Comparison of Two Doses of the GnRH Antagonist, Acyline, for Pregnancy Termination in Bitches

Gonadotropin-releasing hormone (GnRH) antagonists are particularly useful when a rapid inhibitory effect on the gonadal axis is required. The aim of this study was to test the efficacy and clinical safety of a low and high dose of the third generation GnRH antagonist, acyline, on pregnancy termination in female dogs. The effect of the antagonist on the progesterone (P₄) serum concentration was also described. Twenty-one mid-pregnant bitches were randomly assigned to a single subcutaneous (SC) dose of a placebo (PLACE; n = 7), a low (ACY-L; 110 μg/kg; n = 6) or high (ACY-H; 330 μg/kg; n = 8) dose of acyline. The animals were followed up for 15 days. All ACY treated but no placebo-treated animals terminated their pregnancy by abortion (p < 0.01). The ACY-L and ACY-H groups interrupted their pregnancy 7 ± 1.9 and 6.4 ± 1.3   days after treatment, respectively (p = 0.7). A significant interaction between treatment and day was found (p < 0.01) for P₄ serum concentrations when PLACE was compared with both ACY groups. No difference was found for this hormone between both ACY groups (p > 0.05) where P₄ diminished throughout the study. The decreasing rate varied among animals and was closely related to the time of abortion when P₄ reached basal concentrations. In PLACE animals, gestation progressed normally and P₄ did not change throughout the study (p > 0.05). None of the bitches presented side effects. It was concluded that acyline safely terminated mid-pregnancy by permanently decreasing P₄ serum concentrations.     

Effect of Follicle Size and Follicle-Stimulating Hormone on Ovulation Induction and Embryo Recovery in the Mare

The timing of ovulation is an important component to many equine breeding strategies. The action of luteinizing hormone on ovulation induction has been recognized; however, potential effects of follicle-stimulating hormone (FSH) have been less defined. Objectives of this study were to determine whether (1) mares could be induced to ovulate follicles ≤30 mm; (2) equine FSH (eFSH) has a positive effect on ovulation induction, and (3) ovulation of small follicles would affect embryo recovery. Light-horse mares (n = 12) between 4 and 10 years of age were assigned to treatments when they had a dominant growing follicle with a mean diameter of 24, 28, or 35 ± 2 mm and endometrial edema. Treatments were (1) H35, human chorionic gonadotropin (hCG) at 35 ± 2 mm; (2) F35, eFSH at 35 ± 2 mm; (3) H28, hCG at 28 ± 2 mm; (4) FH28, eFSH and hCG at 28 ± 2 mm; (5) D28, deslorelin (gonadotropin-releasing hormone [GnRH] analog) at 28 ± 2 mm; (6) FH24/H24, hCG or eFSH and hCG at 24 ± 2 mm. Mares’ reproductive tracts were scanned at 24 ± 2-hour intervals after treatment to detect ovulation. Mares were inseminated, and embryos were collected. Numbers of mares that ovulated within 48 ± 2 hours after treatment were: H35, 8/8 (100%); F35, 8/14 (57%); H28, 7/12 (58%); FH28, 9/12 (75%); D28, 3/7 (43%) and FH/H24, 4/14 (29%). The number of mares that ovulated in 48 ± 2 hours for H35 was not different from that for FH28 but was higher (P < .05) than all other groups. Embryo recovery rates, diameters, developmental stages, and morphology scores were not different for mares ovulating 48 hours or less versus more than 48 hours after treatment or among treatment groups. Results of this study demonstrate that follicles ≤30 mm can be induced to ovulate with no effect on embryo recovery or quality, as assessed by stereomicroscopy.     

马繁殖季节和非繁殖季节卵泡波变化观察

利用B型超声波诊断仪,对繁殖季节和非繁殖季节母马卵泡发育过程进行连续观察,揭示母马卵泡生长发育的动态模式。在繁殖季节的5—7月随机选取5匹空怀和4匹排卵后配种妊娠的伊犁马为监测对象,在非繁殖季节的8—10月随机选取8匹空怀伊犁马为监测对象,用B型超声波诊断仪每日或隔日扫描两侧卵巢1次,记录卵泡数量并测量卵泡直径,分别持续观测一个发情周期和40 d。结果表明,母马在繁殖季节的一个发情周期内有1～2个卵泡波;在非繁殖季节,马卵巢也有卵泡波的变化;母马不是严格的季节性发情动物。     

The Effect of Sulpiride Treatment During the Periovulatory Period on Prolactin Concentration and Ovulation in the Mare

Sixteen estrous cycles from 10 cyclic mares were randomly assigned to a control or sulpiride group (n = 8 each). All mares received 1,500 IU of human chorionic gonadotropin (hCG) (hour 0) during estrus with a follicular diameter ≥32 mm. Mares were scanned every 12 hours until ovulation. In the treatment group, beginning at hour 0, each mare received 1.5 mg/kg of sulpiride every 12 hours intra-muscularly until ovulation or formation of a luteinized unruptured follicle (LUF). Concentrations of luteinizing hormone (LH) and prolactin (PRL) were measured by radioimmunoassay. In each group, there were 10 preovulatory follicles for the eight cycles. The ovulation rate (9/10, 90%) was similar in the control and sulpiride groups. Two mares formed an LUF, which was first detected at hours 48 and 72 for the sulpiride and control mares, respectively. The interval from hCG to ovulation was 49.5 ± 11.1 and 43.5 ± 5.8 hours, for the control and sulpiride groups, respectively (P > .5). LH followed the typical preovulatory surge pattern, with no difference between groups (P > .5). Sulpiride administration increased PRL concentration in treated mares at 24 (P < .1), 36, and 48 hours (P < .05) after treatment. In conclusion, sulpiride administration every 12 hours increased PRL concentration in treated mares after 24 hours of the beginning of treatment. However, at this time window and concentration, PRL did not have any effect on ovulation. The control mare that developed an LUF had a PRL concentration similar to other ovulatory control mares (always ≤10 ng/mL).     

The Effect of Gonadotrophin Releasing Hormone and Follicle Stimulating Hormone in Conjunction with Pregnant Mare Serum Gonadotrophin on the Superovulatory Response in Crossbred Sheep In India

Thirty-two mature crossbred (Fine Wool Synthetic, FWS) sheep developed for fine wool production in India were treated for superovulation and oestrus synchronization in spring season. The ewes were randomly allocated to four treatment groups in a factorially designed experiment. For induction of superovulation, pregnant mare serum gonadotrophin (PMSG) was administered alone (group 1), in combination with gonadotrophin releasing hormone (GnRH) (group 2) or with follicle stimulating hormone (FSH) (group 3) and with both (group 4). Oestrus was synchronized in all the ewes by two injections of prostaglandin F2 (PGF, 10 mg each) administered at an interval of 10 days. Superovulation treatment started 48 h prior to the second PGF injection. The proportion of ewes in oestrus did not differ significantly (p>0.05) in the four groups. The use of GnRH set the ewes into oestrus earlier than the ewes in the other groups. Treatment with PMSG (800 IU) in conjunction with 4 g of Buserilin (GnRH) increased the ovulation rate (9.1±2.6 corpora lutea (CL)) above that observed when PMSG was used alone (3.0±0.7 CL). The use of FSH (0.5 U ovagen) in conjunction with PMSG was characterized by a decrease in the proportion of ewes with 2 CL (4/8 vs 7/8; p<0.05) and in the number of ovulations, i.e. CL observed (2.4±0.6 vs 9.1±2.6), and a nonsignificant increase in the incidence of large follicles (LF) (4.6±1.28 vs 3.25±0.6; p>0.05). The interaction between treatments of FSH and GnRH was not significant (p>0.05).It is concluded that use of GnRH, in conjunction with either PMSG alone or PMSG plus FSH treatment, advanced the onset of oestrus and increased the ovulation rate in FWS sheep.     

The Use of Equine Follicle Stimulating Hormone to Increase Equine Chorionic Gonadotropin in the Pregnant Mare

Equine chorionic gonadotropin (eCG), obtained from pregnant mares, is used for assisted reproductive technologies in laboratory rodents and livestock. The objective of the present study was to use equine follicle-stimulating hormone (eFSH) to increase the incidence of twin pregnancies, through multiple ovulations, and increase eCG. Nineteen light horse–type mares were enrolled in the study. The control group (n = 9) was bred with fresh or cooled semen and given human chorionic gonadotropin (hCG) at the time of breeding. The second group (n = 10) was given 12.5 mg of eFSH intramuscularly twice a day beginning 5–7 days after ovulation. Prostaglandin F2α was administered intramuscularly the second day of eFSH treatment. Treatment with eFSH continued until follicles were >35 mm in diameter, and mares were then given no treatment for 36 hours. The mares were then bred with fresh or cooled semen from the same stallion as the control group and given hCG. Blood samples were taken weekly from day 35 to day 105 after ovulation. Serum concentration of eCG was obtained, and data were analyzed with multivariate analysis using the mixed procedure. Significance was set at P < .05. Data were combined for all mares carrying twins and compared with those carrying singletons. The group of mares carrying twins had higher peak concentrations of eCG and higher values for area under the curve compared with mares carrying singletons (P < .05). These results suggest inducing twins could be a method used to increase eCG production.     

Pituitary responsiveness to GnRH, hypothalamic content of GnRH and pituitary LH and FSH concentrations immediately preceding puberty in gilts

To determine whether pituitary concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) or hypothalamic content of gonadotropin releasing hormone (GnRH) change before puberty, 40 prepubertal gilts averaging 7 mo of age were slaughtered before or on the second, third or fourth day after relocation and boar exposure. Some gilts responded to relocation and boar exposure as indicated by swollen vulvae, turgid uteri and enlarged ovarian follicles at the time of slaughter. Pituitary concentrations of LH and FSH and hypothalamic content of GnRH were similar between gilts that responded to relocation and boar exposure and gilts that did not respond. In addition, boar exposure and relocation had no effect on pituitary concentrations of LH and FSH or on hypothalamic content of GnRH. To determine whether pituitary responsiveness to GnRH changes before puberty, a third experiment was conducted in which 72 gilts were injected with 400 micrograms of GnRH either before or on the second, third or fourth day after relocation and boar exposure. In gilts that subsequently responded (i.e., ovulated) as a result of relocation and boar exposure, pituitary responsiveness to GnRH was reduced as compared with gilts that failed to ovulate after relocation and boar exposure. Peak concentrations of serum LH after GnRH injection were 4.6 +/- 1.3 vs 9.8 +/- .8 ng/ml for responders vs nonresponders. Peak serum FSH after GnRH injection was also lower for responders than for nonresponders (29.5 +/- 4.2 vs 41.2 +/- 2.4 ng/ml). When compared with controls, relocation and boar exposure did not significantly affect GnRH-induced release of LH and FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basal and GnRH induced secretion of FSH and LH in ovariectomized ewes treated with bovine follicular fluid

Ovariectomized (OVX) ewes were injected with 5 ml of either bovine serum, charcoalextracted bovine follicular fluid (FF), or whole bovine FF. Five hours after this pretreatment, ewes on each pretreatment were injected with either 0, 1, or 5 μg of GnRH. Ewes that were pretreated with either type of FF had decreased concentrations of FSH regardless of dose of GnRH when compared to ewes pretreated with bovine serum. There was no effect of charcoal extraction. There were no differences among the pretreatment groups in LH response to GnRH. In a second experiment, OVX ewes were pretreated (4 ml) with either bovine serum or bovine FF 5 hr prior to GnRH or with bovine FF 42, 30 and 18 hr prior to GnRH. Ewes were injected with either 0 or 5 μg of GnRH. Pretreatment with FF for 5 or 42 hr prior to GnRH resulted in significantly decreased concentrations of FSH both at the time of GnRH treatment and during the following 2 hr. Concentrations of LH did not differ among pretreatment groups. In a third experiment, OVX ewes were pretreated with either bovine serum or bovine FF 30, 18 and 5 hr prior to GnRH. Ewes were injected with either 0, 5 or 50 μg of GnRH. Pretreatment with FF resulted in decreased concentrations of FSH both at the time of GnRH treatment and during the following 2 hr. Concentrations of LH were also decreased at the time of GnRH treatment.     

Suppression of behavioural and physiological oestrus in the mare by vaccination against GnRH

OBJECTIVE: To examine the immunogenicity of an equine immunocontraceptive vaccine and its efficacy in controlling hormone-related behaviour. DESIGN: A total of 24 mares at two sites in Australia were vaccinated with an immunocontraceptive vaccine comprising gonadotrophin releasing hormone (GnRH) conjugated to a carrier protein in immunostimulating complex as an adjuvant. Twelve animals at each site received a placebo of adjuvant alone and served as controls for seasonal oestrus, hormonal and behaviour patterns. Animals were observed for injection site reactions, ovarian and follicular activity, and serum levels of antibody, 17beta-oestradiol and progesterone in the weeks following vaccination. Mares were also examined for oestrous behaviour by teasing with a stallion. RESULTS: All mares responded to vaccination. Two weeks following the second vaccination there was a peak in antibody response to GnRH that declined gradually over the following weeks. Commensurate with the elevated anti-GnRH antibody there was a marked effect on ovarian activity with a reduction in 17beta-oestradiol and progesterone levels in the 24 vaccinated mares. There was also a reduction of oestrus-related behaviour as determined by a teaser stallion. This effect lasted a minimum of 3 months and correlated with the initial level of antibody response. CONCLUSION: Following a conventional two-dose immunisation regime this commercially available equine immunocontraceptive vaccine was effective at inhibiting oestrous behaviour for at least 3 months. This vaccine has a high level of safety since there were no significant local reactions nor were there any adverse systemic responses to vaccination.     

Matrix Metalloproteinase (MMP)-2 and MMP-9 Activity in the Canine Uterus Before and During Placentation

The aim of the present study was to demonstrate the presence and localization of MMP-2 and -9 by means of RT-PCR and immunohistochemistry (IHC) within the canine uterus from the pre-implantation stage until mid-gestation and to determine MMP-2 and -9 activities by means of zymography. For this purpose, samples of the uterus and salpinx from bitches were obtained after ovariohysterectomy. Pre-implantation stages (5-12 days after mating, n = 11) were determined by verifying embryos after flushing the uterus. Further groups were determined as implantation (15-19 days after mating, n = 9), post-implantation (20-30 days after mating, n = 9) and placental stages (30-45 days after mating, n = 3). A non-pregnant group (17-30 days after mating, n = 4) served as control. MMP-2 and -9 positive cells were detected in all specimens from pregnant and nonpregnant bitches, however, with different distributions. MMP-2 was present in endothelium and smooth muscles of blood vessels and the myometrium of pregnant and nonpregnant bitches, additionally in the surface epithelium of the oviduct. The latter also stained positive for MMP-9. During placentation, MMP-2 was detected mainly in fetal blood vessels and trophoblastic cells. Higher MMP-2 activity was observed in the endometrium and myometrium of all pregnant groups compared with the nonpregnant group (p < 0.05). The pregnant groups did not differ significantly from each other (p > 0.05). MMP-9 was present in blood vessels, smooth muscle cells and epithelia, such as maternal surface epithelial cells, uterine crypts and glands. During placentation, the deep uterine glands and the epithelium of the glandular chambers were immunoreactive to MMP-9. Highest MMP-9 activities were reached in the endometrium of the pre-implantation group (23.2% of total MMP-9) and placental parts (33.3%).     

Successful use of a gonadotropin-releasing hormone (GnRH) analog for the treatment of tertiary hypogonadism (GnRH deficiency) in a 5-year-old Belgian Blue bull

A bull was referred for a progressive oligoasthenotheratozoospermia that resulted in a unsuitable seminal quality for the cryopreservation. Breeding soundness evaluation results suggested gonadal dysfunction. Because of the lack of normal ranges for these hormones in the bull, in this study, the hypogonadism and the site of the dysfunction (hypothalamus) were diagnosed by the gonadotropin-releasing hormone (GnRH) stimulation test. The evaluation of pituitary and testicular responsiveness by a GnRH stimulating test revealed a responsiveness of the pituitary and testis, thus a secondary hypogonadism (hypothalamic hypogonadism) was postulated and a therapeutic approach based on the subcutaneous administration of GnRH analog was attempted. An increase in semen volume, concentration and sperm characteristics were detected 9 weeks after the start of the treatment, corroborating the hypothalamic origin of the disease and the useful of the GnRH therapy.     

Function of the Cumulus Oophorus Before and During Mammalian Fertilization

Fertilization encompasses a series of different steps which have to be performed in a well‐orchestrated way to create a new individual. They include sperm capacitation, sperm binding and penetration of the zona pellucida, traversing the perivitelline space, binding and fusion with the oolemma, activation of the oocyte and decondensation of the sperm head to form the male pronucleus. In most mammalian species, cumulus cells surround the oocyte at the time of fertilization. Removal of the cumulus oophorus at this point of time often leads to a drop in fertilization rates. It is not yet known how cumulus cells interact with the oocyte or with spermatozoa to promote fertilization. There are different possibilities:

• 1 cumulus cells cause mechanical entrapment of spermatozoa and guide hyperactivated spermatozoa towards the oocyte, while preventing abnormal spermatozoa to enter the cumulus matrix;
• 2 cumulus cells create a micro‐environment for the spermatozoa which favours their capacitation and penetration into the oocyte;
• 3 cumulus cells prevent changes in the oocyte which are unfavourable for normal fertilization; these changes can be located in the zona pellucida or in the cytoplasm.

In this review, studies in several species are listed to prove the importance of these three cumulus cell functions and the current lines of research are highlighted. Moreover, different ways to improve in vitro fertilization of bovine cumulus‐denuded oocytes are discussed.     

Effects of a single injection of GnRH or equine pituitary extract on plasma LH and FSH concentrations in stallions

Plasma LH and FSH concentrations were measured in mature stallions after administration of synthetic GnRH or equine pituitary extract. GnRH caused significant rises in plasma LH (2-fold) and FSH (1.7-fold). Concentrations of LH remained significantly elevated for 4 hours and FSH remained elevated for 2 hours. Similar increases in plasma LH (1.6-fold) and FSH (1.8-fold) occurred after an injection of equine pituitary extract. LH was significantly elevated for 4 hours and FSH was elevated for 6 hours.     

单色光对肉鸡脑内促性腺激素释放激素分布和表达的影响

24只刚出壳的雄性AA肉鸡分别在白(400～760 nm)、红(660 nm)、绿(560 nm)和蓝色(480 nm) LED灯下饲养7周,光照强度15 lx,光照制度23h∶1 h(L:D).49日龄时取脑,利用免疫组化的方法显示GnRH神经元和神经纤维的分布及比较各光色下GnRH神经元的表达强度.结果显示,GnRH神经元和神经纤维在下丘脑、丘脑和中脑均有分布,且蓝光组下丘脑的视前室周核、视前内侧核、室周核、室旁大细胞核以及丘脑的圆核和卵圆核GnRH神经元表达强度显著高于红、绿光组.结果表明,光色可影响家禽下丘脑和丘脑GnRH的表达与分泌.     

Two Methods for the Treatment of Ureterolithiasis in a Mare

Ureteroliths were removed on two occasions from a mare by different techniques. A 3 cm calculus was removed by right ureterolithectomy during a ventral midline celiotomy. Blood urea nitrogen (BUN) and serum creatinine were measured monthly and remained within normal limits for 5 months. During the fifth postoperative month, after 9 days of lumbar pain, the mare was represented with an increased BUN and creatinine and a ureterolith in the left ureter. Using a Dormia basket stone dislodger, the second ureterolith was removed through a vestibulourethral approach. Fourteen days after surgery, the BUN and creatinine had returned to normal limits and the mare remained asymptomatic for 3 months. The mare then developed severe laminitis after 2 days of strenuous exercise and was euthanized. At necropsy, both kidneys contained multiple calculi.     

雌性哺乳动物FSH及其受体

本文介绍了FSH及其受体的结构,重点讨论了雌性哺乳动物FSH的功能及FSH受体在卵巢中的表达。为进一步的研究工作提供了参考资料。     

Maxillary Granulomatous Inflammation Caused by Halicephalobus gingivalis (Nematoda) in a Connemara Mare in Belgium

A 5-year-old anorexic and lethargic Connemara mare presented with severe facial swelling and dyspnea. No distinct central nervous symptoms were present. Radiographs of the skull showed diffuse radiolucency with loss of definition of the periodontal lamina dura and swelling of the decalcified nasal bones. Given the severe bone damage and its poor general condition, the pony was euthanized. Histological evaluation of the lesion revealed a granulomatous reaction with numerous sections of adult and juvenile nematodes, which were morphologically and molecularly identified as Halicephalobus gingivalis. The position of this facultative parasite within its free-living congeners is reviewed in this article, and the possible infection routes are discussed. This report presents the first record of equine halicephalobiasis in Belgium.