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Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2/neu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (t1/2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g(-1)) than in blood (6.7 %ID ml(-1)) or normal tissue (liver, 2.8 %ID g(-1); lung, 7.1 %ID g(-1); kidney, 5.2 %ID g(-1)). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g(-1) or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g(-1). When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

Contents The ovulation-inducing effect of seminal plasma was first reported in Bactrian camels over 30 years ago, and the entity responsible was dubbed 'ovulation-inducing factor' ( OIF). More recent studies, primarily in llamas and alpacas, characterized the biological and chemical properties of OIF and ultimately identified it as β NGF. This recent discovery has allowed a convergence of knowledge previously separated by discipline and by mechanism; that is, neurobiology and reproductive biology, and autocrine/paracrine vs endocrine. To preserve this link, we have referred to the seminal factor as OIF/ NGF. As a highly conserved protein, the implications of discoveries related to OIF/ NGF in reproductive tissues extend beyond the camelid species, and results of recent studies show that the presence and function of OIF/ NGF in seminal plasma are conserved among species considered to be induced ovulators as well as those considered to be spontaneous ovulators. The abundance of OIF/ NGF in seminal plasma and the effects of seminal plasma on ovarian function strongly support the idea of an endocrine mode of action (i.e. systemic distribution with distant target tissues). This review is intended to provide an update on the progress in our understanding of the nature of OIF/ NGF in seminal plasma and its effects on reproductive function in the female, including the effects of dose and route of administration, evidence for ovarian effects in other species, tissue sources of OIF/ NGF and early findings related to the mechanism of action of OIF. [ABSTRACT FROM AUTHOR]

Antibodies have emerged as significant agents for the treatment of a number of diseases including cancer and autoimmunity. However, most of the antibodies currently used in clinical practice were developed from humanized or chimeric molecules based on mouse monoclonal antibodies. Recent advances in antibody selection and engineering techniques have led to the development of antibodies specific for highly conserved targets, the creation of novel antibody-based structures, significant improvements in affinity for target antigens, enhanced ability to engage immune effector functions, and the creation of fusion proteins with direct cytotoxic properties. This review provides an overview of the techniques that we expect will have the greatest impact on the field of antibody engineering. [ABSTRACT FROM AUTHOR]

Introduction: Persons with Parkinson's disease (PwPD) suffer from motor and non-motor symptoms which significantly affect their quality of life (QoL), and the QoL of their care partners (CP). Tandem cycling reduces PwPD motor symptoms; however, no studies have examined other benefits or included PwPD CP. We conducted an 8-week community virtual reality (VR) tandem cycling intervention to assess the feasibility and efficacy for PwPD and their CP (i.e., PD dyads). We hypothesized that dyadic tandem cycling would improve (1) PwPD motor and non-motor symptoms and (2) dimensions of PD dyads' QoL and physiologic health., Methods: Ten PD dyads were recruited to complete 8 weeks of progressive intensity, bi-weekly tandem cycling. At pre- and post-testing, PwPD were assessed using the Movement Disorder Society-Unified Parkinson's Disease Rating Scale-III (MDS-UPDRS-III), functional gait assessment (FGA), and 10-m gait speed test. PD dyads also completed emotional and cognitive status questionnaires [e.g., Geriatric Depression Scale-Short Form (GDS-SF)], and wore BodyGuard 2 heart rate (HR) monitors for 48 h to assess surrogate measures of heart rate variability. Statistical analyses were conducted using Student's t tests with significance set at p ≤ 0.05., Results: Eight PD dyads and one PwPD completed the intervention. Retention of PwPD (90%) and CP (80%) was adequate, and PD dyad adherence ranged from 91.67 to 97.91%. PwPD demonstrated significant clinical improvements in MDS-UPDRS-III scores (- 7.38, p < 0.01), FGA scores (+ 3.50, p < 0.01), and 10-m gait speed times (+ 0.27 m/s, p < 0.01), in addition to significant self-reported improvements in mobility (- 13.61, p = 0.02), fatigue (- 5.99, p = 0.02), and social participation (+ 4.69, p < 0.01). CP depressive symptoms significantly decreased (- 0.88, p = 0.02), and PD dyads shared a significant increase in root mean square of the successive differences (RMSSD; p = 0.04)., Conclusion: Our pilot study demonstrated feasibility and multiple areas of efficacy supporting further investigation of community VR tandem cycling as a therapeutic intervention for PD dyads., (© 2024. The Author(s).)

Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18-20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dadarwal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recent studies document the LH-releasing pathway of nerve growth factor (NGF) in male camelids and that the LH response to seminal NGF is associated with elevated plasma testosterone concentration. Results provide rationale for the hypothesis that NGF in semen is associated with male fertility. In Experiment 1, the association between the amount of NGF in the ejaculate and characteristics of the male reproductive system was examined in alpacas. The concentration of NGF was measured by radioimmunoassay in semen samples collected from male alpacas (n = 47) and correlated with sperm morphology and motility, and measurements of the male reproductive anatomy. Most ejaculates had NGF concentrations that, based on previous studies, triggered ovulation in female camelids, however, we only found a positive correlation between NGF concentration with sperm concentration, thread formation and total NGF, and a negative correlation with pH. In Experiment 2, a retrospective analysis was carried out to determine if breeding performance during the previous season was related to recent concentrations of seminal NGF in male alpacas (n = 22). Birth rates tended to be correlated with sperm concentration and total amount of NGF in the ejaculate (P = 0.09). Experiment 3 was a prospective study to determine the relationship between seminal NGF (n = 8 male alpacas) and ovulation and pregnancy rates in a breeding trial. No association was detected between seminal NGF concentration and ovulation rate, pregnancy rate, or LH response in the female. We conclude that among the breeding males used in our study, the abundance of seminal NGF was correlated with sperm concentration and thread formation, however, it was not predictive of male fertility in alpacas. Examination of males not previously selected as breeding stock may be expected to include a broader range of seminal NGF and provide a more comprehensive understanding of the relationship between seminal NGF and male fertility., Competing Interests: Declaration of conflict of interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: The purpose of these studies was to develop a nerve growth factor (NGF) radiometal-chelator conjugate to determine the biodistribution and brain uptake of NGF by positron emission tomography/computerized tomography (PET-CT)., Procedures: Purified NGF from llama seminal plasma was conjugated with FITC, and the chelator NOTA or DFO. NGF conjugates were evaluated for bioactivity. NOTA- and DFO-conjugated NGF were radiolabeled with gallium-68 or zirconium-89 ([ 68  Ga]GaCl 3 , half-life = 68 min; [ 89 Zr]Zr(oxalate) 4 , half-life = 3.3 days). [ 89 Zr]Zr-NGF was evaluated for biodistribution (0.5, 1, or 24 h), PET imaging (60 min), and brain autoradiography in mice., Results: Cell-based in vitro assays confirmed that the NGF conjugates maintained NGF receptor-binding and biological activity. Zirconium-89 and gallium-68 radiolabeling showed a high efficiency; however, only[ 89 Zr]Zr-NGF was stable in vitro. Biodistribution studies showed that, as with most small proteins < 70 kDa, [ 89 Zr]Zr-NGF uptake was predominantly in the kidney and was cleared rapidly with almost complete elimination of NGF at 24 h. Dynamic PET imaging from 0-60 min showed a similar pattern to ex vivo biodistribution with some transient liver uptake. Interestingly, although absolute brain uptake was very low, at 24 h after treatment, cerebral cortex uptake was higher than any other brain area examined and blood., Conclusions: We conclude that conjugation of DFO to NGF through a thiourea linkage allows effective radiolabeling with zirconium-89 while maintaining NGF bioactivity. Following intravenous administration, the radiolabeled NGF targets non-neuronal tissues (e.g., kidney, liver), and although absolute brain uptake was very low, the brain uptake that was observed was restricted to the cortex., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

In Brief: Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons., Abstract: Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

Hormonally active tumours are characterized by production and secretion of hormones, irrespective of endogenous feedback mechanisms. An adult llama had exuberant oestrous behaviour, infertility, elevated concentrations of oestradiol and a large ovarian mass. Necropsy revealed the presence of two large abdominal masses, one effacing the right ovary and one in the mesocolon. Considering the clinical and histopathological findings, we conclude that the llama was affected by a granulosa cell tumour. The case suggests that granulosa cell tumours in camelids are hormonally active, and the clinical presentation resembles that of other large animal species. To our knowledge, this is the first case report of an oestrogen-producing, metastatic granulosa cell tumour in a llama., (© 2022 Wiley-VCH GmbH.)

To address the key challenges in the development of next-generation drug delivery systems (DDS) with desired physicochemical properties to overcome limitations regarding safety, in vivo efficacy, and solid tumor penetration, an ultrasmall folate receptor alpha (FRα) targeted silica nanoparticle (C'Dot) drug conjugate (CDC; or folic acid CDC) was developed. A broad array of methods was employed to screen a panel of CDCs and identify a lead folic acid CDC for clinical development. These included comparing the performance against antibody-drug conjugates (ADCs) in three-dimensional tumor spheroid penetration ability, assessing in vitro/ex vivo cytotoxic efficacy, as well as in vivo therapeutic outcome in multiple cell-line-derived and patient-derived xenograft models. An ultrasmall folic acid CDC, EC112002, was identified as the lead candidate out of >500 folic acid CDC formulations evaluated. Systematic studies demonstrated that the lead formulation, EC112002, exhibited highly specific FRα targeting, multivalent binding properties that would mediate the ability to outcompete endogenous folate in vivo , enzymatic responsive payload cleavage, stability in human plasma, rapid in vivo clearance, and minimal normal organ retention organ distribution in non-tumor-bearing mice. When compared with an anti-FRα-DM4 ADC, EC112002 demonstrated deeper penetration into 3D cell-line-derived tumor spheroids and superior specific cytotoxicity in a panel of 3D patient-derived tumor spheroids, as well as enhanced efficacy in cell-line-derived and patient-derived in vivo tumor xenograft models expressing a range of low to high levels of FRα. With the growing interest in developing clinically translatable, safe, and efficacious DDSs, EC112002 has the potential to address some of the critical limitations of the current systemic drug delivery for cancer management.

Kisspeptin modulates GnRH secretion in mammals and peripheral administration of 10-amino acid fragment of kisspeptin (Kp10) induces LH release and ovulation in cattle. Experiments were done to determine if iv administration of kisspeptin will activate GnRH neurons (i.e., after crossing the blood-brain barrier) and if pre-treatment with a GnRH receptor blocker will alter kisspeptin-induced LH release (from gonadotrophs) and ovulation. In Experiment 1, cows (n = 3 per group) were given human-Kisspeptin10 (hKp10; 3 x 15 mg iv at 60-min intervals) or normal saline and euthanized 150 min after treatment was initiated. Every 20th free-floating section (50 μm thickness) from the preoptic area to hypothalamus was double immunostained to colocalize GnRH- (DAB) and activated neurons (cFOS; Nickel-DAB). Kisspeptin induced plasma LH release from 15 to 150 min (P = 0.01) but the proportion of activated GnRH neurons did not differ between groups (5.8% and 3.5%, respectively; P = 0.11). Immunogold electron microscopy detected close contacts between kisspeptin fibers and GnRH terminals in the median eminence. In Experiment 2, pubertal heifers (n = 5 per group) were treated with 1) hKp10 iv, 2) Cetrorelix (GnRH antagonist; im) + hKp10 iv or 3) saline on Day 6 of the follicular wave under low-progesterone condition. A rise in plasma LH concentration was detected from 15 to 240 min in the hKp10 group but not in cetrorelix or control group (P<0.001). Ovulations were detected only in the hKp10 group (4/5; P = 0.02). Cetrorelix treatment was associated with regression of the preovulatory dominant follicle and emergence of a new follicular wave 3.4±0.75 days after the treatment in all five heifers. Results support the hypothesis that the effect of peripheral kisspeptin is mediated downstream of GnRH synthesis and does not involve GnRH-independent LH release from gonadotrophs. Peripheral kisspeptin may release pre-synthesized GnRH from the nerve terminals in areas outside the blood-brain barrier., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Leonardi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The objectives were to investigate the relationships between antral follicle counts and plasma AMH and FSH at the time of follicular wave emergence in prepubertal calves, and to determine the effects of age and duration of gonadotropin treatment on the ovarian superstimulatory response in pre- and post-pubertal heifers. Hereford crossbred prepubertal (Replicate 1 and 2, n = 20) and post-pubertal heifers (Replicates 1, n = 8; Replicate 2, n = 8) were assigned randomly to 2 treatment groups and given FSH for either 4 or 7 d (25 mg pFSH im at 12-h intervals). Prepubertal heifers were first treated at 4 mo and again at 7 mo of age. Blood samples were collected immediately before the first FSH administration, that was initiated 36 h after follicular ablation. An LH treatment (12.5 mg im) was given 12 h after the last FSH injection. Follicular fluid and cumulus-oocyte complexes (COC) were collected 24 h after LH treatment. At wave emergence, the number of follicles ≥1 mm (AFC, 31.1 ± 4.0 vs 16.2 ± 1.8; P < 0.001) and the plasma concentrations of AMH (606.4 ± 90.5 vs 279.6 ± 28.3 pg/mL; P = 0.001) were higher at 4 than at 7 mo of age, while plasma FSH concentrations did not differ between ages. At oocyte collection, a higher number of follicles ≥6 mm were observed in prepubertal calves at 4 mo of age and post-pubertal heifers than in calves at 7 mo of age (32.4 ± 5.4 and 22.0 ± 2.3 vs 14.9 ± 2.0, respectively; P = 0.003). Intrafollicular concentrations of estradiol were lower (23.7 ± 4.5 vs 144.0 ± 29.5 ng/mL; P < 0.0001) and of progesterone tended to be higher (217.5 ± 29.3 vs 157.0 ± 33.9 ng/mL; P = 0.07) in the 7- than in the 4-d groups. A greater number of COC was collected from calves at 4 mo of age and heifers than the 7-mo-old calves (13.4 ± 2.6 and 6.0 ± 1.0 vs 5.8 ± 1.1, respectively; P = 0.008). Overall, the 7-d FSH treatment tended to result in a greater proportion of expanded COC than the 4-d treatment in calves (50.1 ± 7.7 vs 31.9 ± 6.8%; P = 0.07). In summary, there was a positive relationship between AFC and plasma AMH concentrations at the time of wave emergence. A higher AFC was observed in calves at 4- than 7-mo of age, which resulted in greater ovarian response to gonadotropin treatment. Following an exogenous LH stimulus, COC maturation rates were greater in the 7-d than in the 4-d FSH treatment groups, resulting in collection of a higher proportion of fully expanded COC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The study was conducted to test the feasibility of protocols for field collection of cumulus-oocyte complexes (COC) for in vitro embryo production (IVP) in wild bison. The study was done with captive wood bison during the anovulatory season. In Experiment 1, the efficiency of transvaginal ultrasound-guided COC collection was compared between bison restrained in a squeeze chute without sedation vs in lateral recumbency after chemical immobilization using a dart gun ( n  = 8/group). In Experiment 2, a 2 × 2 design was used to examine the effects of superstimulation treatment [single dose of equine chorionic gonodotrophin (eCG) vs multiple doses of follicle stimulating hormone (FSH)] and method of drug administration (manual injection vs field darting) on COC collection and IVP. In Experiment 1, no difference was detected between chute-restrained vs chemically immobilized groups in the time required to complete COC collections, the number of follicles aspirated (11.5 ± 1.9 vs 9.3 ± 1.8; P  = 0.4) or the COC recovery rate [COC recovered/follicle aspirated; 58/92 (63%) vs 44/69 (64%); P  = 0.9]. In Experiment 2, no differences were detected between superstimulation treatments (eCG vs FSH). The total number of follicles available for aspiration did not differ between manual injection and field darting (23.9 ± 2.7 vs 21.6 ± 1.9; P  = 0.4). Compared with the random start unstimulated group, the embryo production rate was higher [18/132 (14%) vs 53/189 (28%); P  = 0.04] after wave synchronization and superstimulation. Results suggest that COC collection is equally feasible in a recumbent position after chemical immobilization as those bison restrained in a standing position in a hydraulic chute. Ovarian superstimulation with a single-dose eCG protocol is as effective as a multiple-dose FSH protocol, and field darting is as effective as chute-side administration of superstimulation treatments. The strategies in the present study are ready to be incorporated into field collections in free-roaming bison herds., (© The Author(s) 2022. Published by Oxford University Press and the Society for Experimental Biology.)

In an effort to develop an effective, minimum-handling protocol for the conservation of wood bison, the present study was designed to determine the effects of ovarian synchronization and superstimulation on cumulus oocyte complex (COC) collection and in vitro embryo production in wood bison during the ovulatory (Exp. 1) and anovulatory seasons (Exp. 2). We tested the hypotheses that COC collection and in vitro embryo production are 1) greater after follicular wave synchronization than at random stages of the follicular wave, 2) repeatable within individuals, 3) greater after ovarian superstimulation with a single dose of eCG than without treatment, and 4) greater during the anovulatory season than the ovulatory season. In Exp. 1, ultrasound-guided COC collection was performed on Day -1 in wood bison to induce follicular wave emergence the following day (Day = 0). Immediately after the COC collection on Day -1, bison were given a single im dose of 2500 IU eCG or saline (n = 6 per group). Subsequent COC collections were on Days 4 and 9. A similar design was used in Exp. 2, with an additional treatment group given 5000 IU eCG (n = 8 per group). In Exp. 1, compared to the saline-treated group, a single dose of 2500 IU eCG resulted in a greater number of ≥8 mm follicles at the time of the Day 4 COC collection (P = 0.03), but not at the Day 9. In Exp. 2, treatment with 5000 IU eCG resulted in a greater number of ≥8 mm follicles than 2500 IU eCG or the saline treatment (37.5 ± 6.9, 17.5 ± 2.0, 16.9 ± 2.0; P = 0.01, respectively). Although the number of embryos produced/COC submitted to IVM was not different among groups (mean = 18.6%), treatment with 5000 IU eCG produced more than twice as many embryos per bison as unstimulated bison (0.8 vs 1.9). In summary, embryo production rates were higher from COC collected subsequent to follicular wave synchronization vs random stages of the wave, and ovarian superstimulation with eCG resulted in a dose-related increase in the number of ≥8 mm follicles, COC collected, and embryos produced. Repeated COC collections after successive wave synchronization resulted in similar follicular counts and embryo production rates within individuals, and the greatest number of follicles aspirated, COC collected, and embryos produced was in the anovulatory season. We conclude that the minimum-handling COC collection protocols in the present study are effective and provide realistic options for embryo production in wild bison., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Experiments were done to determine ovulation synchrony following a 4-day letrozole treatment (Exp 1), compare the efficacy of a letrozole-based protocol with other commonly used synchronization protocols for FTAI (Exp 2), and test a new intravaginal letrozole-releasing device (LRD) with and without pre-synchronization (Exp 3). In Exp 1, heifers and lactating cows at random stages of the estrous cycle were given an LRD for 4 days, PGF at LRD removal, and GnRH at 48 or 60 h after PGF, or no GnRH. In Exp 2, heifers and lactating cows were assigned to three FTAI groups: i) LRD, ii) estradiol+progesterone, iii) 5-d Co-synch+PRID. In Exp 3, heifers were pre-synchronized with PGF or not (control) 8 days before insertion of either an X-LRD or T-LRD for 3 days; FTAI was done 48 h after device removal. In Exp 1, the variation in interval to ovulation in cows, but not heifers, in the GnRH 48-h group was less than half that in other groups (P < 0.05). In Exp 2, the P/AI was lower (P < 0.001) in the LRD group compared to the other groups. In Experiment 3, the X-LRD increased (P < 0.0001) plasma letrozole concentrations compared to the T-LRD; the pregnancy rate was not affected by pre-synchronization or the type of LRD. Although ovulation synchrony was achieved following LRD treatment, the LRD group had the lowest P/AI compared to other protocols, perhaps because of too short an interval between LRD removal and GnRH/FTAI. Drug delivery was enhanced with the new X-LRD., (Copyright © 2022 Elsevier B.V. All rights reserved.)

To elucidate the mechanism by which nerve growth factor (NGF) influences the LH secretory pathway in camelids, a series of experiments were done to determine the involvement of the hypothalamus (Experiment 1), the role of GnRH neurons (Experiment 2), and the effect of progesterone (Experiment 3) on the NGF-induced LH surge and ovulation in llamas. In Experiment 1, the declining phase of the NGF-induced LH surge was used to determine if the decline is a result of pituitary depletion or hypothalamic unresponsiveness. Female llamas were treated with NGF and, 7 h later, assigned to three groups and given a second dose of NGF (n = 5), a dose of GnRH (n = 5), or saline (n = 6). The LH response was attenuated after the second dose of NGF vs GnRH. In Experiment 2, Fos expression (marker of neuronal activation) in GnRH neurons was examined in the hypothalamus of llamas after NGF or saline treatment (n = 3 per group). Despite an LH surge in the NGF group but not in the saline group, no differences were detected between groups in Fos/GnRH co-expression. In Experiment 3, llamas in low-, medium-, and high-plasma progesterone groups (n = 4 per group) were treated with NGF. The NGF-induced LH surge did not differ among treatment groups. Results from the present study show that the induction of a preovulatory LH surge by NGF may be controlled by a novel pathway involving GnRH neuro-terminals downstream of the hypothalamus and is independent of progesterone influence.

Commercially available intravaginal progesterone (P4) devices differ in shape, surface area and P4 load, which may affect the resulting pregnancy per AI (P/AI) following timed-AI (TAI). The objective of this study was to compare two intravaginal P4 devices on estrus rate, follicular dynamics and P/AI in beef cattle subjected to shortened-TAI protocols. In Expt. 1, nulliparous heifers were randomly assigned to a P4-releasing intravaginal device (PRID-Delta, 1.55 g P4) or a controlled internal drug release (CIDR, 1.38 g P4) at the initiation of a J-synch protocol. Heifers that displayed estrus 72 h following device removal were TAI, or if not in estrus given GnRH at 72 h and TAI at 90 h. In Expt. 2, nulliparous heifers and non-suckling cows were randomly assigned to either PRID or CIDR groups and either 1 or 2 mg of estradiol benzoate (EB) at initiation of a J-synch protocol. All cattle were TAI concurrent with GnRH 72 h after device removal. In Expt. 3, nulliparous heifers and suckling cows were randomly assigned to either PRID or CIDR groups and initiated a 5-d Cosynch protocol, with TAI concurrent with GnRH 72 h following device removal. In each experiment, cattle received estrus detection patches at device removal, which were then scored from 0 to 3 based on color change between initial application and TAI; 0 = unchanged, 1 = ≤50% change, 2 = >50% change, 3 = missing. Estrus was defined to have occurred when the patch was scored 2 or 3. Transrectal ultrasonography was used to determine cyclicity, diagnose pregnancy in all experiments, and the size of the ovulatory follicle in Expt. 3. In Expt. 1, the estrus rate was greater (72.0% vs. 61.0%; P = 0.04) in the PRID compared to the CIDR group. In Expt. 2, a parity by EB dose interaction (P = 0.02) was attributed to an increased estrus rate (52.8% vs. 41.4%; P = 0.05) in heifers given 1 vs. 2 mg EB. In Expt. 3, there was no difference in the ovulatory follicle diameter at device removal (P = 0.22) or TAI (P = 0.28) between P4 groups. Treatment with a PRID tended (P = 0.10) to increase the P/AI in cows compared to a CIDR (73.5% vs. 61.0%). In all experiments combined, the overall P/AI tended to increase (55.2% vs. 51.0%; P = 0.08) and P/AI in cattle exhibiting estrus increased (64.4% vs. 59.7%; P = 0.02) in cattle given a PRID compared to those given a CIDR, respectively. In summary, the type of intravaginal P4 device affected estrus response and P/AI following TAI in beef cows., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Antral follicle count (AFC) repeatability at the time of follicular wave emergence and duration of gonadotropin treatment in calves with small and large AFC affects the superstimulatory response of follicles. In Study I, the individual AFC was determined, calves were ranked as having a small, medium or large AFC, and a second count was performed prior to FSH treatments. There was a positive association between the number of follicles ≥1 mm after the first and second counts (r = 0.4; P = 0.003). In Study II, calves with small and large AFC were administered pFSH for 4 or 7 days, pLH 20 h after last pFSH administration and cumulus-oocyte-complexes (COC) were collected. In calves having large as compared with small AFC, number of follicles ≥6 mm were greater (P = 0.01) and COC collected were greater (P = 0.001). The proportion of large-sized follicles (>9 mm) was greater in the 7-day than in the 4-day gonadotropin treatment group (56.4 ± 8.3 and27.8 ± 7.5 %, respectively; P = 0.01). In Study III, there was a positive association between AFC and number of follicles ≥6 mm at the time of COC collection (r = 0.6; P = 0.003). In summary, the number of follicles at the time of follicular wave emergence was associated with the number of follicles recruited during subsequent waves of follicular development and ovarian response following gonadotropin superstimulation. Calves with a large AFC had more COC collected than calves with a small AFC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the female camelid, systemic administration of NGF induces a preovulatory LH surge that results in ovulation, but the effects of seminal NGF in the male are unknown. In the present study, we tested the hypothesis that the LH-releasing pathway of NGF is present in male camelids. In Experiment 1, male llamas and alpacas were treated with NGF or GnRH (n = 2 llamas and 3 alpacas) and blood samples were collected from 1 h before to 3 h after treatment. Plasma LH concentrations increased after treatment in a surge-like fashion in both GnRH- and NGF-treated groups, but concentrations reached a maximum 2.5 times higher and remained elevated for at least 2 h longer in the NGF-treated group (treatment-by-time interaction, P = 0.01). In Experiment 2, we evaluated the LH and testosterone response to NGF vs saline treatment (n = 3 llamas and 3 alpacas). The LH response to NGF was similar to that in Experiment 1, and plasma testosterone concentrations were higher in the NGF group than in the saline group at 2, 4 and 6 h after treatment (P < 0.05). Results support the hypothesis that the LH-releasing pathway for NGF exists in male South American camelids. The LH response to NGF sustained circulating testosterone concentrations in llamas, suggesting a moderate role of NGF in testosterone secretion., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The objective of the study was to characterize the anatomical framework and sites of action of the nerve growth factor (NGF)-mediated ovulation-inducing system of llamas. The expression patterns of NGF and its receptors in the hypothalamus of llamas (n = 5) were examined using single and double immunohistochemistry/immunofluorescence. We also compare the expression pattern of the P75 receptor in the hypothalamus of llama and a spontaneous ovulator species (sheep, n = 5). Both NGF receptors (TrkA and P75) were highly expressed in the medial septum and diagonal band of Broca, and populations of TrkA cells were observed in the periventricular and dorsal hypothalamus. Unexpectedly, we found NGF immunoreactive cell bodies with widespread distribution in the hypothalamus but not in areas endowed with NGF receptors. The organum vasculosum of the lamina terminalis (OVLT) and the median eminence displayed immunoreactivity for P75. Double immunofluorescence using vimentin, a marker of tanycytes, confirmed that tanycytes were immunoreactive to P75 in the median eminence and in the OVLT. Additionally, tanycytes were in close association with GnRH and kisspeptin in the arcuate nucleus and median eminence of llamas. The choroid plexus of llamas contained TrkA and NGF immunoreactivity but no P75 immunoreactivity. Results of the present study demonstrate sites of action of NGF in the llama hypothalamus, providing support for the hypothesis of a central effect of NGF in the ovulation-inducing mechanism in llamas., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Egg yolk, a major semen extender constituent, lacks a defined composition, therefore, there are biosecurity concerns with use of egg yolk. Cryopreservation of bull semen without inclusion of animal protein in the semen extender, therefore, is an important consideration. Cholesterol may be delivered and incorporated into the sperm plasma membrane by cyclodextrins to protect sperm during cryopreservation. The aim of this study was to determine suitability of a cholesterol-cyclodextrin semen extender, without inclusion of egg yolk, for cryopreservation of bull semen. Bull semen was collected and cryopreserved in either egg yolk or with inclusions of three different concentrations of cholesterol-cyclodextrin complex (0.5, 1 or 2 mg/mL semen) in Tris-glycerol (TG) extender. Sperm motion characteristics examined using the computer-assisted sperm analysis, and plasma membrane and acrosome integrity examined using flow cytometry, were similar for all extenders. The inclusion of the greatest concentration of cholesterol-cyclodextrin complex (2 mg/mL semen) followed by dilution in TG extender resulted in lesser pregnancy rates (P <  0.05). There was a pregnancy rate of as great as 56 % when sperm cryopreserved in 0.5 mg/mL cholesterol-cyclodextrin Tris-glycerol extender were used for artificial insemination following imposing of a hormonal treatment regimen for synchrony of timing of ovarian functions among cows for conducting fixed-time artificial insemination (FTAI). Results indicate cholesterol-cyclodextrin Tris-glycerol extender, with a chemically defined composition and without inclusion of egg yolk, may be used to cryopreserve bull sperm with there being acceptable pregnancy rates when this semen is used for FTAI., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of the study was to determine relative effects of dose (200 or 350 mg) and duration (4 or 7 days) of superstimulatory treatment on the ovarian response in prepubertal calves. Calves with similar antral follicular counts at wave emergence (n = 24) were given eight doses of either 25 or 44 mg pFSH every 12 h for 4 days or 14 doses of either 14 or 25 mg pFSH for 7 days beginning at the time of follicular wave emergence and 12.5 mg of pLH im 12 h after the last FSH treatment. On Day 4 of pFSH treatment, calves given 14 mg had fewer follicles ≥3 mm than those given 25 mg (15.1 ± 1.9 and 27.9 ± 3.3, respectively; P = 0.04). At the end of treatment (24 h post-LH), number of follicles ≥9 mm was greater in calves of groups treated with 350 than 200 mg (13.5 ± 1.8 and 8.8 ± 1.3, respectively; P = 0.02) and calves of groups treated for 7 than 4 days (13.3 ± 1.8 and 9.0 ± 1.3, respectively; P = 0.03). The number of spontaneous ovulations was greater in calves of groups treated for 7 than 4 days as was the total number of ovulations (9.7 ± 0.9 and 6.9 ± 1.0, respectively; P ≤ 0.05). In summary, a dose of 25 mg of pFSH per treatment given twice daily for 7 days resulted in a greater ovarian response than other superstimulatory treatments in prepubertal calves., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Successful cryopreservation of bison semen is fundamental for restoration of genetic diversity in Canada's wood bison. Conventional bovine semen extenders contain animal products, such as egg yolk and milk, which are undesirable because of biosecurity risks and undefined composition. In this study, we examined the efficacy of an exogenous protein-free extender containing cholesterol-cyclodextrin complex (CC) to cryopreserve bison semen. The study also provided an opportunity to determine the effectiveness of different ovulation synchronization protocols for fixed-time artificial insemination in bison. Semen was collected from wood bison bulls via electroejaculation and cryopreserved in either Tris-egg yolk-glycerol (called 'TEYG') extender or pretreated with CC (2 mg/mL semen) and diluted in Tris-glycerol (collectively called 'CC-TG') extender. Post-thaw sperm motion characteristics and in vitro fertilization of cattle oocytes confirmed that CC alone without egg yolk protected bison sperm during cryopreservation process. In the first fertility trial, however, no pregnancy was obtained following fixed-time artificial insemination of bison cows with CC-TG extender. In a follow-up trial, low concentration of CC (1 mg/mL semen) resulted in better post-thaw sperm motion characteristics, fertility rate, and birth of live calves following fixed-time artificial insemination. Results showed that 1 mg CC/mL semen completely replaced egg yolk in bison semen extender. In addition, both follicular ablation and steroid treatment protocols provided ovulation synchrony to permit successful application of fixed-time artificial insemination in bison. This is the first report on the birth of live bison calves following fixed-time artificial insemination using semen cryopreserved in a defined extender., Competing Interests: Declaration of competing interest The authors do not have a conflict of interest., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Two experiments were conducted to test the effectiveness of a silicone matrix as an intravaginal drug delivery device for letrozole, an aromatase inhibitor used for synchronization protocols in cattle. A wax dip-coat formulation of the intravaginal device used in previous studies was effective in releasing letrozole but was cumbersome to manufacture and deploy, resulting in unwanted variation in drug delivery and circulating concentrations of letrozole. In Experiment 1, a 3 × 3 design was used to test the release kinetics of letrozole from silicone in vitro. Silicone was mixed with 3 different letrozole drug loads (5%, 10%, 15%) and 3 different mineral oil loads (5%, 10%, 15%), and letrozole release into 62.5% ethanol was compared with the wax dip-coat formulation (positive control) by UV spectrophotometry. Letrozole was released from silicone in a dose-dependent manner, with no effect of mineral oil. Release kinetics were then examined in vivo (Experiment 2) in nulliparous beef heifers assigned randomly to six groups (n = 6/group) and given either a large surface area (LSA) with 5% or 15% drug load, a standard surface area (SSA) intravaginal silicone device impregnated with 10% or 15% drug load, a wax dip-coat device (positive control), or a blank device (negative control). Devices were inserted on Day 3 (Day 0 = ovulation) until Day 11. Blood samples were collected at 0, 30 min, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, and twice daily until Day 11 to determine letrozole plasma concentrations by LC-MS/MS and estradiol plasma concentrations by radioimmunoassay The ovaries were examined once daily from Day 3-13 by ultrasonography to determine follicular and luteal responses to treatment. Letrozole plasma concentrations were higher in heifers given a device with an LSA vs SSA (P < 0.05). Plasma concentrations of estradiol decreased the most in heifers given the 15% LSA device (P = 0.06). The interval between emergence of successive follicular waves was longest (P < 0.05) in the positive control and the 15% LSA groups. As well, the diameter profiles of the dominant follicle and the corpus luteum were largest (P < 0.01) in the positive control and 15% LSA groups. In conclusion, letrozole was released from a silicone matrix in vitro in a dose-dependent manner, and the 15% LSA devices achieved target effects on ovarian function. Results may be used to manufacture a silicone intravaginal device for delivering aromatase inhibitors in a novel synchronization protocol for cattle., Competing Interests: Declaration of competing interest The authors declare that the work described above has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright holder., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor β (TGF-β) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Two experiments were done using a two-by-two design to determine the effects of season and superstimulatory protocol on embryo production in wood bison. In Experiment 1 (in vivo-derived embryos), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with either two or three doses of FSH given every-other-day (FSH × 2 vs. FSH × 3, respectively). Bison were given hCG to induce ovulation, inseminated 12 and 24 hr after hCG, and embryos were collected 8 days after hCG (n = 10 bison/group). In Experiment 2 (in vitro embryo production), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with two doses of FSH, and in vivo maturation of the cumulus-oocyte complexes (COC) was induced with hCG at either 48 or 72 hr after the last dose of FSH. COC were collected 34 hr after hCG, and expanded COC were used for in vitro fertilization and culture. In Experiment 1, the number of follicles ≥9 mm, the proportion of follicles that ovulated, the number of CL, and the total number of ova/embryos collected did not differ between seasons or treatment groups, but the number of transferable embryos was greater (p < .05) in the ovulatory season. In Experiment 2, no differences were detected between seasons or treatment groups for any end point. The number of transferable embryos produced per bison was greatest (p < .05) using in vitro fertilization and was unaffected by season (1.5 ± 0.2 and 1.1 ± 0.3 during anovulatory and ovulatory seasons, respectively), in contrast to in vivo embryo production which was affected by season (0.1 ± 0.01 and 0.7 ± 0.2 during anovulatory and ovulatory seasons, respectively). Results demonstrate that transferable embryos can be produced throughout the year in wood bison by both in vivo and in vitro techniques, but the efficiency of embryo production of in vivo-derived embryos is significantly lower during the anovulatory season., (© 2019 Blackwell Verlag GmbH.)

The objective of the present study was to compare the effect of a single versus multiple doses of a 10-amino acid fragment of human (hKp) or murine (mKp) kisspeptin on LH secretion and the fate of the dominant follicle. In all experiments, a new wave was induced (Day 0) by ultrasound-guided ablation of >5 mm follicles, a progesterone device (CIDR) was placed in the vagina, animals given prostaglandin F 2α analog im on Day 3.5 and 4, and hKp or mKp treatment given on Day 6. The experimental design maintained growth and ovulatory potential of the dominant follicle for 12 days and allowed hypothesis testing during the low-progesterone period (plasma progesterone ≤1.8 ng/ml on Day 6) wherein spontaneous wave emergence and ovulation did not occur between Day 6 and Day 12. In Experiment 1, heifers (n = 10/group) were given single iv dose of 45 mg hKp, 45 mg mKp, or 2 ml normal saline (control). Post-treatment plasma LH concentrations from 15 to 90 min were higher (P < 0.01) in hKp group than in the mKp and control groups. Two heifers ovulated in hKp group versus none in other groups. In Experiment 2, heifers (n = 6/group) were given 45 mg hKp over a 2 h period divided into multiple iv doses treatments or 2 ml normal saline (control). Post-treatment plasma LH concentrations were higher (P < 0.01) in all hKp treatment groups than in the control group. The ovulation rate was higher (P = 0.06) after hKp treatments (11/18) than in the control group (0/6). In Experiment 3, heifers (n = 6/group) were given 45 mg mKp over a 2 h period divided into multiple iv doses treatments or a single iv dose of gonadorelin acetate (positive control). Plasma LH concentration was higher (P < 0.01) and the ovulation rate was greater (P = 0.01) in the GnRH group (5/6) than mKp groups (1/12). In summary, hKp was more effective to induce ovulation than mKp. Human kisspeptin-10 given over a 2 h period induced ovulations at a rate similar to that of GnRH treatment in heifers under a low plasma progesterone state., (Copyright © 2019 Elsevier Inc. All rights reserved.)

An 18-y-old female llama ( Lama glama ) in Saskatoon, Saskatchewan was examined during a routine herd check, and a mass was detected in the ventral cervical area just below the angle of the jaw. No clinical signs were evident except for the mass and chronic loss of body condition. Postmortem examination revealed bilateral enlargement of the thyroid gland with multiple cysts. Histopathology of the thyroid gland revealed follicular compact-cellular carcinoma lesions, with infiltration of neoplastic thyroid follicular cells in regional lymph nodes.

Our goal was to develop an objective computer-assisted volumetric method of assessing vascular flow from colour Doppler ultrasound data of ovarian structures recorded by free-hand movement. We hypothesized that a vascularity index (ratio of the region of blood flood to the region of ovarian structure) obtained from the three-dimensional volumetric analysis would be more precise (less variable) than conventional two-dimensional analysis of single images in estimating the functional status of the preovulatory follicles and corpus luteum. Doppler ultrasound cineloops of water buffaloes (Bubalus bubalis; n = 22) ovaries were recorded daily from 12 h before GnRH treatment to four days after ovulation. Cineloops were processed using Fiji and Imaris software packages for segmenting the area (two-dimensional analysis) and the volume (three-dimensional analysis) occupied by the blood-flow and associated tissue to calculate the vascularity index. For volumetric measurement, all images in a cineloop were used (i.e., no a-priori selection of images) while for two-dimensional analysis, three images from the region with apparent maximum vascularity were selected. The volumetric method was verified with theoretical ellipsoidal volume of the follicle (r = 0.96 P < 0.01) or corpus luteum (r = 0.58 P = 0.02). The variability in the follicular vascularity index among animals was lower using the volumetric method than two-dimensional analysis (0.018 ± 0.002 vs 0.030 ± 0.005, P < 0.01), while the variability for CL vascularity was similar between methods (P = 0.23). An increase in the follicular vascularity index was detected at 12 h after GnRH treatment using both methods (two-dimensional: 0.030 ± 0.008, P < 0.01; three-dimensional: 0.016 ± 0.006, P < 0.02). Buffaloes that ovulated tended to have a greater increase in 3D vascularity index than non-responding buffaloes (P = 0.06); the two-dimensional method was not able to detect these changes. Using the three-dimensional method, a moderate positive correlation (r = 0.59; P = 0.02) was evident between the follicular vascularity index at 14-16 h after GnRH treatment and follicular diameter. In conclusion, an objective volumetric method for assessing relative ovarian blood flow changes was developed using Doppler ultrasound cineloops recorded by free-hand movement. The 3-dimensional method eliminates the need for a-priori selection of images and is more precise as a result of decreased technical variability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The type of stimuli triggering GnRH secretion has been used to classify mammalian species into two categories: spontaneous or induced ovulators. In the former, ovarian steroids produced by a mature follicle elicit the release of GnRH from the hypothalamus, but in the latter, GnRH secretion requires coital stimulation. However, the mechanism responsible for eliciting the preovulatory LH surge in induced ovulators is still not well understood and seems to vary among species. The main goal of this review is to offer new information regarding the mechanism that regulates coitus-induced ovulation. Analysis of several studies documenting the discovery of β-NGF in seminal plasma and its role in the control of ovulation in the llama and rabbit will be described. We also propose a working hypothesis regarding the sites of action of β-NGF in the llama hypothalamus. Finally, we described the presence of β-NGF in the semen of species categorized as spontaneous ovulators, mainly cattle, and its potential role in ovarian function. The discovery of this seminal molecule and its ovulatory effect in induced ovulators challenges previous concepts about the neuroendocrinology of reflex ovulation and has provided a new opportunity to examine the mechanism(s) involved in the cascade of events leading to ovulation. The presence of the factor in the semen of induced as well as spontaneous ovulators highlights the importance of understanding its signaling pathways and mechanism of action and may have broad implications in mammalian fertility.

The objectives of the study were to compare the presence and localization of ovulation-inducing factor (OIF)/nerve growth factor (NGF) in male reproductive organs and determine the abundance in ejaculates of species representative of both spontaneous and induced ovulators. We hypothesized that the protein is a widely conserved component of semen among mammals, but is most abundant in camelids. Immunohistochemical analysis was performed on tissues from the male reproductive system of llamas, rats, cattle, bison, elk, and white-tailed deer (n = 2 males/species), and the abundance of OIF/NGF in the seminal plasma of camelids (llamas and alpacas), cattle, horses, and pigs (n = 69, 53, 24, and 16 ejaculates, respectively) were quantified by radioimmunoassay. Based on immunoreactivity in both the glandular epithelium and glandular lumen, the prostate gland was the main source of seminal OIF/NGF in llamas, the vesicular gland and ampullae in bovids (cattle and bison), and the ampullae and prostate in cervids (elk and white-tailed deer). Camelid and bovine seminal plasma induced dendritic growth in the PC12 differentiation bioassay, but no effect was observed with equine or porcine seminal plasma. The concentration of OIF/NGF was 10 times higher in camelid than bovine seminal plasma (1.2 ± 0.21 vs. 0.10 ± 0.03; P < 0.05); OIF/NGF was not detected in equine or porcine ejaculates by radioimmunoassay. Based on tissue localization, abundance, and bioactivity, we conclude that OIF/NGF is a common protein within the male accessory glands among species, and its abundance in camelids, bovids, and cervids suggests an important role in the mechanisms of ovulation in both induced and spontaneous ovulators.

Two experiments were carried out to determine the effect of Kisspeptin-10 on plasma LH concentrations and follicular dynamics during the luteal phase in cattle. We tested the hypothesis that a single treatment of Kisspeptin-10 will increase plasma LH concentration and the diameter of the dominant follicle, and induce ovulation during the luteal phase of the estrous cycle in cattle. In the Experiment 1, Hereford-cross heifers (n = 28, 14-16 mo) were given PGF2α im to induce luteolysis and ovulation. On Day 5 (Day 0 = ovulation), a new follicular wave was induced by ultrasound-guided follicular ablation. Heifers were treated on Day 10 (4 days after wave emergence) with 100 μg GnRH im (n = 9), 2 mL saline im (n = 7), 1 mg Kisspeptin-10 im (Kp im, n = 6) or 1 mg Kisspeptin-10 iv (Kp iv; n = 6). Blood samples were collected at -60, -15, 0, 5, 15min (0 min = time of injection) and every 15 min thereafter until 3 h. Transrectal ovarian ultrasonography was performed at 12 h intervals from Day 10-14. In Experiment 2, non-lactating beef cows on Day 5 were treated with 100 μg GnRH im (n = 9), saline im (n = 5), 10 mg of Kisspeptin-10 iv (Kp 10 mg; n = 5) or 15 mg of Kisspeptin-10 iv (Kp 15 mg; n = 5). Blood samples were collected at -15, 0, 15, 30, 60, 120, 180 min and twice daily ovarian ultrasonography was done from Day 5-10. In Expt 1, plasma LH concentrations increased for 1 h following Kp iv administration. The peak concentration occurred at 15 min and was higher in the Kp iv group than in the Kp im group (P = 0.01). The LH peak was 3.5-folds higher in the GnRH group than the Kp iv group (P < 0.0001). In Expt 2, GnRH induced higher (P < 0.001) plasma LH concentrations for all time-points than other groups. Kp 15 mg at peak (15min), 30 and 60 min induced higher (P < 0.0001) plasma LH concentrations than Kp 10 mg and saline. Kisspeptin-treated animals did not ovulate in either experiment while GnRH induced ovulation (n = 5/9 in Expt 1; 9/9 in Expt 2). The diameter of the dominant follicle was greater (P = 0.02) at 12-48 h after kisspeptin treatment (Kp groups combined) than the Saline group in Expt 2. In conclusion, Kisspeptin-10 increased plasma LH concentrations and follicle size, and although plasma LH concentrations were higher after iv than im administration, but at the doses used, Kisspeptin-10 did not induce ovulation during the luteal phase in cattle., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus., Methods: Mature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH., Results: Cells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p < 0.05) in a pattern similar to that of TrkA. A low proportion of GnRH neurons were immunoreactive to TrkA (2.5% of total GnRH cells), and no co-localization between GnRH and p75 was detected. GnRH neuron fibers were detected only occasionally in proximity to TrkA immunopositive neurons., Conclusions: Results do not support the hypothesis that the effect of OIF/NGF is driven by a direct interaction with GnRH neurons, but rather provide rationale for the hypothesis that interneurons exist in the hypothalamus that mediate OIF/NGF-induced ovulation.

The four species of New World camelids and 2 species of Old World camelids derived from a common ancestor in North America. The reproductive characteristics, particularly those involving ovarian function and ovulation, are remarkably similar among the 6 living species of camelids, so much so that interspecies hybrids of nearly all possible combinations have been documented. Camelids are induced-ovulators, triggered by an ovulation-inducing factor in seminal plasma. The timing and mechanism of endocrine events leading to ovulation are discussed, as well as the discovery, identification and mode of action of the seminal factor responsible. The applied aspects of our present understanding are discussed with specific reference to controlled induction of ovulation, ovarian synchronization, and superovulation. Emphasis has been given to the literature on llamas and alpacas, with some reference to studies done in wild species of South American camelids and Old World camels.

Gonadotropin-releasing hormone (GnRH) is a decapeptide involved in the regulation of reproduction in all mammals, but the distribution of GnRH neurons within the brain varies widely among species. The objective of the present study was to characterize the number and distribution of GnRH neurons in the hypothalamus and preoptic area of llamas, an induced ovulator. The brains of female llamas (n = 4) were fixed, frozen and sectioned serially every 50 µm in the transverse (coronal) plane. Every 10th section was stained for immunohistochemical detection of GnRH-positive neuron cell bodies and fibers by incubation with 3,3'-diaminobenzidine. The number of counted immunoreactive cells ranged from 222 to 250 (≈241 ± 13 cells in the preoptic area and hypothalamus per animal) and were localized in the medio-basal hypothalamus (44.3%), anterior hypothalamus (27%), preoptic area (14.9%), diagonal band of Broca/medial septum (13.4%), and mammillary area (0.5%). The immunoreactive cells were not localized in specific hypothalamic nuclei, but rather appeared to be distributed diffusely. The highest concentration of immunoreactive neuron fibers was in the median eminence (P < 0.05), but fibers were identified in most of the areas analyzed, including the neurohypophysis. The GnRH neurons within the hypothalamus displayed monopolar (33%), bipolar (39%), and multipolar (28%) morphologies. The bipolar type was most common in the medio-basal region (40%; P < 0.05). We conclude that GnRH neurons and fibers form a network within the anterior and medio-basal hypothalamus of llamas, suggesting the central location of mechanisms controlling reproductive processes in llamas (i.e., induced ovulation)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This study evaluated the feasibility of using an embryo transfer protocol in an alpaca farm in Canada. Alpaca donors and recipients were synchronized with 2 doses of gonadotrophin-releasing hormone (GnRH), 12 days apart. In donors ( n = 5), superstimulation was induced with follicle stimulating hormone (FSH) given daily (40 mg) for 5 days beginning 2 days after the second GnRH treatment. Cloprostenol was given on the last day of FSH, the donors were bred 2 days later, embryos were collected 7 days after breeding. In recipients ( n = 8), the second dose of GnRH was given the day before donor mating, and embryos were transferred on the day of donor collection. On average (± SEM), 5.2 ± 1.4 corpora lutea were detected and 2.5 ± 1.2 transferable embryos were collected in the donors. A mature corpus luteum was detected in 6/8 synchronized recipients and a single embryo was transferred to each. One recipient alpaca became pregnant and delivered a healthy baby 349 days after embryo transfer. This is the first report of successful embryo transfer in alpacas in Canada.

Numerous treatments and protocols have been used to control the reproductive cycle in cattle, with varying effectiveness and many involving the administration of steroid hormones. Steroid hormones, such as estradiol, are perceived as having a negative impact on consumer health. This internationally shared opinion has led to a ban on the use of steroid hormones in food producing animals in many countries (i.e., European Union, New Zealand, and Australia). Letrozole, a non-steroidal aromatase inhibitor, inactivates the aromatase enzyme responsible for the synthesis of estrogens by reversibly binding to the "heme" group of the P450 subunit. Letrozole is approved as an adjuvant or first-line treatment for hormone-dependent breast cancer in post-menopausal women, but has been used increasingly for ovulation induction in the treatment of infertility in women. Using the bovine model to determine the effects on ovarian function, letrozole treatment was found to extend the lifespan of the dominant follicle and thereby delay emergence of the next follicle wave and/or ovulation. Letrozole treatment also had a luteotrophic effect; that is, larger CL and/or higher circulating concentrations of progesterone were detected in letrozole-treated heifers. Results of the initial studies in cattle provided the impetus for the development of aromatase inhibitor-based synchronization and fertility treatment in cattle. Biologically active concentrations of letrozole were achieved via intravenous, intramuscular or intravaginal administration, but the intravaginal route of administration is of particular interest because it permits extended and defined treatment periods, is minimally invasive, and reduces animal handling. Recent results revealed that irrespective of the stage of the cycle, a 4-day letrozole-based protocol induced ovulation in a significantly greater proportion of animals and with significantly greater synchrony than the control treatment. Evidence and reasons for the increasing use of programmed breeding and fixed-time artificial insemination are discussed in this review as a background to current development of an innovative aromatase inhibitor-based protocol as a safe and effective method of controlling the estrous cycle and ovulation in cattle., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Prolongation of superstimulatory treatment appears to be associated with a greater superovulatory response and with greater oocyte maturation in cattle. A genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells collected from ovarian follicles after differing durations of the growing phase induced by exogenous FSH treatment. Cows were given a conventional (4-day) or long (7-day) superstimulatory treatment (25 mg FSH im at 12-h intervals; n = 6 per group), followed by prostaglandin treatment with last FSH and LH treatment 24 h later. Granulosa cells were harvested 24 h after LH treatment., Results: The expression of 416 genes was down-regulated and 615 genes was up-regulated in the long FSH group compared to the conventional FSH group. Quantification by RT-PCR of 7 genes (NTS, PTGS2, PTX3, RGS2, INHBA, CCND2 and LRP8) supported the microarrays data. Multigene bioinformatic analysis indicates that markers of fertility and follicle maturity were up-regulated in the long FSH group., Conclusion: Using the large gene expression dataset generated by the genomic analysis and our previous associated with the growth phase and gene expression changes post LH, we can conclude that a prolonged FSH-induced growing phase is associated with transcriptomic characteristics of greater follicular maturity and may therefore be more appropriate for optimizing the superovulatory response and developmental competence of oocytes in cattle.