Ovulating induction methods in rabbit does: the pituitary and ovarian responses

The aim of this study was to compare the pituitary and ovarian responses in rabbit does subjected to different methods of ovulation induction. Forty-eight receptive females were randomly distributed into six groups (N = 8) and were inseminated with standard glass catheters. Buserelin intramuscular (BM) does were inseminated using a pool of fresh heterospermic semen and an intramuscular injection of 1 μg of buserelin acetate to induce ovulation. Buserelin intravaginal (BV) does were inseminated in a similar way, but ovulation was induced with the GnRH analogue (10 μg of buserelin acetate) combined with 0.5 mL of semen extender. The raw semen (R) and saline groups (S) were inseminated with undiluted semen or saline, respectively, without any inducer of ovulation. Another group (A) received lumbar anaesthesia (1.5 mL of 2% lidocaine), and only the empty catheter was introduced into the vagina. The AR does were treated the same way as group A but were inseminated with raw semen instead of an empty catheter. Blood samples were collected to determine the LH concentrations before and after AI (30, 60, 90, and 120 minutes). Ovulation, pregnancy, and conception rates were determined after euthanasia on day 14 post AI. Ovulating does had higher mean LH concentrations than nonovulating does (197.9 vs. 45.9 ng/mL; P < 0.05). The ovulation rates of buserelin intramuscular and intravaginal does were 100%, and the pregnancy rates were 87.5% and 100%, respectively. Rabbit does in groups A and AR did not ovulate and had similar mean plasma LH concentrations after 60 minutes compared with the S group (49.4 and 49.2 ng/mL vs. 41.6 ng/mL, respectively), which reached ovulation and pregnancy rates of 37.5%. Does inseminated only with raw semen had an ovulation rate of 75% and a pregnancy rate of 62.5%; they also demonstrated higher plasma LH concentrations than does of the S, A, and AR groups. In conclusion, ovulation in rabbit does can be induced by exogenous GnRH administration (im and intravaginal). The high plasma LH concentration and ovulation rate in the R group with respect to the S and A groups could weakly indicate the presence of some molecules in the seminal plasma that could act on or be absorbed by vaginal mucosa. Sensory stimulation and “seminal factors” probably exert a synergy on the ovulation response as demonstrated by the comparison of LH release and the ovulation response in the R, S, RA, and A groups.     

Attenuation of the estradiol-induced surge release of luteinizing hormone by antihistamine in ovariectomized ewes

Ovariectomized ewes received intramuscular (i.m.) injections of an H₁-histamine receptor antagonist, diphenhydramine, or saline during the anestrous and breeding seasons to determine if histamine may regulate the estradiol-induced surge release of LH in ewes. In addition, concentrations of histamine and GnRH in hypothalamic regions and histamine and LH in the pituitary gland were determined during the estradiol-induced surge of LH. Pretreatment mean, basal, and estradiol-induced secretion of LH did not differ (P > 0.05) among seasons. However, the quantity of LH (ng) measured during the estradiol-induced surge of LH was less (P < 0.05) in ewes treated with diphenhydramine (411 ± 104) than saline (747 ± 133). Treatment with diphenhydramine did not (P > 0.05) influence steady-state concentrations of histamine in hypothalamic or pituitary gland tissues, hypothalamic concentrations of GnRH, or anterior pituitary concentrations of LH during the estradiol-induced surge of LH. It is concluded that histamine may modulate the estradiol-induced surge release of LH in ewes by affecting the secretion of GnRH.     

Effect of intracerebroventricular infusion of leptin on the secretory activity of the GnRH/LH axis in fasted prepubertal lambs

Leptin is believed to link metabolic status to reproductive processes. The aim of the present study was to investigate the effect of exogenous leptin on the secretory activity of GnRH/LH system in acutely undernourished prepubertal, female lambs. Merino lambs were randomly divided into four groups, two standard-fed and two fasted for 72 h. One standard and one fasted groups were infused intracerebroventricularly (i.c.v.) with the vehicle; the remaining standard and fasted groups were infused with leptin (25 μg/120 μl/h). Leptin was administered in series of four 1-h infusions at 30-min intervals for 3 consecutive days from 08:30 to 14:00 h. Blood samples were collected on day 0 (before infusions) and on day 3 every 10 min over a 6-h period. Immediately after the experiment, the sheep were slaughtered and brains fixed in situ. Hypothalamic and pituitary tissues were prepared for further immunohistochemical and hybridization in situ analysis. In fasted sheep, increased GnRH levels in the median eminence (P < 0.001) and LHβ levels in the pituitary cells (P < 0.001) plus decreased LHβ mRNA and LH pulsatility in blood plasma were observed (P < 0.05). In leptin-infused fasted sheep, GnRH levels in the median eminence decreased (P < 0.001), LHβ mRNA hybridization signal increased, LHβ levels decreased in the pituitary cells (P < 0.001) and LH pulsatility increased (P < 0.05) in the blood plasma. These results indicate that, in prepubertal sheep, the GnRH/LH axis is sensitive to the fasting signal, that influence of which can be reversed by leptin. Leptin cancels out the suppressing effect of fasting on LH secretion by augmentation of GnRH.     

大鼠GTH细胞内cAMP的改变对LH分泌的影响

将GTH细胞用FSK(cAMP兴奋剂)或SO22,536(cAMP抑制剂兴奋剂)处理后,用GnRH脉冲刺激,再用ELISA法检测其LH分泌量,并与空白对照组比较。结果表明,FSK能显著提高GTH细胞中cAMP含量,SO22,536能显著降低GTH细胞中cAMP含量,FSK和SO22,536都不会影响GTH细胞的PKC活性,GTH细胞cAMP含量显著影响LH的分泌,LH随着cAMP的升高而升高,随着cAMP的降低而降低。cAMP-PKA是GnRH脉冲刺激所引起LH分泌受体后的信号转导途径。     

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

The hour of transition into luteolysis in horses and cattle: a species comparison

Hourly blood sampling in both horses and cattle indicate that the transition between the end of preluteolysis and the beginning of luteolysis occurs within 1 h, as manifested by a change in progesterone concentrations. Each species presents a separate temporality enigma on the relationship between pulses of a prostaglandin (PG) F2α metabolite (PGFM) and the hour of the progesterone transition. In horses, relatively small pulses of PGFM occur during preluteolysis (before transition) and at transition. Oxytocin, but not estradiol, increases and decreases concomitantly with the small PGFM pulse at transition but not with previous pulses and may account for the initiation of luteolysis during the small PGFM pulse. In cattle, the last PGFM pulse of preluteolysis occurs hours before transition (e.g., 4 h), and the next pulse occurs well after transition (e.g., 9 h); unlike in horses, a PGFM pulse does not occur at transition. During the last PGFM pulse before transition, progesterone concentration decreases during the ascending portion of the PGFM pulse. Concentration then rebounds in synchrony with an LH pulse. The rebound returns progesterone to the concentration before the PGFM pulse. During luteolysis, an LH-stimulated progesterone rebound may occur after the peak of a PGFM pulse, but progesterone does not return to the concentration before the PGFM pulse. A similar LH-stimulated progesterone rebound does not occur in horses, and therefore progesterone fluctuations are more shallow in horses than in cattle.     

Effect of intrauterine administration of gonadotropin releasing hormone on serum LH concentrations in lactating dairy cows

The objectives were to compare: (1) preovulatory serum LH concentrations, and (2) synchronization of ovulation, after im or iu administration of the second GnRH treatment of Ovsynch in lactating dairy cows. Lactating cows (N = 23) were presynchronized with two injections of PGF_2α given 14 days apart (starting at 34 ± 3 days in milk), followed by Ovsynch (GnRH-7 d-PGF_2α-56 h-GnRH) 12 days later. At the time of the second GnRH of Ovsynch (Hour 0), cows were blocked by parity and randomly assigned to 1 of 3 groups: (1) control group (CON; N = 7) were given 2 mL sterile water im; (2) intramuscular group (IM; N = 8) received 100 μg of GnRH im; and (3) intrauterine group (IU; N = 8) had 100 μg GnRH infused in the uterus (2 mL). Blood samples for serum LH concentrations were collected at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Furthermore, ultrasonography was performed twice daily (12-h intervals) from Hours 0 to 60 to confirm ovulation. The LH concentrations were greater (P < 0.05) in the IM than IU and CON groups at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Although LH concentrations were numerically higher in the IU group, LH concentrations within the IU and CON groups did not change over time. More cows ovulated in the IM (8/8) and IU (7/8) groups within 60 h after the second GnRH administration compared with the CON (2/7) group. In summary, serum LH concentrations were lower in the IU versus IM group, but the proportion of cows that ovulated within 60 h was similar between these two groups. Therefore, iu administration of GnRH may be an alternative route of delivery to synchronize ovulation in beef and dairy cattle.     

Leptin inhibits the reproductive axis in adult male Syrian hamsters exposed to long and short photoperiod

The aim of the study was to investigate the effects of acute leptin treatment of adult Syrian hamsters exposed to a long (LP, eugonadal males) and short photoperiod (SP, hypogonadal males). Animals were exposed to LP (L:D 14:10) or SP (L:D 10:14) for 10 weeks. Afterwards, both LP and SP hamsters were allocated to a control (SP-C, LP-C) or leptin-treated group (SP 3, SP 10, SP 30 or LP3, LP 10, LP 30). One hour before sacrifice, a single dose of leptin (3, 10 or 30 μg/kg) or vehicle was administered (i.p.) to the males. Testis weight, serum and pituitary luteinizing hormone (LH) concentrations, as well as the hypothalamic concentration of gonadotropin-releasing hormone (GnRH) were recorded. Histological analysis of the testis was performed and GnRH concentration in the culture medium of hypothalamic explants was examined. A dramatic regression of testicular weight and histological atrophy of seminiferous tubules, as well as a decrease in serum and pituitary LH concentrations were found in SP males. All doses of leptin significantly reduced serum LH levels and medium GnRH concentrations in both photoperiod groups. Pituitary LH and hypothalamic GnRH concentrations were not affected by leptin. In conclusion, we demonstrated that leptin inhibited the reproductive axis of Syrian male hamsters exposed to LP and SP and fed ad libitum.     

Effects of intracerebroventricular administration of irisin on the hypothalamus–pituitary–gonadal axis in male rats

Irisin is a product of fibronectin type III domain-containing protein 5 (FNDC5) and plays an important role in energy homeostasis. In this study, we aimed to determine effects of intracerebroventricular administration of irisin on the hypothalamus–pituitary–gonadal axis by molecular, biochemical, and morphological findings. Fourty male Wistar-Albino rats were used and divided into four groups including control, sham (vehicle), 10, and 100 nM irisin infused groups (n = 10). Hypothalamic gonadotropin releasing hormone (GnRH) level and serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were determined. Testicular tissue histology and spermiogram analysis were also performed. Both irisin concentrations significantly reduced hypothalamic GnRH messenger RNA (mRNA) and protein levels (p < 0.05). It was found that serum LH, FSH, and testosterone levels and Sertoli and Leydig cell numbers were decreased by irisin administration (p < 0.05). In addition, irisin administration reduced sperm density and mobility (p < 0.05). However, it did not cause any change in testicular and epididymis weights and tubular diameter. Our results reveal that irisin can play a role in the central regulation of reproductive behavior and also reduces testosterone levels by suppressing LH and FSH secretion. These results suggest that the discovery of irisin receptor antagonists may be beneficial in the treatment of infertility.     

Effects of 72 hr calf removal and/or gonadotropin releasing hormone on luteinizing hormone release and ovarian activity in postpartum beef cows

A study was conducted to determine the pituitary and ovarian responses to 72 hr calf removal (CR) and/or gonadotropin releasing hormone (GnRH) in beef cows. Forty-eight Angus, Simmental, and Charolais crossbred cows in moderate body condition were allotted to an experiment of 2 x 2 factorial design involving CR and GnRH. At 30 to 32 days postpartum, calves were removed for 72 hr from the CR and CR plus GnRH groups. All cows were injected (i.m.) with saline or 200 mug of GnRH at 33 to 35 days postpartum. Saline or GnRH was injected 5 hr before calf return. Plasma luteinizing hormone (LH) was measured in blood samples collected every 30 min for 5.5 hr beginning 30 min prior to injection of saline or GnRH. Plasma progesterone was measured in blood samples collected 0, 7, and 14 days after GnRH injection and 7 and 14 days following the first detected estrus. There were no differences (P>0.05) in the interval to peak LH release or the magnitude of the LH release between the GnRH and CR plus GnRH groups; however, the GnRH induced release of LH was greater (P<0.05) over time when preceded by CR. Plasma progesterone concentrations were increased on day 7, compared to day 0, after GnRH injection in 57% and 50% of the animals in the GnRH and CR plus GnRH groups, respectively. However, behavioral estrus was not observed in any of the cows between days 0 and 7 after GnRH injection. A higher (P<0.05) percentage of the cows injected with GnRH formed luteal tissue compared to cows injected with saline; however, the luteal lifespan following GnRH injection was decreased relative to the luteal lifespan following the first observed estrus. The mean interval from calving to first estrus was decreased (P<0.05) by 17 days in the CR group relative to the other groups, and calf removal had no detrimental effect on milk production at 80 days postpartum or on calf weaning weights at approximately 7 months of age. In summary, 72 hr CR decreased the postpartum interval and increased the pituitary responsiveness to GnRH. Pretreatment with 72 hr CR did not alter circulating progesterone concentrations or luteal lifespan of corpora lutea induced by GnRH.     

Importance of estrus on pregnancy per insemination in suckled Bos indicus cows submitted to estradiol/progesterone-based timed insemination protocols

The objective was to evaluate the effect of estrus occurrence (based on removal of tail-head marks) on ovarian responses and pregnancy per AI (P/AI; 30 d after AI) in suckled Bos indicus beef cows submitted to timed AI (TAI) protocols. Cows received an intravaginal device containing 1.0 g progesterone, and 2.0 mg estradiol benzoate im; 8 d later, the intravaginal device was removed, and they were given PGF_2α (0.25 mg of cloprostenol sodium) and 300 IU of eCG, with TAI 48 to 52 h later. In Experiment 1, cows were assigned to receive one of three treatments: 1 mg of estradiol cypionate (ECP) im at progesterone (P4) device removal (N = 178); 10 μg of GnRH im at TAI (N = 190); or both treatments (N = 172). In cows given estradiol (ECP or ECP + GnRH), more displayed estrus (P = 0.002) and became pregnant (P < 0.0001) compared with those receiving only GnRH. In Experiment 2, the effect of the occurrence of estrus on ovarian responses was evaluated in cows (N = 53) synchronized using ECP at device removal. Cows that displayed estrus had a greater diameter of the largest follicle (LF) at device removal (P < 0.0001), a greater diameter at TAI (P < 0.0001), a greater ovulation rate (P = 0.02), a larger CL (P = 0.02), and a greater P4 concentration (P < 0.0001) than cows that did not display estrus. In Experiment 3, the effect of GnRH treatment on P/AI at TAI was evaluated in cows that received ECP at device removal, and either displayed, or did not display, estrus (N = 726). There was no estrus by GnRH interaction (P = 0.22); the P/AI was greater (P < 0.0001) in cows that displayed estrus (61.9%) than cows that did not display estrus (41.4%). However, GnRH did not improve (P = 0.81) P/AI (GnRH = 53.7% vs. no GnRH = 52.6%). In conclusion, exogenous estradiol at device removal increased both the proportion of suckled Bos indicus cows that displayed estrus and P/AI. Cows that displayed estrus had better ovarian responses (i.e., larger follicles at TAI, a greater ovulation rate, larger CL, and greater P4 concentrations) following an estradiol/P4-based synchronization protocol. Although occurrence of estrus improved pregnancy outcomes, GnRH at TAI did not improve P/AI in suckled Bos indicus cows treated with ECP, regardless of estrus occurrence.     

不同剂量的抗孕-53和18-甲基炔诺酮对大鼠垂体机能反应性影响的比较

为了研究甾体避孕药对垂体生殖激素的影响,本实验以动情前期大鼠受二次静脉注射GnRH诱发的LH激发效应(priming effect)为模型,观察经肌肉注射不同类型、不同剂量的避孕药(抗孕-53或18-甲基炔诺酮)后垂体对GnRH的敏感性反应。抗孕-53的剂量为3,1.5,0.75,0.375和0.19mg;l8-甲基炔诺酮的剂量为4,1和0.5mg,各溶于0.5ml茶油;对照组为0.5ml茶油。实验结果表明,不同剂量的抗孕-53均促进血浆LH浓度升高,其中以3mg组的反应最显著,与对照组比较P<0.01。不同剂量的18-甲基炔诺酮对垂体敏感性均有显著的抑制作用,使血浆LH浓度降低(P<0.01)。大鼠经肌肉注射乙炔雌二醇21μg和10.5μg后,其垂体对GnRH的反应也有促进作用,但不能完全模拟抗孕-53的效应。上述结果表明,不同类型的甾体避孕药能增加或降低大鼠垂体对GnRH的敏感性。     

Historical perspectives and recent research on superovulation in cattle

Superovulation protocols have evolved greatly over the past 40 to 50 years. The development of commercial pituitary extracts and prostaglandins in the 1970s, and partially purified pituitary extracts and progesterone-releasing devices in the 1980s and 1990s have provided for the development of many of the protocols that we use today. Furthermore, the knowledge of follicular wave dynamics through the use of real-time ultrasonography and the development of the means by which follicular wave emergence can be controlled have provided new practical approaches. Although some embryo transfer practitioners still initiate superstimulatory treatments during mid-cycle in donor cows, the elective control of follicular wave emergence and ovulation has had a great effect on the application of on-farm embryo transfer, especially when large groups of donors need to be superstimulated at the same time. The most common treatment for the synchronization of follicular wave emergence for many years has been estradiol and progestins. In countries where estradiol cannot be used, practitioners have turned to alternative treatments for the synchronization of follicle wave emergence, such as mechanical follicle ablation or the administration of GnRH to induce ovulation. An approach that has shown promise is to initiate FSH treatments at the time of the emergence of the new follicular wave after GnRH-induced ovulation of an induced persistent follicle. Alternatively, it has been suggested recently that it might be possible to ignore follicular wave status, and by extending the treatment protocol, induce small antral follicles to grow and superovulate. Recently, the mixing of FSH with sustained release polymers or the development of long-acting recombinant FSH products have permitted superstimulation with a single or alternatively, two gonadotropin treatments 48 hours apart, reducing the need for animal handling during superstimulation. Although the number of transferable embryos per donor cow superstimulated has not increased, the protocols that are used today have increased the numbers of transferable embryos recovered per unit time and have facilitated the application of on-farm embryo transfer programs. They are practical, easy to administer by farm personnel, and more importantly, they eliminate the need for detecting estrus.     

A secondary surge of prolactin on the estrus afternoon

It has been described that throughout the estrous cycle of the rat, plasma prolactin (PRL) is basal except on proestrus afternoon when a preovulatory surge occurs. However, there have been controversies about PRL levels on the estrus day. Thus, the aim of this study was to evaluate the existence of a secondary surge of PRL on estrus afternoon and correlate it with plasma estradiol levels. The jugular vein of cycling rats was cannulated at 14:00 h on proestrus and a blood sample was withdrawn at 17:00 h for plasma LH measurement and determination of the preovulatory LH surge occurrence. In order to exclude the regular cycling rats that do not present the gonadotropins preovulatory surge and do not ovulate, only rats showing the LH surge on proestrus were considered in this study. Blood samples were collected hourly during estrus from midnight to 9:00 h (group 1) and from 10:00 to 18:00 h (group 2). In group 1, PRL showed a descending profile from midnight to 9:00 h, whereas the estradiol concentrations were constant. In group 2, a secondary surge of PRL was observed in 20 of 25 (80%) rats and plasma estradiol remained constant, but was higher in animals with the PRL surge. Thus the present data suggest the occurrence of a secondary surge of PRL in the afternoon of estrus that seems to be related to plasma estradiol levels of estrus day, which might exert only a permissive role in this surge generation.     

Plasma luteinizing hormone concentrations in cows given repeated treatments or three different doses of gonadotropin releasing hormone

We hypothesized that: (i) repeated GnRH treatments would increase the magnitude and duration of the LH surge and would increase progesterone (P4) concentrations after ovulation; and (ii) the release of pituitary LH would be greater in response to larger doses of GnRH. In Experiment 1, ovary-intact cows were given an intravaginal P4 (1.9 g) insert (CIDR) for 10 d and 500 μg cloprostenol (PGF) at CIDR removal to synchronize estrus. On Days 7 or 8 after estrus, cows received two PGF treatments (12 h apart) and 100 μg GnRH at 36 (Control), 36 and 38 (GnRH38), or 36 and 40 h (GnRH40) after the first PGF. Mean plasma LH concentration (ng/mL) was greater (P < 0.05) in GnRH38 (8.8 ± 1.1) than in Control (5.1 ± 1.3), with that in GnRH40 (5.8 ± 1.3) being intermediate. Although the duration (h) of the LH surge was longer in GnRH40 (8.0 ± 0.4) than in either GnRH38 (P < 0.05; 7.0 ± 0.3) or Control (P < 0.09; 7.1 ± 0.4), mean postovulatory P4 (ng/mL) was greater (P < 0.01) in Control (4.2 ± 0.7) than in GnRH38 (2.9 ± 0.6) or GnRH40 (3.0 ± 0.7) cows. In Experiment 2, ovariectomized cows were given a CIDR for 10 d and 2 mg of estradiol cypionate im at CIDR insertion. Thirty-six hours after CIDR removal, cows received, 50, 100, or 250 μg of GnRH. Cows given 250 μg GnRH released more LH (9.4 ± 1.4 ng/mL) than those given 50 or 100 μg (6.1 ± 1.3 and 5.4 ± 1.4 ng/mL, respectively), and had an LH surge of longer duration than those given 50 μg (6.8 ± 0.4 vs. 5.1 ± 0.3 h). In summary, ovary-intact cows in the GnRH38 group had greater mean and peak LH concentrations, but subsequent plasma P4 concentrations were lower than in Control cows. Ovariectomized cows given 250 μg GnRH had a greater pituitary release of LH.