrations >2ng/mL in a timely manner (Mozzaquatro et al., Reprod Domest Anim. 2012; 47:288-92). The objective of the present study was to determine if hCG administered 24h before ablation of dominant follicles <30 or ≥30mm increases the luteinization rate and results in progesterone concentrations >2ng/mL to support pregnancy. Cycling mares were randomly assigned to four ablation groups: 1) ≥25 (ablation of follicles 25-29mm, n=9), 2) hCG+≥25 (2500 IU hCG, i.v. 24h before ablation of follicles 25-29mm, n=11), 3) ≥30 (ablation of follicles ≥30mm, n=13), and 4) hCG+≥30mm (2500 IU hCG, i.v. 24h before ablation of follicles ≥30mm, n=10). Subsequent to PGF2α – induced luteolysis in all mares and administration of hCG to respective groups, D0 marked the day whenthe dominant follicle reached 25-29mm or ≥30mm and follicular fluid was collected. Blood samples were collected daily until D15, fresh embryos were transferred when plasma plasma progesterone (PP4) was >2ng/mL on D7. Follicular fluid progesterone (FFP4) and PP4 were measured by a commercial RIA kit. Wilcoxon test was used to evaluate FFP4. Repeated measures models and Tuckey test were used to evaluate PP4. Regardless of follicle size on D0, mean FFP4 but not PP4 was higher (P<0.05) in mares that received hCG (115.0ng/mL) vs those that did not (53.0ng/mL). Based on PP4 >2ng/mL, luteinization rate was 43 vs 75% in the ≥25 vs hCG+≥25 group (P<0.05) and 77 vs 67% in the ≥30 vs hCG+≥30 group (P>0.05). From D0, daily PP4 gradually increased and on D7 mean concentrations did not differ among the ≥25, hCG+≥25, ≥30, and hCG+≥30 groups, respectively. Regardless of follicle size, embryo transfer resulted in pregnancy rates of 43% (3/7) for the follicle ablation alone and 43% (6/14) for the hCG + ablation groups. In conclusion, administration of hCG before ablation of follicles 25-29mm but not ≥30mm enhanced the luteinization rate with PP4 >2ng/mL at D7 among groups. This was sufficient to support pregnancy after embryo transfer.

In vitro derived embryos produced from mares treated with intravenous clomiphene citrate

Optimizing the number of follicles available for ovum pick-up and in vitro embryo production (OPU-IVP) would increase the efficiency of OPU-IVP sessions. In other species, clomiphene citrate (CC) has been credited with enhancing follicular recruitment through augmented GnRH pulsatility. Our goal was to describe the number of follicles, reproductive hormone secretion patterns, and oocyte developmental potential in mares treated with CC and subjected to OPU-IVP. Six light-breed mares underwent follicle ablation (Day 0) to induce a new follicular wave, followed by administration of 250mcg of cloprostenol (PGF2-α) on Day 6. On Days 10-14, a five-day protocol of 0.5% intravenous CC (Annaji.et.al. AAPS PharmSciTech. 2023; 24:48) was administered at a rate of 2grams/mare/once daily; all mares subsequently underwent OPU on Day 17. Collected oocytes were shipped counter-to-counter to a laboratory for fertilization via intracytoplasmic sperm injection (ICSI). Ovarian follicle counts were performed via transrectal ultrasonography immediately prior to each daily administration of CC (Days 10-14) and on the day of OPU (Day 17); the total number of follicles, in addition to the diameter of the largest follicle (mm) were recorded. Serum samples were collected to quantify and characterize systemic reproductive hormone patterns (i.e., LH, FSH, AMH, and P4); sample collection started on the day of ablation (Day 0) and concluded one week following OPU (Day 24).CORR and PRINQUAL procedures of SAS version 9.4 (SAS Institute, Cary, North Carolina) were utilized to evaluate correlation amongst hormones. The development rate of embryos originating from CC treated mares were compared to development rates of commercial embryos produced during the same time period at the ICSI laboratory. Parametric (T-test) and nonparametric (NPAR1WAY) procedures (SAS) were utilized to compare embryo development rates. For all statistical analyses, significance level was set at p≤0.05. Upon completion of CC treatment, the mean (+/- SEM) diameter of the largest follicle and the mean (+/- SEM) total number of follicles was 40 mm (+/- 5.1 mm) and 11.3 (+/- 1.2), respectively. Significant negative correlations were found between LH and FSH (p=0.007), FSH and the largest follicle (p<0.001), and FSH and total follicle number (p=0.01). Conversely, significant positive correlations were found between LH and the largest follicle (p=0.003), AMH and total follicle number (p=0.01), and AMH and P4 (p=0.008). The overall embryo development rate was 50% for CC treated mares (15/30 injected oocytes) and 36.8% (14/38 injected oocytes) for commercial embryos (p=0.56). Although experimental control mares were not utilized in this pilot study, our results warrant continued investigation to identify the impact(s) of intravenous CC administration on follicular dynamics, OPU-IVP and foal production.

The effect of different FSH administration before ovum pick up on superstimulation response and oocyte yield.

This study aims to evaluate the effect of synchronization and different superstimulation protocols on oocyte yield before ovum-pick up (OPU) to provide a homogeneous follicle population. Animals in all study groups except the control group applied a synchronization protocol including modified ovsynch+progesterone and dominant follicle ablation (DFA, on the 6th day following synchronization). In group 1, oocytes were retrieved by ultrasonography only on the 4th day after DFA. In group 2, 250 μg pFSH (100 μg IM, 150 μg SC) was administered as a single dose on the 2nd day following DFA, and oocytes were retrieved on the 2nd day following this injection. In group 3, 250 μg pFSH was administered IM in four equal doses 12 h apart on the 1st and 2nd days following DFA, and oocytes were retrieved 2 days after the last FSH injection. In group 4, 250 μg pFSH (IM) dissolved in Montanide ISA 206 adjuvant was administered as a single dose on the 2nd day following DFA, and oocytes were retrieved 2 days after this treatment. In the control group (group 5), oocytes were retrieved from animals without any hormonal treatment on a random day of the oestrus cycle. In order to assess the follicle population in the ovary on the day of OPU, the number of follicles according to their diameter was determined by ultrasonography in all groups. The ratio of medium-sized follicles (3-8 mm) was higher in the synchronized groups (Groups 1, 2, 3 and 4) than in the control group (Group 5) (p < .05). It was determined that the total number of oocytes obtained after OPU and the number of oocytes of suitable quality (Grade A and B) in in vitro embryo production were higher in the superstimulated groups (Groups 2, 3 and 4) compared to the control group. It was found that the number of Grade-A quality oocytes was higher in the superstimulated groups (Groups 2, 3 and 4) than the other groups. As a result, it was found that the synchronization and superstimulation treatments prior to the OPU increased the ratio of medium-sized follicles and the total number of oocytes obtained. In addition to the synchronization protocol, it was determined that superstimulation treatments increased the oocyte quality obtained with OPU. Furthermore, it was observed that a single dose of FSH dissolved in Montanide ISA 206 adjuvant produced a superstimulation response similar to that produced by repeated doses of FSH.

Temporality of ovarian steroids and LH/FSH pulse profiles encompassing selection of the dominant follicle in heifers†.

The tested hypotheses were (1) LH/FSH pulses and F2 diameter are diminished by P4 and, (2) E2 increases during the transition to deviation and alters LH/FSH pulses. On Day 5 (Day 0 = ovulation), heifers were randomized into an untreated group (HiP4, n = 11), and a prostaglandin analog treated group (NoP4, n = 10). On Day 6, a follicular wave was induced by follicle ablation. Ultrasound and blood collections were performed every 12h from Days 7 to 11. Blood was collected every 15min for 10h on Day 9 (largest follicle expected to be ~7.5mm). Estradiol was ~75% greater (0.36 ± 0.14 vs 0.63 ± 0.19pg/mL) in heifers with F1 ≥ 7.2mm than in heifers with F1 < 7.2mm. The HiP4 had smaller second largest follicle (F2) diameter, lower estradiol (P = 0.06), LH pulse baseline and peak concentrations (P < 0.007), in addition to half the frequency of LH/FSH pulses (4.1 ± 0.3 vs 9.6 ± 0.7 in 10h) than the NoP4. Within HiP4, heifers with F1 ≥ 7.2mm had ~25% fewer (P = 0.03) LH pulses compared to heifers with F1 < 7.2mm. In contrast, within the NoP4, heifers with F1 ≥ 7.2mm had ~75% greater LH (P = 0.05) and FSH (P = 0.08) pulse amplitude. We propose that greater F2 diameter at deviation in low P4 is related to greater LH baseline and peak concentrations, and greater frequency of LH/FSH pulses. A greater increase in E2 after F1 reaches ~7.2mm results in further stimulation of LH/FSH pulse amplitude. Elevated P4 not only diminished frequency of LH/FSH pulses but also converted an E2 increase into a negative feedback effect on LH/FSH pulse frequency leading to smaller F2 at deviation.

Resolution of Mild Pilonidal Disease in Adolescents Without Resection.

Pilonidal disease is a common condition of the gluteal crease, affecting around 1 in 2,000 adolescents and young adults. 1 Traditional options for management of pilonidal disease include improved hygiene with or without hair removal or resection of the nidus. Given a high recurrence rate associated with hygiene alone, nidus resection is often recommended, even for patients with mild pilonidal disease, despite significant postoperative morbidity. We present a consecutive series of patients with mild pilonidal disease managed in a dedicated Pilonidal Care Clinic using an alternate approach directed toward source control: improved hygiene to limit debris in the gluteal crease, excision of midline pilonidal pits under local anesthesia to prevent intrusion of debris with drainage of any nidus present, and laser ablation of midline follicles to prevent new pits from forming. 2. Data on demographics, disease severity, symptom scoring, treatments provided, and outcome for consecutive new patients presenting to an outpatient pilonidal care clinic with mild disease between August 2017 and September 2020 were analyzed. One hundred two patients presented; their mean age was 16.3 years, 56 (55%) were female, and 42 (41%) had undergone prior nidus incision and drainage. Twenty-four were lost to follow up, and 77 of the 78 who continued care had resolution of their pilonidal disease after a mean of 3 ± 2.5 laser epilations and 1.3 ± 1 pit excisions during 4 ± 2 clinic visits over a treatment duration of 30 ± 19 weeks. Mild pilonidal disease may be resolved with improved hygiene, pit excision, and laser epilation with minimal morbidity and no activity restrictions. Adoption of this approach may keep a large number of patients with pilonidal disease from undergoing unindicated resection.

Effect of follicular ablation and gonadotropin priming on the recovery and quality of oocytes in Boran cows.

The objective of the study was to evaluate the effect of follicle ablation, exogenous FSH application, and different coasting time prior to ovum pick-up (OPU) on the number of follicles suitable for aspiration, oocyte quality, and cleavage rate in Ethiopian Boran cows. The experiment was carried out in three parts, (I) cows were synchronized using 500µg PGF2α given 11days apart. Cows were then subjected to a biweekly ovum pickup session before ovulation (n = 5) or starting day 7 after ovulation (n = 4) for 3weeks. (II) Cows were synchronized, and all visible follicles were ablated on the first days of overt estrus, and cows were grouped into those that received a divided dose of 350IU FSH (n = 5) or 175IU FSH (n = 5) over 3days. In both groups, OPU was carried out weekly starting 48h after the last FSH for 6weeks. (III) Protocol was similar to part II, but in group with 350IU FSH (n = 5), coasting period was increased to 72h. The covariates of follicles and oocyte were not affected (P > 0.05) by corpus luteum presence at OPU. The mean number of medium (7.36 ± 0.57) and large (8.28 ± 0.96) follicles were significantly higher (P < 0.05) in the group that received divided 350IU FSH. Similarly, the mean number of grade-1 (4.19 ± 0.24) and grade-2 (4.32 ± .27) oocytes, maturation rate (70.41%), and cleavage rate (47.5%) were significantly higher (P < 0.05) in the group that received 350IU FSH. COC quality was significantly (P < 0.05) influenced by coasting period. However, both maturation and cleavage rates were not affected by the coasting period. This study demonstrated that follicular ablation and treatment with FSH improves follicular population and oocyte recovery rate in Boran cows.

Synchronization of follicle wave emergence before ovarian superstimulation with FSH and ovum pick-up improves in vitro embryo production in pregnant heifers

The objective of the present study was to evaluate effects of synchronization of timing of follicle wave emergence, before ovarian superstimulation and ovum pick-up (OPU), on ovarian response and embryo production in pregnant heifers. Pregnant (47–69 days of gestation) Holstein heifers (n = 64), 19.0 ± 0.3 months of age, were assigned in a completely randomized design to one of two groups: synchronization of follicular wave emergence using follicle ablation (Synchronized) or untreated control (Non-synchronized). Superstimulatory treatments consisting of 160 mg (280 IU) of porcine follicle stimulating hormone (p-FSH), administered in four decreasing dose treatments 12 h apart, were initiated 36 h after follicle ablation or at random stages of the follicular wave in heifers of the Synchronized and Non-synchronized group, respectively. Ovum pick-up was performed in all heifers 40 h after the last p-FSH administration and retrieved cumulus oocyte complexes (COCs) were subjected to in vitro embryo production (IVEP) procedures. Ultrasonography was performed immediately before OPU to determine number and size of ovarian follicles. Differences in treatment responses between groups were evaluated using generalized linear mixed models. Total number of follicles at the time of OPU was not different between treatment groups (P = 0.61), however, the number of small follicles (<6 mm) was greater (P = 0.05) in heifers of the Non-synchronized group, whereas number of medium size follicles (6–10 mm) tended (P = 0.09) to be greater in heifers of the Synchronized group. Heifers in the Synchronized group had a greater (P = 0.01) percentage of medium sized follicles and a lesser (P = 0.01) percentage of smaller sized follicles than heifers in the Non-synchronized group. There were no differences (P > 0.15) in total number of recovered COCs, or number of viable COCs between groups. Cleavage percentage (84.5% and 72.8%) and blastocyst percentage (48.2% and 33.4%) were greater (P < 0.01) in heifers of the Synchronized than Non-synchronized group, respectively. As a result, mean number of blastocysts per OPU/heifer was greater (P = 0.006) in the heifers of the Synchronized (8.9 ± 1.0) than the Non-synchronized (5.5 ± 0.9) group. In conclusion, synchronizing the time of follicle wave emergence in pregnant heifers, prior to ovarian superstimulation with FSH and OPU results in a greater superstimulatory response and oocyte competence leading to greater embryo production.

Activity-dependent feedback regulation of thalamocortical axon development by Lhx2 in cortical layer 4 neurons.

Establishing neuronal circuits requires interactions between pre- and postsynaptic neurons. While presynaptic neurons were shown to play instructive roles for the postsynaptic neurons, how postsynaptic neurons provide feedback to regulate the presynaptic neuronal development remains elusive. To elucidate the mechanisms for circuit formation, we study the development of barrel cortex (the primary sensory cortex, S1), whose development is instructed by presynaptic thalamocortical axons (TCAs). In the first postnatal weeks, TCA terminals arborize in layer (L) 4 to fill in the barrel center, but it is unclear how TCA development is regulated. Here, we reported that the deletion of Lhx2 specifically in the cortical neurons in the conditional knockout (cKO) leads to TCA arborization defects, which is accompanied with deficits in sensory-evoked and spontaneous cortical activities and impaired lesion-induced plasticity following early whisker follicle ablation. Reintroducing Lhx2 back in L4 neurons in cKO ameliorated TCA arborization and plasticity defects. By manipulating L4 neuronal activity, we further demonstrated that Lhx2 induces TCA arborization via an activity-dependent mechanism. Additionally, we identified the extracellular signaling protein Sema7a as an activity-dependent downstream target of Lhx2 in regulating TCA branching. Thus, we discovered a bottom-up feedback mechanism for the L4 neurons to regulate TCA development.

Fertility response following ablation-induced follicular wave emergence and ovulation induction in anestrous buffaloes

The fertility response following induction of follicular wave emergence by follicle ablation combined withprogesterone based ovulation synchronization in postpartum anestrous buffaloes was assessed. Postpartum anestrousbuffaloes (n=24) with absence of overt estrus signs categorized as treatment group (Group 1; n=15) and controlgroup (Group 2; n=9). Group 1 was subjected to transvaginal ultrasound-guided follicle ablation on a random dayof the estrous cycle (Day=0) and insertion of CIDR. Seven days later, CIDR was removed and prostaglandin F2αanalogue was given intramuscularly. GnRH analogue was injected on day 9.5 and fixed-time AI was done 12 h and24 h later. Ultrasonography was performed to follow ovarian structures on different occasions and on day 38 postovulationfor detecting pregnancy. Group 2 did not receive any treatment however, estrus detection in this groupwas done daily by bull parading and visual monitoring followed by AI if found in standing estrus. In treatmentgroup, the follicular wave emergence occurred by 24 h following ablation in 60% buffaloes and in 40% remaininganimals by 48 h. The estrus exhibition was 100 % in group 1 versus 22.2 % in group 2. Mean interval from CIDRremoval to ovulation was 81.2±3.08 h and from wave emergence to ovulation was 9.92±0.18 days. The first servicepregnancy rate in treatment group was higher than control (66.67% vs 22.2%). The induction of follicular waveemergence through ultrasound guided follicle ablation and progesterone based ovulation synchronization can beused to improve pregnancy rates in anestrous buffaloes.

Effect of follicular ablation and gonadotropin priming on the recovery and quality of oocytes in Boran cows

Genetic differences have been suggested as a possible cause for variation in responses to exogenous hormones. Here we evaluated the effect of follicle ablation, exogenous FSH and coasting time prior to ovum pick-up (OPU) on the number of follicles suitable for aspiration, oocyte quality, and cleavage rate in Ethiopian Boran cows. The experiment was carried out in three parts, I) Cows were synchronized using 500µg PGF2α given 11 days apart. Cows were then subjected to a biweekly ovum pickup session before ovulation (n=5) or starting Day 7 after ovulation (n=4) for three weeks. II) Cows were similarly synchronized and all visible follicles were ablated on the first days of overt estrus which were then further grouped into cows that received a divided dose of 350IU FSH (n=5) or 175IU FSH (n=5) over three days. In both groups OPU was carried out weekly starting 48h after the last FSH for six weeks. III) A similar protocol as in part II was carried out but coasting period was increased to 72hrs for cows that received 350IU FSH as divided dose (n=5) and 48hrs coasting period for single 350IU FSH dose (n=5). The covariates of follicles and oocyte were not affected (P&gt;0.05) by corpus luteum presence at OPU. The mean number of medium (7.36±0.57) and large (8.28±0.96) follicles were significantly higher (P&lt;0.05) in group that received divided 350IU FSH. Similarly, the mean number of Grade-1 (4.19±0.24) and Grade-2 (4.32±.27) COC, maturation rate (70.41%) and cleavage rate (47.5%) were significantly higher (P&lt;0.05) in group that received 350IU FSH. COC quality was significantly (P&lt;0.05) influenced by costing period. However, both maturation and cleavage rates were not affected by the coasting period. This study demonstrated that follicular ablation and treatment with FSH improves follicular population and oocyte recovery rate in Boran cows.

Active immunization against GnRH as an alternative therapeutic approach for the management of Bos indicus oocyte donors diagnosed with chronic cystic ovarian disease

The aim of this study was to evaluate the effect of active immunization against GnRH in Nelore (Bos indicus) cows repeatedly used as oocyte donors that developed chronic cystic ovarian disease (COD). In the first experiment, ovarian and uterine features were first compared between COD cows (n = 15) and healthy cows (n = 22, cycling control group) from the same breed and herd. Cows with COD had a greater number of large (P < 0.0001) and medium follicles (P < 0.01) but lesser small follicles (P < 0.05) than cycling controls. Mucometra was diagnosed in 73.3% of COD cows, but in none of the controls. No difference in average thickness of the endometrium was detected between groups; however, endometrial thickness and mucometra score were negatively correlated (R = −0.73, P = 0.0029) in COD cows. In the second experiment, COD cows were randomly allocated into two experimental groups, which received two 1.0 mL SC injections of either an anti-GnRH vaccine (COD immunized group, n = 8) or saline (COD control group, n = 7), given 28 days apart. Cows were examined weekly by transrectal ultrasonography during nine consecutive weeks after the first injection to evaluate the number and distribution of follicles among size classes, endometrial thickness, and presence of clinical mucometra. Vaccination against GnRH resulted in a progressive suppression of follicle growth and a reduction in the average size of the largest follicle, as well as in the number of large follicles (P < 0.01) in COD immunized cows compared with COD controls from week 7 onwards. Conversely, the number of small follicles in the COD immunized group increased after week 5 and was greater (P = 0.0023) than controls on week 9. Endometrial thickness and mucometra score were not affected (P > 0.05) by immunization against GnRH. In the third experiment, the COD immunized cows with effective suppression of follicle growth four weeks after the 2nd injection (n = 6) were submitted to three consecutive ovum pick-up (OPU) sessions (weeks 10, 11, and 12) for in vitro embryo production (IVEP). Cumulus-oocyte complexes (COC) collected from slaughterhouse ovaries were used as controls for IVEP. COD cows with produced 25.0 ± 3.8 COC per OPU session with no apparent detrimental effect of anti-GnRH vaccine on oocyte developmental potential in vitro, i.e., we observed similar cleavage rate (P = 0.5914) and greater blastocyst rate (P = 0.0177) in immunized cows compared with COC from slaughterhouse controls. Finally, in the fourth experiment wave emergence and follicular dynamics after follicle ablation were compared between COD immunized cows with effective suppression of follicle growth and a subset (n = 6) of the cycling, control group. No follicles grew beyond 4 mm diameter after follicle ablation in the COD immunized group, whereas a normal follicular wave emergence occurred in cycling controls. Antral follicle count was similar between cycling controls and COD immunized groups at 24 h and 96 h post-follicle ablation (P > 0.05), but greater in cycling controls at 48 h and 72 h post-follicle ablation (P < 0.05). In summary, our results suggest that active immunization against GnRH is effective to induce the regression of follicular cysts as well as increase the number of small follicles growing on the ovaries, in oocyte donors diagnosed with chronic COD, with no apparent negative effect on oocyte developmental potential in vitro.

83 Response to follicle-stimulating hormone superstimulation in heifers with ovarian activity suppressed by active immunization against gonadotrophin-releasing hormone

In the present study, we evaluated the ovarian response to exogenous FSH stimulation in the absence of endogenous LH, using as experimental model heifers immunized against GnRH. Pubertal, cycling Nelore (Bos indicus) heifers were allocated into three experimental groups: (1) non-immunized, FSH stimulated (B−FSH+, n=5), (2) immunized, FSH stimulated (B+FSH+, n=5), and (3) immunized, nonstimulated (B+FSH−, n=5). Active immunization was obtained by 3 subcutaneous injections of 1.0mL anti-gonadotrophin-releasing hormone vaccine (Bopriva, Zoetis), given at 20-day intervals. Effective immunization was characterised by the absence of growing follicles &amp;gt;4mm or corpora lutea (CL) on the ovaries. Follicular wave emergence was synchronized in groups B+FSH+ and B+FSH− by follicle ablation, and in group B−FSH+ by using of a protocol consisting of an injection of 2mg of oestradiol benzoate and 0.5mg of sodium cloprostenol, and insertion of an intravaginal progesterone (P4) device (1g). Four days later (Day 0), groups B−FSH+ and B+FSH+ received 100mg of NIH-FSH-P1 (Folltropin-V, Vetoquinol), injected twice-a-day in 8 decreasing doses, and group B+FSH− received saline. Transvaginal ultrasonography (7.5MHz) was performed daily from Days 0 to 4 and the number and size of follicles were recorded. P4 devices of group B-FSH+ were removed at Day 3. All heifers underwent ovum pickup (OPU) at Day 4, and the cumulus–oocyte complexes (COC) recovered were graded for quality. Viable COC were used for invitro embryo production. The heifers were re-evaluated at Day 11 (7 days after OPU). The GLIMMIX procedure from SAS (SAS Institute Inc.) with repeated-measure statement was used to analyse the effects of group, day, and interactions; and the Chi-squared method was used to analyse binomial data. The results are shown as mean±s.e.m. A progressive increase in average follicle size was observed in groups B−FSH+ and B+FSH+ (P&amp;lt;0.0001), whereas no follicle growth was observed in group B+FSH− (P&amp;gt;0.05). Follicle growth rate was similar between groups B−FSH+ and B+FSH+, and both were greater than group B+FSH− (1.2±0.2 and 1.1±0.1 vs. 0.0±0.1 mm/d; P&amp;lt;0.0001). However, the smaller follicle size in group B+FSH+ at Day 0 resulted in smaller follicle size at Day 4, compared with group B−FSH+ (2.4±0.1 vs. 3.6±0.2 and 6.9±0.7 vs. 8.2±0.6mm, respectively; P&amp;lt;0.05). There was no (P&amp;gt;0.05) difference in the number of COC recovered among groups. The group B+FSH+ yielded fewer (P&amp;lt;0.01) COC of grades I and II and more (P&amp;lt;0.01) degenerated oocytes than groups B−FSH+ and B+FSH− (41.2% vs. 80.0% and 68.0%, and 34.0% vs. 19.8 and 7.0%, respectively). Nevertheless, blastocyst rates were similar (P&amp;gt;0.05) for B−FSH+, B+FSH+, and B+FSH− (57.1%, 45.9% and 44.2%, respectively). Residual follicles or luteal tissue were observed after OPU only in group B−FSH+, resulting in a significant difference in the size of ovaries between Days 0 and 11, compared with that of groups B+FSH+ and B+FSH− (3.7±1.4 vs. 0.2±0.2 and −0.2±0.2cm2, respectively; P&amp;lt;0.05). In summary, exogenous FSH supported follicle growth but did not improve oocyte quality in heifers immunized against GnRH. This research was funded by CAPES.

144 Proof of concept for bilateral twin pregnancies in Trio allele carriers

Abstract Bovine twin birth is associated with detriments including increased embryo/fetal losses, malpresentation and dystocia. Incidence of these is lessened in bilateral versus unilateral twin pregnancy. This study was undertaken to assess use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In Experiment 1, carriers (n = 30) and non-carriers (n = 10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (&amp;gt; 5 mm) in excess of one per ovary were aspirated ~16h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 weeks post-TAI to assess progesterone (P4) concentrations; fetal count was determined by ultrasound 6 weeks post-TAI. P4 concentration post-TAI was significantly (P &amp;lt; 0.001) associated with both genotype and subsequent pregnancy status (pregnant non-carriers, 7.06 ± 0.68 ng/mL; pregnant carriers, 5.54 ± 0.55 ng/mL; non-pregnant non-carriers, 5.22 ± 1.05 ng/mL; non-pregnant carriers, 3.13 ± 0.42 ng/mL). Experiment 2 was undertaken to offset negative effects of follicular aspiration on subsequent P4 concentration observed in Experiment 1. Carriers (n = 38) and non-carriers (n = 32) were submitted to fixed-TAI and follicle ablation as described. Additionally, accessory corpora lutea were induced by administration of hCG (carriers) at d6 post-TAI. Consequently, P4 concentration post-TAI was significantly (P &amp;lt; 0.05) associated with subsequent pregnancy status (pregnant, 8.48 ± 0.61 ng/mL; non-pregnant, 6.70 ± 0.63 ng/mL) but not with genotype (carrier, 8.01 ± 0.59 ng/mL; non-carrier, 7.17 ± 0.64 ng/mL). Fetus number was greater in carriers (Exp1, 1.64 ± 0.81; Exp2, 1.45 ± 0.09) versus non-carriers (1.00 ± 0.00, both Experiments). Singles, twins, and triplets occurred in carriers in Experiment 1, whereas multiples in Experiment 2 were limited to twins. Genotype effects on pregnancy rate were not significant (P &amp;gt; 0.10) in either experiment. Results suggest follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires induction of accessory corpora lutea to offset the negative effects of follicular aspiration on subsequent P4 concentration.

An attempt to potentiate the ovarian superstimulatory response in cattle by co-treatment with an aromatase inhibitor

Letrozole is used for the treatment of subfertility in women undergoing ovarian superstimulation, but the mechanism of action has not been investigated critically. The objective was to test the hypothesis that treatment with letrozole will potentiate the superstimulatory response following gonadotropin treatment by increasing the number of follicles present at ovarian follicular wave emergence in cattle. In Experiment 1, ovarian follicular wave emergence was synchronized among beef heifers (n = 8) by transvaginal ultrasound-guided follicle ablation. On Day 0 (wave emergence), a letrozole-releasing device (LRD) was placed intravaginally for 5 days, followed again by transvaginal follicle ablation on Day 5. The number of follicles ≥3 mm was recorded by transrectal ultrasonography on Days 0 and 6.5 (i.e., pre- vs. post-LRD treatment). In Experiment 2, non-lactating dairy cows were assigned randomly to one of two groups (n = 15/gp) after follicle ablation-induced synchronization of wave emergence (Day 0), and given either an LRD or sham device for 5 days. Superstimulatory treatment was initiated on Day 0, consisting of 8 doses of 50 mg of porcine FSH im at 12 h intervals, and luteolytic doses of prostaglandin on Days 3 and 3.5. The LRD/sham devices were removed on Day 3.5, GnRH was given im on Day 5, estrus response was determined on Days 5 and 6, and the ovarian response was recorded by ultrasonography on Days 0, 3.5, 5, 6.5, and 12. In Experiment 1, no difference was detected in the number of antral follicles at wave emergence pre- vs. post-LRD treatment (23.2 ± 3.2 vs. 23.5 ± 3.8 follicles; P = 0.67; mean ± SEM). In Experiment 2, the interval from prostaglandin treatment to estrus was longer (50.3 ± 1.1 vs. 40.7 ± 2.0 h; P < 0.001) and less variable (residuals: 3.1 ± 0.5 vs. 6.7 ± 0.9 h; P < 0.01) in the LRD vs. sham group. The proportion of ovulations (number of CL on Day 12 over the number of follicles ≥3 mm on Day 0) did not differ (0.65 ± 0.02 vs. 0.70 ± 0.02; P = 0.15) nor did the number of CL on Day 12 (15.9 ± 2.5 vs. 19.0 ± 2.0; P = 0.32) between the LRD and sham groups. In summary, treatment with letrozole did not increase the number of antral follicles at wave emergence or the superstimulatory response, but increased precision in the interval to estrus and may be useful for artificial insemination at a fixed time in superstimulatory protocols.

Optimal imaging windows of indocyanine green-assisted near-infrared dental imaging with rat model and its comparison to X-ray imaging.

In this study, we used rat animal model to compare the efficiency of indocyanine green (ICG)-assisted dental near-infrared fluorescence imaging with X-ray imaging, and we optimized the imaging window for both unerupted and erupted molars. The results show that the morphology of the dental structures was observed clearly from ICG-assisted dental images (especially through the endoscope). A better image contrast was easily acquired at the short imaging windows (<10 minutes) for unerupted and erupted molars. For unerupted molars, there is another optimized imaging window (48-96 hours) with a prominent glow-in-the-dark effect: only the molars remain bright. This study also revealed that the laser ablation of dental follicles can disrupt the molar development, and our method is able to efficiently detect laser-treated molars and acquire the precise morphology. Thus, ICG-assisted dental imaging has the potential to be a safer and more efficient imaging modality for the real-time diagnosis of dental diseases.

175 Use of equine chorionic gonadotrophin in a minimum-handling protocol for oocyte collection in bison

Wood bison (Bison bison athabascae) and plains bison (Bison bison bison) are threatened subspecies native to North America. The creation of a germplasm biobank will connect valuable and inaccessible genetics from geographically distant herds in a biosecure manner. Protocols that are feasible in field conditions are required for cumulus-oocyte complex (COC) collection for the purpose of invitro embryo production (IVP). The efficacy of a single dose of equine chorionic gonadotrophin (ECG) was tested in an effort to develop a minimum-handling ovarian superstimulation protocol for bison. The experimental design enabled comparison between ECG-treated and non-superstimulated bison. Transvaginal ultrasound-guided follicle ablation was performed in mature wood bison (n=24) during May (anovulatory season) to induce follicular wave emergence the following day. Immediately after ablation, the bison were assigned to one of three groups (n=8 per group) and treated intramuscularly with 5000IU of ECG (Folligon, Merck), 2500IU of ECG, or saline (control). Transvaginal COC collection was performed 5 days later. Follicular and COC data were recorded, and only grade 1 and 2 COC were used for IVP. The COC were matured invitro for 25-28h at 38.8°C, fertilised (2×106 spermmL−1), and co-incubated at 38.8°C in 5% O2, 5% CO2, and 90% N2 for 18h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O2, 5% CO2, and 90% N2. Nominal data were compared among groups using analysis of variance, and proportional data were compared using GLIMMIX. The total number of follicles ≥3mm on the day of COC collection was greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (37.5±6.9, 17.5±2.0, and 16.9±2.0, respectively; P&amp;lt;0.005). The number of follicles 5-8mm was also greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (12.5±2.1, 7.6±1.0, and 5.8±0.9, respectively; P&amp;lt;0.01), as was the number of follicles &amp;gt;8mm (21.1±5.4, 3.3±1.2, and 0.9±0.2, respectively; P&amp;lt;0.0005). The proportion of grade 1 and 2 COC/total COC recovered was greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (84/124 (68%), 39/76 (51%), and 37/75 (49%), respectively; P&amp;lt;0.05). The proportion of cleaved zygotes/number of COC matured, assessed at 56h after fertilisation, was similar among the 5000-IU ECG, 2500-IU ECG, and control groups (42/84 (50%), 27/39 (69%), and 21/37 (57%), respectively; P=0.20). The proportion of embryos of IETS grades 1-3/number of COC matured was also similar among the 5000-IU ECG, 2500-IU ECG, and control groups (15/84 (17.9%), 8/39 (20.5%), and 7/37 (18.9%), respectively; P=0.94), but the bison in the 5000-IU ECG group produced twice as many embryos than those in the other groups. In summary, a single dose of 5000IU of ECG increased the number and size of follicles available for COC collection, more than doubled the number of COC collected for IVP, and resulted in the production of more embryos than the other groups. A single dose of 5000IU of ECG is effective in a minimum-handling protocol for ovarian superstimulation and IVP in bison. This research was supported by NSERC.

Hormonal mechanisms regulating follicular wave dynamics II: Progesterone decreases diameter at follicle selection regardless of whether circulating FSH or LH are decreased or elevated

Selection of a single dominant follicle is morphologically manifested by diameter deviation between the future dominant follicle (F1) and the future largest subordinate follicle (F2). Conventional deviation is defined as F2≥7 mm when F1 reaches ∼8.5 mm whereas, undersized deviation is if F2<7 mm when F1 reaches ∼8.5 mm. Greater frequency of undersized deviation has been temporally associated with greater circulating progesterone (P4) and greater FSH but reduced LH in observational studies. Experiment 1 was conducted to directly test if elevating P4 increased the likelihood of undersized deviation and altered circulating concentrations of LH and FSH. Experiment 2 was conducted to test if increasing LH action by treatment with exogenous porcine LH or human chorionic gonadotropin (hCG) in the presence of elevated P4, would stimulate growth of F2 and increase the likelihood of conventional deviation. Ovaries were evaluated by ultrasound and blood samples collected every 12 h after development of a new wave following follicle ablation on D6 (D0 = ovulation). Data were normalized to F1≥7.5 mm and compared using SAS software. In experiment 1 (n = 20), the CL was regressed by prostaglandin F2α treatment and heifers were randomized on D6 into control (no P4 treatment) or P4 treatment (75 mg every 12 h for 5.5 d) beginning when F1 reached ∼3 mm (P4-3 mm group) or ∼6 mm (P4-6 mm group). The P4 treatment significantly increased the frequency of undersized deviation from 0% (controls) to 54%, decreased LH by 44%, and increased FSH by 32%. In experiment 2 (n = 27) heifers were randomized on D6 into control (saline) or treatment with the LH analogs - pLH (1.25 mg porcine LH/12 h) or hCG (160 IU initially and subsequently 96 IU/24 h).Treatment with LH analogs significantly increased P4 (control, 4.6 ± 0.3 ng/mL; pLH, 6.6 ± 0.4 ng/mL; and hCG, 8.9 ± 0.4 ng/mL) and decreased FSH (control, 0.46 ± 0.03 ng/mL; combined-pLH/hCG, 0.34 ± 0.02 ng/mL). However, F1 and F2 diameter and frequency of conventional (37%) and undersized (48%) deviations were similar between the control and combined-pLH/hCG groups. In conclusion, elevated P4 was directly linked to undersized deviation but the P4 effect on decreasing F2 diameter occurred independently of the P4 effects on FSH and LH concentrations.

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.

Changes in thyroid hormone activity disrupt photomotor behavior of larval zebrafish

High throughput in vitro, in silico, and computational approaches have identified numerous environmental chemicals that interfere with thyroid hormone (TH) activity, and it is posited that human exposures to such chemicals are a contributing factor to neurodevelopmental disorders. However, whether hits in screens of TH activity are predictive of developmental neurotoxicity (DNT) has yet to be systematically addressed. The zebrafish has been proposed as a second tier model for assessing the in vivo DNT potential of TH active chemicals. As an initial evaluation of the feasibility of this proposal, we determined whether an endpoint often used to assess DNT in larval zebrafish, specifically photomotor behavior, is altered by experimentally induced hyper- and hypothyroidism. Developmental hyperthyroidism was simulated by static waterborne exposure of zebrafish to varying concentrations (3–300 nM) of thyroxine (T4) or triiodothyronine (T3) beginning at 6 h post-fertilization (hpf) and continuing through 5 days post-fertilization (dpf). Teratogenic effects and lethality were observed at 4 and 5 dpf in fish exposed to T4 or T3 at concentrations >30 nM. However, as early as 3 dpf, T4 (> 3 nM) and T3 (> 10 nM) significantly increased swimming activity triggered by sudden changes from light to dark, particularly during the second dark period (Dark 2). Conversely, developmental hypothyroidism, which was induced by treatment with 6-propyl-2-thiouracil (PTU), morpholino knockdown of the TH transporter mct8, or ablation of thyroid follicles in adult females prior to spawning, generally decreased swimming activity during dark periods, although effects did vary across test days. All effects of developmental hypothyroidism on photomotor behavior occurred independent of teratogenic effects and were most robust during Dark 2. Treatment with the T4 analog, Tetrac, restored photomotor response in mct8 morphants to control levels. Collectively, these findings suggest that while the sensitivity of photomotor behavior in larval zebrafish to detect TH disruption is influenced by test parameters, this test can distinguish between TH promoting and TH blocking activity and may be useful for assessing the DNT potential of TH-active chemicals.