Gonadotropin-releasing Hormone Treatment Research Articles

Fertility of lactating dairy cows treated with gonadotropin-releasing hormone at AI, 5 days after AI, or both, during summer heat stress

Objectives of this experiment were to evaluate pregnancy per AI (P/AI) and progesterone (P4) concentrations after AI of lactating dairy cows under heat stress conditions treated with 100 μg gonadotropin-releasing hormone (GnRH) at AI, 5 d after AI, or both. Lactating Holstein cows from two herds were enrolled in the study during summer. Cows detected in estrus based on tail paint removal were allocated to four treatments: (1) control (CON = 722); (2) GnRH treatment at AI (G0 = 739); (3) GnRH treatment 5 d post-AI (G5 = 697); or (4) GnRH treatment at AI and 5 d post-AI (G0+G5 = 697). Blood samples were collected from a subgroup of cows at AI (study d 0), study d 5 and 12 to determine P4 concentrations. Pregnancy diagnosis was conducted at study d 36 and 94. Temperature and relative humidity were collected from a meteorological station nearest the herds and temperature-humidity index (THI) was calculated. Average THI during study enrollment was 83.8 ± 0.1 and THI at study d 0 tended (P = 0.09) to be associated with P/AI at study d 36, but it was not (P = 0.33) associated with P/AI at study d 94. Treatment with GnRH at AI, 5 d after AI, or both, did not affect (P ≥ 0.64) P/AI at study d 36 or 94. An interaction, however, was detected (P = 0.06) between treatment and lactation number. Such an interaction was observed because, among ≥ third-lactation cows, P/AI was greater for G0+G5 (28.6%) and G5 (25.3%) than CON cows (17.3%) at study d 36. Pregnancy per AI of ≥ third-lactation G0 cows did not (P ≥ 0.11) differ from CON, G5, and G0+G5 cows. No differences in P/AI were detected among treatments in first- and second-lactation cows. Overall, treatments with GnRH at AI, 5 d after AI, or both were not (P = 0.94) associated with P4 concentrations on study d 0, 5 or 12. Among cows diagnosed pregnant at study d 36, progesterone concentration on d 5 was not affected (P ≥ 0.24) by treatments with GnRH at AI, 5 d after AI, or both, but progesterone concentration on d 12 was greater for cows treated with GnRH at AI and 5 d after AI compared with G0 cows. Lactating dairy cows in their third or greater lactation exposed to heat stress conditions with compromised fertility may have increased P/AI after treatment with GnRH 5 d after AI or the combined treatment of GnRH at AI and 5 d after AI because of reduced early embryonic loss.

Impacts of GnRH, PMSG and HCG Treatments on Follicular Diameter, Conception and Lambing Rates of Egyptian Ewe Lambs Using Intravaginal Sponges

ABSTRACTThe influence of gonadotropin releasing hormone (GnRH), Pregnant Mare SerumGonadotropin (PMSG) and human Chronic Gonadotropin (hCG) on some reproductive responses ofEgyptian ewe lambs was evaluated. Twenty five animals (aged 8 – 12 months and weighed 27 - 33Kg) were divided into 5 groups ( 5 ewe lambs each). Group (1) served as a control, Group (2),received vaginal sponges impregnated with MAP (60 mg medroxy progesterone acetate) for 14 days,during this period, animals fed 3 kg Berseem, (Trifolium alexandrinum) plus 500 gm concentrate /head/ day. Group (3), received vaginal sponges impregnated with (60 mg, MAP) for 14 days, on theday of sponge removal; each animal injected by PMSG (250 IU, I/M). Group (4) animals received1.25 ml Receptal (0.5 μg, GnRH) I/M, on day 0, seven days later, ewe lambs injected with 0.5 mlEstromate (125 μg. PGF2α) I/M, after 48 hours, animals treated with a second dose of GnRH. Group(5) received two injections of (0.5 ml Estromate) at days 0 and 7, respectively, after 72 hours from thesecond dose of PGF2α ewe lambs were injected 0.2 ml hCG. Heat detection was performed and transrectalultrasound scanning also performed to confirm estrus, pregnancy and follicular diameter. Theresults revealed that, administration of PMSG (group 3) reduced the interval to estrus (the onset ofheat /hours) significantly (P< 0.01) compared with control and other groups. Regarding conceptionand lambing rates, groups 2, 3, & 4 had affected significantly (P˂ 0.01) compared to groups 1 & 5.Follicular diameter (mm) tended to increase significantly in treated groups than the control one.Results suggest that, tested hormones have effect on selected reproductive responses of Egyptian ewelambs while administration of PMSG (Group 3) has the best response compared to other hormonaltreatments.

Priming anoestrous Corriedale ewes with progesterone and gonadotrophin-releasing hormone causes cervical tissue remodelling due to metalloproteinase-2 (MMP-2) activity

The aim was to obtain experimental evidence of cervical collagen degradation in anoestrous Corriedale adult ewes induced to ovulate with progesterone (P) and gonadotrophin-releasing hormone (GnRH), at the expected time of induced ovulation and early luteal phase. In Experiment 1, anoestrous ewes were treated with P for 10 days (P, n = 4), with nine micro-doses of GnRH followed by a GnRH bolus injection (GnRH, n = 4) or with P plus GnRH treatments (P+GnRH, n = 3), and cervices were obtained either without treatment (A, n = 4), when P was removed, or 24 h after the GnRH bolus injection. In Experiment 2, cervices were obtained 1 (group P+GnRH, n = 5) or 5 (P+GnRH 5, n = 6) days after the GnRH bolus injection with P pretreatment. MMP-2 activity was detected in all samples; however, MMP-9 activity was only detected in 15% of the samples. The activity of the latent (L) form of MMP-2 in the cranial zone of group A was higher than in the cranial zone of groups P, GnRH and P+GnRH, and was also higher than that in the caudal zone of the same group (P &lt; 0.05). The collagen concentration was lower in group P+GnRH 1 than in group P+GnRH 5 (P &lt; 0.04). The activity of the activated (A) form of MMP-2 and the A/L MMP-2 ratio were higher in group P+GnRH 1 than in group P+GnRH 5 (P &lt; 0.05). Data suggest that the L form of MMP-2 was expressed mainly in a constitutive form in the cervix of anoestrous ewes and that an oestrogen-dependent activation mechanisms due to the GnRH treatment may be responsible for the lowest collagen content at the moment of the induced ovulation. This work provides evidence about cervical collagen remodelling in anoestrous ewes treated with P + GnRH.

Hormonal Aspects of the Pathogenesis and Treatment of Cryptorchidism.

A normal functioning hypothalamic-pituitary-testicular axis is required for normal testicular descent. The percentage of cases that result from a disturbance in this axis remains controversial. Much has yet to be learnt about cryptorchidism, but is seems that the existence of A dark spermatogonia (Ad spermatogonia) is essential for later fertility. Bilateral cryptorchid patients have a high risk of later infertility, even though they undergo early surgery for cryptorchidism. It is possible today to distinguish-to a certain extent-between three different groups of cryptorchid patients based on testicular histology, gonadotropins, and inhibin B at the time of early surgery: Group 1, patients suspected of prepubertal transient hypothalamic-pituitary-testicular hypofunction and a high risk of later infertility; Group 2, patients with hypergonadotropic hypogonadism and a primary testicular dysfunction; and Group 3, patients with normal histology and normal serum levels of inhibin B and gonadotropins at the time of early surgery and a low risk of later infertility. Given the potential adverse effects of hormonal treatment, attention should be directed toward small doses of adjuvant gonadotropin-releasing hormone (GnRH) treatment for those who might benefit the most, that is, bilateral cryptorchid boys at early surgery without evidence of normal maturation of gonocytes into Ad spermatogonia. Optimally, gonadotropin levels in such patients should be measured to ensure that levels are not compensatory elevated, thereby supporting the suspicion of hypothalamic-pituitary-testicular hypofunction. Studies of GnRH-supplementary treatment should include testicular biopsy at surgery and at follow-up in childhood as well as examinations of fertility potential in adulthood.

Pulsatile gonadotropin-releasing hormone treatment

Hintergrund Fur die hormonelle Stimulation bei zentralen hypothalamischen Funktionsstorungen steht mit der pulsatilen Gonadotropin-Releasing-Hormon(GnRH)-Behandlung eine Option zur Verfugung, die haufig aufgrund des vermeintlichen Aufwands erst als Zweitlinientherapie eingesetzt wird.

Gonadotropin-releasing hormone analogues lead to pro-inflammatory changes in T lymphocytes.

We aimed to investigate the effect of gonadotropin-releasing hormone (GnRH) analogues on T-cell immunity. TNF-α(+) -, INF-ɣ(+) -, IL-10(+) -, and IL-17(+) -expressing T cells in peripheral blood mononuclear cells (PBMCs) were treated with various concentrations (0.1, 1, 5, and 10μm) of GnRH agonist (buserelin acetate) and antagonist (cetrorelix acetate) for 4hours in vitro and they were analyzed with flow cytometry. TNF-α(+) /IL-10(+) T helper (TH) cell ratios were increased in PBMCs treated with 1, 5, and 10μm GnRH agonist when compared to controls (P=.006, P=.014 and P=.030, respectively). IFN-ɣ(+) /IL-10(+) TH cell ratios were significantly increased with 0.1, 1, 5, and 10μm GnRH agonist as compared with controls (P=.046, P=.004, P=.013, and P=.011, respectively). TNF-α(+) TH cell levels, and IFN-γ(+) /IL-10(+) TH cell ratios were significantly different (P<.001 and P<.004, respectively) between GnRH agonist- and antagonist-treated cells. GnRH analogues induce pro-inflammatory TH1 shift in T-cell immunity, in vitro. GnRH treatment during assisted reproductive technology cycle might explain a possible cause of inflammatory flare in women with inflammatory conditions.

Pattern of induced estrus and conception rate following Ovsynch and Ovsynch based gonadotropin-releasing hormone treatments initiated on day 6 of estrous cycle in repeat breeding crossbred cows.

Aim:The aim was to evaluate the estrus response, incidence of accessory corpus luteum formation and fertility following different hormonal protocols in repeat breeding crossbred cows.Materials and Methods:This study was carried out on 24 repeat breeding crossbred cows allotted into four groups. Cows of Group I was not given any treatment, Group II was treated with gonadotropin-releasing hormone (GnRH) injection on day 6 post-estrus, Group III was treated with Ovsynch protocol, and Group IV was treated with Ovsynch based GnRH treatment. Estrus responses such as duration, onset, percentage, and intensity of estrus were recorded during the study. The incidence of accessory corpus luteum was recorded per rectally on day 7 after first and additional GnRH of Ovsynch treatment. The conception rate for all groups was calculated by the absence of estrus and on day 45 after artificial insemination (AI) per rectum. Serum samples were collected at AI and day 12 post-AI in Group I and II. Serum samples were also collected at GnRH, Prostaglandin F2α (PGF2α), timed AI (TAI) and day 12 post-TAI in Group III and IV.Results:Ovsynch and Ovsynch based GnRH treatments are resulted in 100.00% induction of estrus after the PGF2α injection. Onset of induced estrus after the PGF2α injection for Group III and IV was recorded as 48.750±0.713 and 51.472±1.989 h, respectively, and it was not significant. There was no significant difference in duration of estrus among the groups. The incidence of intermediate estrus intensity was found to be highest. All the cows showed the incidence of formation of accessory corpus luteum subsequent to GnRH treatment on day 6 of the estrous cycle in Group II, III, and IV. The conception rate was 0.00%, 16.67%, 50.00%, and 50.00% in Group I, II, III, and IV, respectively.Conclusion:Ovsynch and Ovsynch based GnRH treatments initiated on day 6 of estrous cycle capable of responding with a higher percentage of ovulation and formation of accessory corpus luteum which helped in higher conception rate over single post-AI GnRH treatment in repeat breeders. These treatments responded with better estrus response but did not significantly improve estrus intensity.

Responsiveness of prepubertal crossbred bull calves to exogenous GnRH and its impact on reproductive hormones under tropical conditions.

This study investigated the age related variations in luteinizing hormone (LH), androstenedione, testosterone, and total estrogens response to exogenous gonadotropin-releasing hormone (GnRH) in Holstein–Friesian (HF) × Tharparkar bull calves. Fifteen bull calves were selected and, based on their age, were divided into Group I (14–16 months, n = 5), Group II (9–12 months, n = 5), and Group III (6–8 months, n = 5). All bull calves were administered with 10 µg of GnRH intramuscularly. Blood samples were collected at an interval of 30 min commencing 1 h prior to GnRH treatment until 4 h post-GnRH treatment and thereafter, at an interval of 1 h for the next 3 h. Endocrine response in terms of pretreatment values, peak values, area under curve, and time taken to attain peak values for LH, androstenedione, testosterone, and total estrogens was evaluated in all the bull calves. Significant differences were observed in pretreatment values, peak concentrations, and area under curve for androstenedione and testosterone between the groups; with response being higher in Group I bull calves. The results indicated that the HF × Tharparkar bull calves of 14 months age and above respond to exogenous GnRH by secreting significant amounts of testosterone.

GnRH regulates trophoblast invasion via RUNX2-mediated MMP2/9 expression

We hypothesized that Runt-related transcription factor 2 (RUNX2), matrix metalloproteinase (MMP)2 and MMP9 are involved in basal and gonadotrophin-releasing hormone (GnRH)-induced human extravillous trophoblast (EVT) cell invasion. Our finding indicates that GnRH-induced RUNX2 expression enhances the invasive capacity of EVT cells by modulating the expression of MMP2 and MMP9. GnRH is expressed in first-trimester placenta and exerts pro-invasive effects on EVT cells in vitro. RUNX2 regulates MMP2 and MMP9 expression and is often associated with invasive phenotypes. First-trimester human placenta (n = 9) was obtained from women undergoing elective termination of pregnancy. The localization of RUNX2, MMP2 and MMP9 in first-trimester human placenta was examined by immunohistochemistry. Primary or immortalized (HTR-8/SVneo) EVT cells were treated alone or in combination with GnRH, GnRH antagonist Antide, MAPK kinase inhibitor PD98095, phosphatidylinositol 3-kinase inhibitor LY294002, MMP2/9 inhibitor or small interfering RNAs (siRNAs) targeting RUNX2, MMP2 and/or MMP9. Protein and mRNA levels were measured by western blot and RT-PCR, respectively. Cell invasiveness was evaluated by transwell Matrigel or collagen I invasion assays. RUNX2, MMP2 and MMP9 were detected in the cell column regions of human first-trimester placental villi. GnRH treatment increased RUNX2 mRNA and protein levels in HTR-8/SVneo cells and primary EVTs, and these effects were attenuated by co-treatment with Antide, PD98095 or LY294002. Down-regulation of RUNX2 by siRNA reduced basal and GnRH-induced MMP2/9 expression and cell invasion. Moreover, pharmacological inhibition or siRNA-mediated knockdown of MMP2/9 reduced basal and GnRH-induced cell invasion. The lack of an in vivo model is the major limitation of our in vitro study. Our findings provide important insight into the functions of the GnRH - GnRH receptor system in early implantation and placentation. Not applicable. This research was supported by Canadian Institutes of Health Research (Grant #143317) to P.C.K.L. The authors have nothing to disclose.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.

Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

Luteinizing hormone receptors are expressed in rat myenteric neurons and mediate neuronal loss.

Clinical observations have suggested repeated gonadotropin-releasing hormone (GnRH) exposure to cause intestinal dysfunction and loss of enteric neurons. This has been further studied and confirmed in a rat in vivo model involving iterated GnRH treatments. Mechanisms behind are enigmatic since no GnRH receptors are found to be expressed in enteric neurons neither in man nor rat. Both species, however, harbor substantial subpopulations of luteinizing hormone (LH) receptor-immunoreactive myenteric neurons which suggests that intestinal GnRH-induced neuropathy may be mediated by LH release. To reveal if exposures of GnRH or LH to rat myenteric neurons in vitro cause neuronal loss. Primary cultured adult rat myenteric neurons were exposed to single or repeated treatments of the GnRH analog buserelin or the LH analog lutrotropin alpha, and neuronal survival was determined by cell counting. Possible presence of GnRH- or LH receptor -immunoreactive neurons was determined by immunocytochemistry. Exposure to the LH, but not the GnRH, analog caused significantly reduced neuronal survival. LH, but not GnRH, receptors were found to be expressed on cultured myenteric neurons. Myenteric neurons express LH receptors in vitro and LH exposure causes reduced neuronal survival. This suggests that GnRH-induced enteric neuropathy in vivo is mediated by way of LH release and activation of enteric neuronal LH receptors.

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1-7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.

Gonadotropin‑releasing hormone inhibits the proliferation and motility of nasopharyngeal carcinoma cells.

Gonadotropin-releasing hormone (GnRH), or its analogues have been demonstrated to exhibit anti-proliferative effects on tumour cells in ovarian, endometrial and breast cancer through GnRH-receptors (GnRH-R). However, the role of GnRH in nasopharyngeal carcinoma (NPC) remains to be elucidated. In order to investigate the effects of GnRH in NPC, the present study examined the expression of the GnRH-R transcript in NPC and investigated the phenotypic changes in HK1 cells, a recurrent NPC-derived cell line, upon receiving GnRH treatment. Firstly, the GnRH-R transcript was demonstrated in the NPC cell lines and four snap frozen biopsies using reverse transcription-quantitative polymerase chain reaction. In addition, immunohistochemistry revealed the expression of GnRH-R in two of the eight (25%) NPC specimens. Treatment with GnRH induced a rapid increase in intracellular ionised calcium concentration in the NPC cells. GnRH and its agonists, triptorelin and leuprolide, exerted anti-proliferative effects on the NPC cells, as determined using an MTS assay. GnRH did not induce any cell cycle arrest in the HK1 cells under the conditions assessed in the present study. Time-lapse imaging demonstrated a reduction in cell motility in the GnRH-treated cells. In conclusion, GnRH, or its analogues may have antitumour effects on NPC cells. The consequences of alterations in the levels of GnRH on the progression of NPC require further examination.

Effect of natural mating or laparoscopic artificial insemination in superovulated Santa Inês ewes on superovulatory response, fertility and embryo viability

This study evaluated the effect of two mating methods (GNM: natural mating or GAI: laparoscopic artificial insemination) on superovulatory response, fertility and embryo yield in superovulated ewes. Fifteen non-pregnant Santa Inês ewes were superovulated and either mated by GNM or GAI in a crossover design. Oestrus was synchronised using intravaginal progestagen sponges for 6 days and on Day 5, 300 IU eCG and 0.0375 mg d-cloprostenol were given. Twelve hours after sponge removal, 0.025 mg gonadotropin-releasing hormone was administered. Superovulation started 48 h after gonadotropin-releasing hormone treatment, using 5 IU/kg follicle-stimulating hormone (pFSH). At the first pFSH dose, new sponges were inserted. At the fifth dose, 0.0375 mg cloprostenol was administered and the sponges were removed. The GNM was mated with rams every 12 h, until the end of oestrus. The ewes of GAI were laparoscopic inseminated with frozen–thawed semen 36 and 48 h after sponge removal. Ultrasonography was performed every 24 h from the beginning of oestrus synchronisation treatment and every 12 h from the second sponge removal to 2 days after the last pFSH dose. Six to seven days after mating, the number of corpora lutea (CL) was evaluated by laparoscopy and the females with &gt; 4 CL were subjected to embryo collection. The interval from sponge removal to ovulation was shorter (P &lt; 0.05) in the GNM. The overall superovulatory response was 63.3% (19/30), with 60.0% and 66.7% in GNM and GAI, respectively (P &gt; 0.05). The number of recovered structures (6.4 ± 2.4 vs 4.5 ± 3.0), recovery rate (74.0 ± 16.0 vs 52.3 ± 26.5%), number of transferable embryos (3.0 ± 2.9 vs 3.6 ± 2.0) and viability rate (47.2 ± 45.3 vs 77.4 ± 37.1%) did not differ between GAI and GNM (P &gt; 0.05). However, the GAI group showed a higher (P &lt; 0.05) number of unfertilised oocytes (3.1 ± 3.1) and a higher non-fertilisation rate (47.1 ± 45.3%) than the GNM (0.9 ± 2.1 and 11.5 ± 21.5%). The mating method did not affect the superovulatory response, and production of viable embryos although the non-fertilisation rate has been inferior for the AI group.

Luteinizing hormone, testosterone and total estrogens response to exogenous GnRH in crossbred bulls with differing semen quality

The present study aims to investigate the Luteinizing hormone (LH), testosterone and total estrogens response to exogenous gonadotropin-releasing hormone (GnRH) in adult crossbred bulls with differing semen quality. Fourteen adult crossbred bulls of differing semen quality were selected and treated with 10µg of GnRH (Buserelin acetate) intramuscularly. Blood samples of the bulls were collected at an interval of 30min commencing 1h prior to GnRH treatment until 4h post-GnRH treatment and thereafter, at an interval of 1h for the next 3h. The endocrine response in terms of peak values, area under the curve, and the time taken to attain peak values for LH, testosterone, and total estrogens were evaluated in all the bulls. The mean±SEM peak levels of LH, testosterone, and total estrogens were found to be 150±24.1, 5.3±0.69, and 0.07±0.01ng/mL, respectively. The mean±SEM area under the curve of LH, testosterone, and total estrogens were found to be 392±51.3, 23.5±3.4, and 0.32±0.04ng/mL×h, respectively. The relationships between hormonal responses (LH, testosterone, and total estrogens) and semen quality were analyzed using the linear regression method, which provided non-significant (P>0.05) results. This study indicated that the gonadal and pituitary hormonal response to single exogenous GnRH treatment may have no relationship with the semen quality of crossbred bulls.

Functional and structural recovery of the injured spinal cord in rats treated with gonadotropin-releasing hormone.

Several studies have shown that gonadotropin-releasing hormone (GnRH) have extra-pituitary roles, including neurotrophic effects. This study was to evaluate the effects of GnRH treatment on the spinal cord injury (SCI) of rats. Ovariectomized rats were divided into: sham SCI surgery (Sham), SCI treated with saline solution (SCI + SS), and SCI treated with GnRH (SCI + GnRH). The SCI was induced by compression. One day after the lesion, SCI + GnRH group was injected with GnRH (60 µg/kg/twice/day; i.m.) for 15 days and the other groups with saline solution. To kinematic gait analysis, length and velocity of the stride were measured. In spinal cord, axonal morphometry and spared white and gray matter were analyzed by histochemistry. Protein expression of spinophilin was evaluated by western blot. The results showed that, 5 weeks after the injury, the group of animals treated with GnRH, significantly increased the length and velocity of the stride compared to SCI + SS group and they were similar to Sham group. In spinal cord, GnRH treatment increased the number and caliber of nerve axons and in the case of white matter, spared tissue was significantly higher than those animals treated with saline solution. The expression of spinophilin in spinal cord of SCI + GnRH group was slightly increased with respect to those not treated. In conclusion, GnRH treatment improves recovery of gait and decreases histopathological damage in the injured spinal cord of rat. These findings suggest that GnRH acts as a neurotrophic factor and can be used as a potential therapeutic agent for treatment of SCI.

Severe gastrointestinal dysmotility developed after treatment with gonadotropin-releasing hormone analogs

Background. Sporadic cases of abdominal pain and dysmotility has been described after treatment with gonadotropin-releasing hormone (GnRH) analogs. The aim of the present study was to scrutinize for patients with severe gastrointestinal complaints after treatment with GnRH analogs, to describe the expression of antibodies against progonadoliberin-2, GnRH1, GnRH receptor (GnRHR), luteinizing hormone (LH), and LH receptor in serum in these patients, and to search for possible triggers and genetic factors behind the development of this dysmotility. Methods. Patients suffering from prolonged gastrointestinal complaints after treatment with GnRH analogs at the Department of Gastroenterology, Skåne University Hospital, were included. GnRHR and LH receptor (LHCGR) genes were exome-sequenced. Serum was analyzed by enzyme-linked immune sorbent assays for the presence of antibodies. Healthy blood donors and women treated with GnRH analogs because of in vitro fertilization (IVF) were used as controls. Results. Seven patients with severe gastrointestinal complaints after GnRH treatment were identified, of whom six suffered from endometriosis. Several variants were found within the 11 exons of LHCGR. The minor allele G, at the single nucleotide polymorphism rs6755901, was detected in homozygosity in two patients (28.5%) who had developed chronic intestinal pseudo-obstruction and in 5.5% of the IVF controls. Three patients expressed IgM antibodies against progonadoliberin-2 and three against GnRH1 (42.9%) when cut off was set to a titer >97.5th percentile in blood donors. Conclusion. A high prevalence of endometriosis, polymorphism in the LHCGR and GnRH1 and progonadoliberin-2 antibodies in serum was found among the patients with severe dysmotility after treatment with GnRH analogs.

Outcome of Gonadotropin Releasing Hormone Treatment 12 Days Post AI and Double Insemination on Conception Rate of Repeat Breeding Cows in Mekelle, Tigray, Ethiopia

The aim of the present study was to determine outcome of gonadotropin releasing hormone analogue given 12 days post AI and double inseminations during estrous period of dairy cattle and improvement of conception rate. Field trial was carried out on repeat breeding cross breed dairy cows which are lactating, apparently healthy and exhibits estrus regularly. A total of Sixty three repeat-breeding cows from twenty eight herds were selected and assigned randomly in to three equal groups G (1, 2 and 3); two treated and one control group. Group1 (n=21) cattle were treated intramuscularly with 10 µg buserelin acetate 12 days post AI. Group2 (n=21) cattle were inseminated twice during estrus exhibition with 6-8 hours interval. Group3 (n=21) cattle were receive no treatment, inseminated once as heat was detected and considered as a control group. Dairy cows were examined for pregnancy after three months through rectal palpation. Pregnancy rates recorded were 52%, 38% and 28% in G1, G2 and G3 respectively. Conception rates of treated groups (G1 and G2) exceed control group by 24% and 10% respectively and showed a significant statistical variation (P<0.05) as compared to control groups. Comparison between treated groups also indicated that G1 has statistical significant difference from G2. From this experiment, it is recommended that use of GnRH 12 days can improve pregnancy rate as compared to double insemination and single service.

12 EFFECT OF HUMAN CHORIONIC GONADOTROPIN (hCG) ADMINISTRATION ON DAY 2 OR DAY 5 AFTER OESTRUS ON PREGNANCY RATE IN HIGH-YIELDING DAIRY COWS

In cattle, ~40% of embryonic loss occurs in the period from Day 8 to Day 16 of pregnancy. A significant proportion of embryo loss may be due to inadequate circulating progesterone (P4) concentrations. Low P4 concentrations have also been implicated as a causative factor in the low pregnancy rates (PR) observed in high-yielding dairy cows. Administration of hCG during the early luteal phase stimulates hypertrophy of the original corpus luteum (CL) and, depending on the day of administration, induces ovulation of the first-wave dominant follicle and formation of a functional accessory CL, which increases circulating P4 concentrations. The aim of this study was to examine whether administration of hCG on Day 2 or Day 5 after oestrus after timed AI (TAI) would lead to an increase in pregnancy rates in dairy cattle. Lactating Holstein cows (n = 194) from 12 commercial dairy herds in Southern Spain (37.8833° N, 4.7667° W) with an average milk production at 37.8 L/cow per day and typically with a PR to first AI of ~30% were randomly assigned based on their body condition score (2.65 ± 0.05; mean ± SEM), parity (2.60 ± 0.09), and days in milk (75.06 ± 0.63) to 1 of 3 treatments and administered a single intramuscular injection of 3000 IU of hCG (4 mL of Veterin Corion) either (1) on Day 2 = 36 h after TAI (n = 65; hCG2 group), (2) Day 5 = 108 h after TAI (n = 64; hCG5 group), or (3) 4 mL of saline on Day 2 = 36 h after TAI (n = 65; control group). Cows were synchronized using a 7-day Ovsynch TAI protocol that included a P4-releasing intravaginal device (PRID DELTA 1.55 g). First, gonadotropin-releasing hormone (Cystorelin 100 mg) treatment was administered at PRID insertion (Day 0) followed by 25 mg Dinoprost (prostaglandin F2α: Enzaprost T) on Day 7 at PRID withdrawal. Then, 56 h later, the second gonadotropin-releasing hormone (100 mg) treatment was administered and all cows were inseminated 16 h later. Pregnancy was diagnosed by ultrasonography 28 to 32 days after TAI. Logistic regression model and chi-squared test were used to analyse data. Pregnancy rate to AI was significantly higher in the hCG2 and hCG5 groups than in the control group (43.1 and 45.3%, v. 27.7%; P &lt; 0.05). A treatment-by-parity interaction was observed; while pregnancy rate for primiparous cows was not affected by treatment, multiparous cows from the hCG2 group had greater pregnancies per AI than those in the control group (47.2% v. 21.1%, respectively; P &lt; 0.05). In conclusion, these preliminary results suggest that hCG administration on Day 2 and 5 after oestrus increases PR at first postpartum AI in Holstein cows. In addition, hCG on Day 2 increases the fertility in multiparous cows.This study was funded by the Spanish Ministry of Science and Innovation (AGL2012–37510) and partially supported by Ceva Salud Animal S.A., Spain – synchronization protocol and DFV, Spain – hCG treatments.

102 SUPPLEMENTATION WITH ESTRADIOL CYPIONATE AT THE ONSET OF A SYNCHRONIZED PROESTRUS ALTERS THE UTERINE GENE EXPRESSION OF SUCKLED ANESTROUS BEEF COWS

The present study evaluated the effect of oestradiol cypionate (ECP) supplementation at the onset of a proestrus [i.e. at progesterone (P4) device removal of the synchronization of ovulation protocol] on the global endometrial gene expression of suckled anestrous beef cows. A total of 12 suckled cows presenting absence of corpus luteum detected by ultrasound received 2 mg of oestradiol benzoate IM and an intravaginal P4 device. Eight days later, P4 devices were removed, and the cows received 500 mg of sodium cloprostenol IM. Cows were blocked according to body condition score and diameter of largest follicle (LF) at P4 device removal and were randomly assigned to receive an IM treatment with 1 mg of ECP (ECP, n = 6) or not (CON, n = 6) at the P4 device removal. All cows received 10 μg of buserelin acetate 48 h after P4 device removal and were timed AI immediately after. Cows presenting a new corpus luteum formed 6 days after the gonadotropin-releasing hormone treatment had an endometrial fragment collected by transcervical biopsy. RNA-Seq analysis was performed and the transcriptome profile was obtained. The significance of differential gene expression was assessed with the package DESEqn 2 v.1.2.10 and the differential expression estimation was based on the GLM followed by the Wald test. The significance threshold was established as an FDR- Benjamini-Hochberg-adjusted P-value of &lt;0.1. The integrated analysis was performed using DAVID database. In total, 135 transcripts were differentially expressed between ECP and CON groups, of which 73 genes were up-regulated by ECP supplementation and 62 genes were up-regulated in the CON cows. Two pathways were overrepresented by the ECP-induced transcripts: pathways in cancer (n = 5 genes) and small cell lung cancer (n = 3 genes). On the other hand, ECP-inhibited transcripts indicated the enrichment of 3 pathways: Parkinson's disease (n = 3 genes), oxidative phosphorylation (n = 3 genes), and Alzheimer's disease (n = 3 genes). More specifically, ECP-induced transcripts associated with pathways in cancer [gene symbol (fold change); respectively] were LAMC3 (1.55), PTCH1 (1.51), PTCH2 (1.52), PIK3R3 (1.22), and PIAS1 (1.18), whereas ECP-inhibited transcripts associated with oxidative phosphorylation were ATP5F1 (1.18), ATP5J (1.24), and NDUFB3 (1.37). Therefore, ECP supplementation at onset of the synchronized proestrous slightly alters the uterine transcriptome. The enriched pathways affected by the ECP supplementation described in this work need to be studied more but these results show candidate pathways that can be associated with uterine environment and receptivity and with possible regulation by oestradiol supplementation given at the onset of the proestrous.