A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows

To compare 2 hormonal protocols for submission of lactating dairy cows for timed artificial insemination (TAI), nonpregnant lactating Holstein cows (n = 269) >60 d in milk were randomly assigned to each of 2 treatments to receive TAI (TAI = d 0). Cows assigned to the first treatment (Ovsynch, n = 134) received 50 microg of GnRH (d -10), 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) beginning at a random stage of the estrous cycle. Cows assigned to the second treatment (Presynch, n = 135) received Ovsynch but with the addition of 2 PGF2alpha (25 mg) injections administered 14 d apart beginning 28 d (d -38 and -24) before initiation of Ovsynch. All cows received TAI 16 to 18 h after the second GnRH injection. Ovulatory response after each GnRH injection for a subset of cows (n = 109) and pregnancy status 42 d after TAI for all cows were assessed using transrectal ultrasonography. Based on serum progesterone (P4) profiles determined for a subset of cows (n = 109), P4 concentrations decreased for Presynch cows after the first 2 PGF2alpha injections, and Presynch cows had greater P4 concentrations at the PGF2alpha injection on d -3 compared with Ovsynch cows. Although the proportion of cows ovulating after the first and second GnRH injections did not differ statistically between treatments (41.1 and 69.6% vs. 35.9 and 81.1% for Ovsynch vs. Presynch, respectively), pregnancy rate per artificial insemination (PR/AI) at 42 d post TAI was greater for Presynch than for Ovsynch cows (49.6 vs. 37.3%). Parity, DIM, and body condition score (BCS) at TAI did not affect PR/AI to TAI. These data support use of this presynchronization protocol to increase PR/ AI of lactating dairy cows receiving TAI compared with Ovsynch.     

Effects of GnRH administered to cows at the onset of estrus on timing of ovulation,endocrine responses,and conception

Two experiments examined effects of GnRH administered within 3 h after onset of estrus (OE) on ovulation and conception in dairy cows. In experiment 1, 46 cows received either saline, 250 microg of GnRH, or 10 microg of the GnRH analogue, Buserelin. Cows were observed for estrus, blood samples were collected, and ovulations were monitored by ultrasound. In controls, 76% of cows had intervals from estrus to ovulation of < or = 30 h and 24% had intervals > 30 h. Treatment with either GnRH or GnRH analogue (data combined) increased magnitude of LH surges and decreased intervals from estrus to LH surge or to ovulation. Treated cows all ovulated < or = 30 h after OE. Among control cows, plasma estradiol concentrations before estrus correlated positively with amplitudes of LH surges. Higher plasma progesterone was observed in the subsequent estrous cycle in GnRH-treated cows compared to control cows with delayed ovulations. Experiment 2 included 152 primiparous and 211 multiparous cows in summer and winter. Injection of GnRH analogue at OE increased conception rates (CR) from 41.3 to 55.5% across seasons. In summer, GnRH treatment increased CR from 35.1 to 51.6%. Across seasons, GnRH increased CR from 36.0 to 61.5% in cows with lower body condition at insemination and GnRH increased CR (63.2 vs. 42.2%) in primiparous cows compared to controls. Use of GnRH eliminated differences in CR for cows inseminated early or late relative to OE and increased CR in cows having postpartum reproductive disorders. In conclusion, GnRH at onset of estrus increased LH surges, prevented delayed ovulation, and may increase subsequent progesterone concentrations. Treatments with GnRH increased conception in primiparous cows, during summer, and in cows with lower body condition.     

Immunization against gonadotropin-releasing hormone in dairy cattle: Antibody titers,ovarian function,hormonal levels,and reversibility

Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle not intended for breeding. A cattle-specific anti-GnRH vaccination (Bopriva, Zoetis Australia Ltd., West Ryde, Australia) is approved for use in heifers and bulls in New Zealand, Australia, Mexico, Brazil, Argentina, Turkey, and Peru. Eleven healthy, cyclic Swiss Fleckvieh cows were included in the study and vaccinated twice with Bopriva 4 wk apart. Injection site, rectal body temperature, and heart and respiratory rates were recorded before and 3 d following each vaccination. Blood samples were taken weekly for progesterone and estrogen analysis and to determine GnRH antibody titer. Ovaries were examined weekly, using ultrasound to count the number of follicles and identify the presence of a corpus luteum. Thirty weeks after the first vaccination, the cows were subjected to a controlled internal drug-releasing device-based Select-Synch treatment. The GnRH antibody titers increased after the second vaccination and peaked 2 wk later. Estrogen levels were not influenced by vaccination, and progesterone level decreased in 7 of 11 cows up to 3 wk after the second vaccination and remained low for 10 to 15 wk following the second vaccination. The number of class I follicles (diameter ≤5 mm) was not influenced by vaccination, whereas the number of class II follicles (diameter 6–9 mm) decreased between 7 and 16 wk after the first vaccination. Class III follicles (diameter >9 mm) were totally absent during this period in most cows. The median period until recurrence of class III follicles was 78 d from the day of the second vaccination (95% confidence interval: 60–92 d). After vaccination, all cows showed swelling and pain at the injection site, and these reactions subsided within 2 wk. Body temperature and heart and respiratory rates increased after the first and second vaccinations and returned to normal values within 2 d of each vaccination. The cows in our study were not observed to display estrus behavior until 30 wk after the first vaccination. Therefore, a Select-Synch protocol was initiated at that time. Ten cows became pregnant after the first insemination (the remaining cow was reinseminated once until confirmed pregnancy). Bopriva induced a reliable and reversible suppression of reproductive cyclicity for more than 2 mo. The best practical predictor for the length of the anestrus period was the absence of class III follicles.     

Chemogenetic Depletion of Hypophysiotropic GnRH Neurons Does Not Affect Fertility in Mature Female Zebrafish

The hypophysiotropic gonadotropin-releasing hormone (GnRH) and its neurons are crucial for vertebrate reproduction, primarily in regulating luteinizing hormone (LH) secretion and ovulation. However, in zebrafish, which lack GnRH1, and instead possess GnRH3 as the hypophysiotropic form, GnRH3 gene knockout did not affect reproduction. However, early-stage ablation of all GnRH3 neurons causes infertility in females, implicating GnRH3 neurons, rather than GnRH3 peptides in female reproduction. To determine the role of GnRH3 neurons in the reproduction of adult females, a Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) line was generated to facilitate a chemogenetic conditional ablation of GnRH3 neurons. Following ablation, there was a reduction of preoptic area GnRH3 neurons by an average of 85.3%, which was associated with reduced pituitary projections and gnrh3 mRNA levels. However, plasma LH levels were unaffected, and the ablated females displayed normal reproductive capacity. There was no correlation between the number of remaining GnRH3 neurons and reproductive performance. Though it is possible that the few remaining GnRH3 neurons can still induce an LH surge, our findings are consistent with the idea that GnRH and its neurons are likely dispensable for LH surge in zebrafish. Altogether, our results resurrected questions regarding the functional homology of the hypophysiotropic GnRH1 and GnRH3 in controlling ovulation.     

基因工程菌株BLG8900的遗传稳定性

目的研究基因工程菌株BLG8900的遗传稳定性。方法在有选择压力(加氨苄西林)的条件下,测定pYC2质粒在大肠杆菌BLG8900株[含有质粒pYC2的BL21(DE3)]中的遗传稳定性。结果工程菌连续传10、25、50和100代后质粒具有良好的稳定性,而且经BamHⅠ/EcoRⅠ双酶切,酶切图谱相同。第100代菌株重组质粒的GnRH/TRS序列与原代菌株相同。原代与第10、25、50和100代菌株经IPTG诱导表达,GST-GnRH/TRS融合蛋白表达水平、菌体蛋白的SDS-PAGE图谱鉴定无明显差异。Western blot表明各代菌株表达产物均具有GnRH抗原特异性。结论质粒pYC2在大肠杆菌BL21(DE3)宿主菌中有较好的遗传稳定性。     

Leydig Cells in Immunocastrated Polish Landrace Pig Testis: Differentiation Status and Steroid Enzyme Expression Status

Porker immunocastration against gonadoliberin (GnRH) secretion has been utilized since 2009; however, consumers are still skeptical of it. This is due to not having full information available on the problem of a boar taint, as well as a lack of research on morphological and molecular changes that may occur in the animal reproductive system and other body systems. The present study aimed to explore the functional status of steroidogenic Leydig cells of the testicular interstitial tissue in immunocastrated Polish Landrace pigs. Analyses were performed using Western blot, immunohistochemistry for relaxin (RLN), insulin-like 3 protein (INSL3), pelleted growth factor receptor α (PDGFRα), cytochrome P450scc, 3β- and 17β-hydroxysteroid dehydrogenases (3β-HSD, 17β-HSD), cytochrome P450arom, and 5α-reductase (5α-RED). Immunoassay ELISA was used to measure the androstenone, testosterone, and estradiol levels in the testis and serum of immunocastrates. We revealed disturbances in the distribution and expression of (i) RLN, indicating an inflammatory reaction in the interstitial tissue; (ii) INSL3 and PDGFRα, indicating alterations in the differentiation and function of fetal, perinatal, or adult Leydig cell populations; (iii) P450scc, 3β-HSD, 17β-HSD, P450arom, and 5α-RED, indicating disturbances in the sex steroid hormone production and disturbed functional status of Leydig cells; as well as (iv) decreased levels of androstenone, testosterone, and estradiol in testicular tissue and serum, indicating the dedicated action of Improvac to reduce boar taint at both the hypothalamic–hypophysis–gonadal axis and local level (Leydig cells). In summary, our study provides a significant portion of knowledge on the function of Leydig cells after immunocastration, which is also important for the diagnosis and therapy of testis dysfunction due to GnRH action failure and/or Leydig cell differentiational–functional alterations.     

Ovarian hormones and fasting differentially regulate pituitary receptors for estrogen and gonadotropin‐releasing hormone in rabbit female

To investigate the mechanisms by which caloric restriction affects reproductive function in female rabbits, we measured, in animals intact or ovariectomized (OVX) estrogen‐primed and fed ad libitum or fasted for 48 h, the adenohypophysial expression of estrogen receptor‐alpha (ESR1) and gonadotropin releasing hormone receptor (GnRHR) and the dynamic secretion of LH following GnRH stimulation. Fasting increased the number of GnRHR‐immunoreactive (‐IR) cells in intact animals, whereas reduced the density of ESR1‐IR cells in OVX rabbits. Estrogen priming decreased the number of ESR1‐IR cells in fasted and OVX animals. Ovariectomy increased the number of ESR1‐IR cells in fed rabbits, but caused an opposite effect in both fed and fasted animals treated with estrogen. Fasting down regulated the mRNA levels for ESR1 and GnRHR. Estrogen‐priming reduced the abundance for ESR1 mRNA in both fed and fasted rabbits, and that for GnRHR in fasted rabbits. Ovariectomy halved ESR1 mRNA levels independently of treatment and feeding condition, whereas increased (P < 001) that for GnRHR in estrogen‐primed rabbits. In all rabbits, an LH surge occurred 30 min after GnRH injection but the lowest levels were found in intact fasted rabbits and the highest in fasted, estrogen‐primed animals. The LH profile was similar in intact and OVX rabbits and neither fasting nor estrogen priming modified it. In conclusion, fasting differentially modifies the ESR1 and GnRHR expression in the pituitary, depending on the presence of gonadal hormones, indicating complex interactions between metabolic signals and ovarian steroids. Microsc. Res. Tech. 77:201–210, 2014. © 2013 Wiley Periodicals, Inc.     

Discovery of a Lead Brain-Penetrating Gonadotropin-Releasing Hormone Receptor Antagonist with Saturable Binding in Brain

We report the synthesis, radiosynthesis and biological characterisation of two gonadotropin-releasing hormone receptor (GnRH−R) antagonists with nanomolar binding affinity. A small library of GnRH−R antagonists was synthesised in 20–67 % overall yield with the aim of identifying a high-affinity antagonist capable of crossing the blood–brain barrier. Binding affinity to rat GnRH−R was determined by autoradiography in competitive-binding studies against [¹²⁵I]buserelin, and inhibition constants were calculated by using the Cheng–Prusoff equation. The radioligands were obtained in 46–79 % radiochemical yield and >95 % purity and with a molar activity of 19–38 MBq/nmol by direct nucleophilic radiofluorination. Positron emission tomography imaging in rat under baseline conditions in comparison to pretreatment with a receptor-saturating dose of GnRH antagonist revealed saturable uptake (0.1 %ID/mL) into the brain.     

Silicon analogues of the nonpeptidic GnRH antagonist AG-045572: syntheses, crystal structure analyses, and pharmacological characterization

AG‐045572 (CMPD1, 1 a ) is a nonpeptidic gonadotropin‐releasing hormone (GnRH) antagonist that has been investigated for the treatment of sex hormone‐related diseases. In the context of systematic studies on sila‐substituted drugs, the silicon analogue disila‐AG‐045572 ( 1 b ) and its derivative 2 were prepared in multi‐step syntheses and characterized by elemental analyses (C, H, N), NMR spectroscopic studies (¹H, ¹³C, ²⁹Si), and single‐crystal X‐ray diffraction. The pharmacological properties of compounds 1 a , 1 b , and 2 were compared in terms of their in vitro potency at cloned human and rat GnRH receptors. Compounds 1 a and 2 were also examined in regard to their pharmacokinetics and in vivo efficacy in both castrated rat (luteinizing hormone (LH) suppression) and intact rat (testosterone suppression) models. The efficacy and pharmacokinetic profiles of 1 a and its silicon‐containing analogue 2 appear similar, indicating that replacement of the 5,6,7,8‐tetrahydronaphthalene ring system by the 1,3‐disilaindane skeleton led to retention of efficacy. Therefore, the silicon compound 2 represents a novel drug prototype for the design of potent, orally available GnRH antagonists suitable for once‐daily dosing.     

Inducing ovulation early postpartum influences uterine health and fertility in dairy cows

The objective of the current study was to evaluate the effect of GnRH early postpartum on induction of ovulation, uterine health, and fertility in dairy cows. Holstein cows without a corpus luteum (CL) at 17 ± 3 DIM were assigned randomly to receive i.m. GnRH (n = 245) at 17 ± 3 and 20 ± 3 DIM or remain as controls (n = 245). Ovaries were scanned by ultrasonography twice weekly totaling 4 examinations. Ovulation was characterized by the appearance of a CL ≥20 mm at any ultrasound or CL <20 mm in 2 consecutive examinations. Clinical and cytological endometritis were diagnosed at 35 DIM. Compared with control, GnRH increased ovulation up to 3.5 d after the last treatment (78.7 vs. 45.0%) and did not affect the prevalence of clinical endometritis (23.9 vs. 18.6%) or cytological endometritis (30.9 vs. 32.8%). Prevalence of clinical endometritis increased in cows that had calving problems (32.6 vs. 15.9%) and metritis (40.6 vs. 15.8%). Metritis increased prevalence of cytological endometritis (50.7 vs. 23.5%). Treatment with GnRH did not affect pregnancy per artificial insemination at 32 (37.6 vs. 38.6%) or 74 d after artificial insemination (35.0 vs. 31.5%), but reduced pregnancy loss (6.8 vs. 18.1%). No overall effect of GnRH treatment on hazard of pregnancy was observed; however, an interaction between GnRH treatment and ovulation showed that GnRH-treated cows that ovulated had increased hazard of pregnancy by 300 DIM compared with GnRH-treated and control cows that did not ovulate (hazard ratio = 2.0 and 1.3, respectively), but similar to control cows that ovulated (hazard ratio = 1.1). Gonadotropin-releasing hormone early postpartum induced ovulation without affecting uterine health, but failed to improve pregnancy per artificial insemination or time to pregnancy, although it reduced pregnancy loss.