Passive immunoneutralization of endogenous inhibin increases ovulation rate in miniature Shiba goats

We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2alpha injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin alpha (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 microg PGF2alpha 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17beta increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 +/- 2.2) than in the control group (2.2 +/- 0.6), and the mean number of follicles > or = 4 mm in diameter was 10.0 +/- 0.8 in the inhibin-AS group compared with 2.4 +/- 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2alpha injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.     

卵泡抑制素主动和被动免疫及基因免疫研究进展

抑制素是由动物卵巢颗粒细胞或睾丸间质细胞分泌的糖蛋白激素 ,对垂体FSH的分泌具有抑制作用。用抑制素或其片断主动或被动免疫可以提高家畜的排卵率和产仔数 ;用抑制素基因免疫可望得到类似效果。本文通过分析抑制素的理化特性、生物学作用及作用机制、主动和被动免疫对家畜繁殖力的影响 ,阐述了抑制素基因免疫的原理与方法及应用前景     

Physiological roles of inhibin in regulation of FSH secretion and follicular development during early pregnancy in goats

The aim of the current study was to clarify the physiological role of inhibin in controlling FSH secretion and follicular development during the early pregnancy in goats. Eight goats investigated sonographically on Days 19-21 (Day 0=day of mating) for pregnancy were assigned into control (n=3) and treated (n=5) groups. The ovaries of all animals were daily scanned with ultrasound for follicles 2mm or more in diameter from 1 day before to 5 days after treatment. On Day 25 postbreeding; animals received either 10 ml, of normal goat serum or antiserum against [Tyr (30)]-inhibin alpha (1-30). Jugular blood samples were collected every 6 h starting 24 h before and until 120 h after treatment. The plasma concentration of FSH increased at 6 h and remained at significantly high levels until 120 h in treated vs. control group. The plasma concentrations of estradiol showed a marked increased at 66 h, with peak levels at 120 h after treatment of antiserum. The basal concentrations of LH and the pattern of plasma concentrations of progesterone were not significantly different between the two groups. The number of medium size (3.5-5.0 mm) follicles increased considerably from Day 2, whereas small (3.5 mm or less) and large (5 mm or more) follicles increased noticeably from Day 3, as compared with pre-treatment and controls. These results clearly indicated that inhibin is a key hormone in regulation of follicular development through regulation of endogenous FSH secretion during early pregnancy in goats.     

Antiserum to an inhibin alpha-chain peptide neutralizes inhibin bioactivity and increases ovulation rate in sheep.

A synthetic fragment representing the N-terminal 25 amino acid residues of the alpha-subunit of ovine inhibin (alpha-IF) was coupled to human alpha-globulin (h alpha-G) and used as an antigen. In Exp. 1, ovine antiserum generated against alpha-IF-h alpha-G was shown in vitro to neutralize inhibin bioactivity contained in ovine follicular fluid. In Exp. 2, 18 lambs were immunized with .3, .6 and 1.2 mg alpha-IF-h alpha-G or equivalent doses of h alpha-G. Antibody titer to alpha-IF was detected only in serum from lambs immunized against alpha-IF-h alpha-G and was first detected 27 +/- 2 d after primary immunization. Thereafter, antibody titers increased steadily. The degree of antibody responses was unrelated to antigen dose and differed among lambs. Plasma FSH concentrations were unchanged, whereas LH concentrations were lower (P less than .001) in sheep immunized against alpha-IF-h alpha-G. Ovulation rate was increased (3.5 +/- .5 vs 1.5 +/- .1; P less than .01) in lambs immunized against alpha-IF-h alpha-G. Ovulation rate was similar among animals receiving different antigen doses and increased with time after primary immunization (P less than .01). At estrous periods occurring approximately 34, 50, 74 and 107 d after primary immunization, respective ovulation rates were 157, 169, 207 and 450% of control values. Ovulation rate and antibody titer were correlated positively (pooled r = .95; P less than .01) within lambs. In Exp. 3, three lambs were immunized with .25 mg unconjugated alpha-IF; this was nonantigenic. In conclusion, the use of a synthetic fragment of the alpha-subunit of ovine inhibin as a hapten elicits an antibody capable of neutralizing inhibin bioactivity in vitro and increasing ovulation rate in vivo.     

牛卵泡抑制素抗体对小白鼠和大白鼠卵巢与子宫的作用

从用牛卵泡抑制素α－亚基片段免疫过的健康鸡所产蛋的卵黄液中提取抗体。得粗提物：然后用不同剂量的粗提物被动免疫２０只性成熟小白鼠和２０只发情周期大白鼠。并以生理盐水处理作为对照。结果发现,小白鼠用０．３ｍｌ抗体处理后,卵巢与子宫增重显著高于对照组（Ｐ〈０．０５）。大白鼠用０．６ｍｌ抗体处理后,卵泡平均数显著高于对照组（Ｐ〈０．０５）。这些结果表明,牛卵泡抑制素抗体被动免疫对小白鼠和大白鼠的子宫和卵泡     

Evaluation of ovulation rate and ovarian phenotype in puberal heifers from a cattle population selected for increased ovulation rate

Long-term selection for increased ovulation rate (1984 to 2002) has resulted in a unique ovarian phenotype in the MARC Twinner cattle population. Ovulation rate and frequency of bilateral ovulations were examined by rectal palpation in 29,547 estrous cycles for 3,910 heifers (12 to 18 mo of age) in this population. Bilateral ovulations (one corpus lutuem [CL] on each ovary) were of interest because bilateral twin pregnancies result in decreased dystocia and increased calf survival. Ovulation rate increased linearly at a rate of 0.026 CL per year, and it currently averages 1.48 +/- 0.04 CL per estrous cycle. Concurrent with the increase in ovulation rate, the frequency of triplet ovulations increased from 0% to 2.3 +/- 0.8% (P < 0.001). Ovulation rate of both the right and left ovary increased equally at a rate of 0.013 CL per year, and mean ovulation rate of the right ovary remained greater than mean ovulation rate of the left ovary throughout the study (0.66 vs. 0.55 +/- 0.003 CL per estrous cycle; P < 0.001). Although correlations were low, ovulation rate of one ovary was negatively correlated (P < 0.001; r = -0.07) with ovulation rate of the same ovary in the previous estrous cycle, but positively correlated (P < 0.001; r = 0.13) with the contralateral ovary of the previous estrous cycle. The proportion of bilateral ovulations averaged 55.7 +/- 0.7%, a value greater than the predicted 49.5% (P < 0.001). In addition to dystocia and retained placenta, triplet pregnancies increase the incidence of pregnancies gestating fetuses of opposite sexes and subsequent incidence of freemartins; thus, selection pressure on ovulation rate may need to be adjusted in the MARC Twinner population. The proportion of bilateral ovulations in the population is greater than expected, and this may be an economically important trait, which will respond to selection and be beneficial for improving bovine reproductive efficiency. Understanding factors controlling the increased functional activity of the right ovary and bilateral ovulations may provide further insights into the mechanisms controlling follicle selection and methods to improve reproductive management of cattle.     

Effect of active immunization of pony mares against recombinant porcine inhibin alpha subunit on ovarian follicular development and plasma steroids and gonadotropins

Two pony mares were immunized against recombinant porcine inhibin alpha subunit three times with 39 day intervals. Clinical findings and endocrinological changes before immunization were taken as the control. The first significant rise in the anti-inhibin titre (P<0.05) in the circulation was found 27 days after the first injection. Maximum binding activity was reached by the 12th day after the second booster dose. The number of small, medium and large sized follicles had increased significantly compared to before immunization (11.75 +/- 4.30, 2.75 +/- 0.69 and 2.51 +/- 0.63 vs 6.50 +/- 1.43, 1.83 +/- 0.44 and 1.33 +/- 0.38, respectively), but the ovulation rate remained unchanged after immunization. The average plasma concentration of FSH and estradiol-17beta during the estrous cycle increased significantly (P<0.05) after immunization. These results suggest that immunization against inhibin is a useful tool to increase the number of ovarian follicles during the estrous cycle of pony mares. Moreover, the present study supported the concept that inhibin plays a major role in the control of follicular growth through its inhibitory effect on FSH secretion synergistically with steroid hormones.     

非繁殖季节羊诱导发情排卵的研究进展

处于乏情状态的绵山羊,不管是泌乳性乏情还是季节性乏情,卵巢上都无发育成熟至可以排出的卵泡,也无功能黄体存在.这显然是由于垂体促性腺激素活动低下,FSH与LH的水平低,这种情况下,如果用外源的生殖激素(神经生殖激素、促性腺激素、性腺激素)或创造人工气候环境或公羊效应等其它方法引起内源促性腺激素分泌与释放,可使卵巢恢复活动,引起卵泡发育成熟至排卵.     

Effect of purified equine follicle-stimulating hormone on follicular development and ovulation in transitional mares

Horse owners want to have their mares bred as early as possible in the breeding season after February 1. Numerous medical treatments, such as progesterone, dopamine antagonists, and gonadotropin-releasing hormone have been administered to anestrous or transitional mares in an attempt to induce follicular development. Some of these treatments are ineffective or impractical, so there is a need in the horse industry to develop alternative techniques to stimulate follicular development and ovulation early in the breeding season. Twenty transitional mares were assigned to one of two treatment groups. Mares in group 1 (n = 10) served as untreated controls, and mares in group 2 (n = 10) were administered 12.5 mg of purified equine follicle-stimulating hormone (eFSH) (Bioniche Animal Health USA, Inc., Athens, Ga) intramuscularly twice daily for a maximum of 15 consecutive days. Mares were considered to be in transition when the diameter of the largest follicle was ≥25 mm. Once one or more follicles >35 mm were detected, eFSH treatment was discontinued and human chorionic gonadotropin was administered intravenously. The percentage of mares ovulating during the 15-day observation period was compared by means of chi-square analysis. The interval to ovulation and the number of ovulations per mare were compared between the two groups by Student t test. In 8 of 10 mares treated with eFSH follicles developed and ovulation occurred during the 15-day observation period, compared with 0 of 10 control mares. Interval from onset of treatment to ovulation was 7.6 ± 2.4 days for these eight mares. The eight mares were treated for an average of 5.2 ± 1.3 days with eFSH. Thus, the eFSH treatment was effective in advancing the first ovulation of the year in transitional mares.     

A dynamic model for ovulation rate reveals an effect of the estrogen receptor genotype on ovarian follicular development in the pig

A mathematical model that describes the recruitment and growth of ovarian follicles was fitted to data on ovulation rate and the measurements of plasma estradiol collected at times during the estrous cycle for individual gilts. The method of least squares was used to obtain estimates of the parameters of the mathematical model. The estimated model parameters were the maximum estradiol production for a follicle, development of each follicle after commitment, and a function describing the initial estradiol production of committed follicles. The estimated parameters for each pig were classified by estrogen receptor (ER) genotype (AA or BB) and analyzed using a multivariate analysis of variance. There were differences between genotypes (P < 0.05) for the parameter that described the initial distribution of individual follicles at recruitment. Gilts with ER genotype BB recruited follicles that varied more in size but had fewer very small follicles, indicating that the ER gene affects the relative estradiol secretion of the follicles at commitment. This analysis is an example of a general approach to genetic studies that uses a mathematical model of the physiology as a statistical basis for estimating gene action.     

Enhancement of ovulation rate in gilts by increasing dietary energy and administering insulin during follicular growth

Two experiments were conducted to examine influences of dietary energy and insulin on ovulation rate and patterns of luteinizing hormone (LH), follicle stimulating hormone (FSH), glucose, insulin and estradiol in gilts during 6 d before estrus. In Exp. 1, 36 gilts were given altrenogest for 14 d to synchronize estrus. In a factorial arrangement, gilts were fed one of two levels of dietary energy (5,771 or 9,960 kcal metabolizable energy (ME)/d), and given one of two levels of porcine insulin (0 or .1 IU/kg body weight iv every 6 h). Dietary treatments began 4 d before and insulin treatments began 1 d after the last day of altrenogest, respectively, and lasted until 24 h after estrus. Main effect means for number of corpora lutea were 14.0 +/- 1.3 and 17.6 +/- .9 for 5,771 and 9,960 kcal ME (P less than .05), and 14.6 +/- 1.0 and 17.0 +/- .9 for 0 and .1 IU insulin (P less than .05). Number of LH peaks on d 3 was greater for gilts that received 9,960 kcal than 5,771 kcal (3.3 +/- .2 vs 2.7 +/- .2; P less than .05), and for .1 than 0 IU insulin (3.2 +/- .2 vs 2.7 +/- .2; P less than .05). During the first 24 h of sampling, concentrations of LH and FSH were greater (P less than .05) in gilts receiving 9,960 kcal ME plus insulin than for other treatment combinations. Concentrations of estradiol were not affected by treatments. In Exp. 2, two formulations of insulin were evaluated for influence on ovulation rate. All gilts received altrenogest and 9,960 kcal ME/d as in Exp. 1. Then on the first day after altrenogest, seven gilts each received short-acting insulin (as in Exp. 1), long-acting insulin (zinc suspension, 1.0 IU/kg body weight every 18 to 24 h), or served as controls. Ovulation rates were increased (P less than .05) by both insulin preparations (15.6, control; 19.1, short-acting; 18.5, long-acting; SE = 1.2). Concentrations of LH tended to be greater after short-acting insulin, but differences were not significant (P = .13). We conclude that increases in ovulation rate produced by dietary energy and insulin are not necessarily accompanied by changes in gonadotropins or estradiol.     

The effects of exogenous growth hormone on follicular steroid secretion and ovulation rate in sheep.

Growth hormone (GH) has diverse actions in many tissues, including the follicle. This paper summarizes three experiments that examined the effects of GH and insulin-like growth factor (IGF)-I on the ovary. Ewes given oGH and pregnant mane serum gonadotrophin were compared with control and pregnant mane serum gonadotrophin-treated ewes. Ewes, with synchronized cycles, were given varying doses of pregnant mane serum gonadotrophin and/or oGH to determine if oGH is able to augment ovulation rate (Experiment 1). Experiments 2 and 3 used the ovarian autotransplant model. Ewes were infused via the ovarian artery with oGH (Experiment 2) or insulin-like growth factor I (IGF-I) (Experiment 3). Both were administered for 12 hr on Day 10. In Experiment 2, ewes were given intravenous gonadotropin releasing hormone (150 ng i.v.) at -2.5 and 10.5 hr relative to infusion. Ovarian and jugular venous blood was collected every 15 min from -30 to 150 min relative to gonadotropin releasing hormone. In Experiment 3, luteolysis was induced at the end of infusion. Ovarian and jugular venous blood was collected every 3 hr from before and until 84 hr after the infusion. Estradiol and androstenedione were assayed in ovarian venous plasma and GH in jugular venous plasma. In Experiment 1, treatment with oGH increased the jugular venous concentration of GH. However, in Experiment 2 treatment with oGH via the ovarian artery did not increase jugular venous GH but did increase ovarian venous GH. Treatment with oGH had no effect on ovulation rate (Experiment 1) or the secretion of androstenedione and estradiol (Experiment 2). Infusion of IGF-I (Experiment 3) increased the secretion of estradiol during the follicular phase. These data show that short-term treatment of sheep with GH had no in vivo effects on the follicle and that IGF-I was a potent stimulator of follicular steroidogenesis in vivo.     

Initial results of genomic scans for ovulation rate in a cattle population selected for increased twinning rate

Genomic scans were conducted with 273 markers on 181 sires from a cattle population selected for increased twinning rate to identify chromosomal regions containing genes that influence ovulation rate. Criteria used for selecting markers were number of alleles, ease of scoring, and relative position within linkage group. Markers were multiplexed or multiple-loaded on the gels to reduce the costs and labor required to obtain genotypic data. This approach reduced the number of gels by 45% when compared with running each marker independently. Male animals selected for the genomic scan sired the majority of the population. A modified interval analysis was used in a granddaughter design to compare effects of each allele within sire for 10 different sire families. The midparent deviation of the son's estimated breeding value for ovulation rate was used as the phenotype. Forty-one potential peaks were identified with a nominal significance level < or = 0.05. The 10 peaks with the highest significance levels (P < 0.02) were selected for further analysis. Markers were genotyped across daughters of the sire where nominal significance was found for each of the 10 peaks. One peak (BTA5, relative position 40 cM) was found to be nominally significant in the daughters. The nominal significance levels were P = 0.01 for the sons (n = 32) and P = 0.02 for the daughters (n = 94) of sire 784403. A combined genomewide significance value (P = 0.07) was calculated that accounted for the 10 analyses with sons and the 10 analyses with daughters. These results strongly suggest that this region contains a gene(s) that is involved in the follicular recruitment and development process.     

Ovarian follicular dynamics in lines of sheep (Finn, Merinos) selected on ovulation rate

Ewes from selected lines of sheep from each of two breeds, Finns (high ovulation rate, low ovulation rate and control lines with respective ovulation rates of 5.4, 2.7 and 3.3) and Merinos (T Merinos selected for increased ovulation rate and control Merinos with respective ovulation rates of 1.9 and 1.2) were used to examine how selection to alter ovulation rate had altered follicle development. Ovarian antral follicles were counted, measured, classified as nonatretic or atretic (more than five pyknotic bodies). The growth of ovulatory follicles in vivo, followed by repeated follicle ink marking, also was compared in the three lines of Finns. Regardless of breed, ewes selected for high ovulation rate had a similar number of antral follicles and a similar extent of atresia compared with their controls. Alterations induced by selection were located in the last stages of folliculogenesis. T Merinos exhibited a lower proportion of atretic follicles among follicles greater than 3 mm and a larger diameter of the largest healthy follicle when preovulatory follicles were excluded. High-line Finn ewes recruited more follicles, which produced smaller preovulatory follicles, each containing a smaller number of granulosa cells compared with either the low- or control-line ewes. Hence, physiological selection for high ovulation rate raised it by different methods in Merino than in Finn ewes.     

Effect of exogenous administration of oxytocin on postpartum follicular dynamics,oestrous rate and ovulation in Nili-Ravi buffaloes

Based on different surveys, dairy farmers are concerned about extensive use of exogenous oxytocin in buffaloes, which is being held responsible for reproductive problems including irregular oestrous cycle and delayed ovulation. For these concerns, effects of oxytocin injection on postpartum follicular dynamics, postpartum oestrous interval (PEI), oestrous length, the interval from onset of estrus to ovulation and blood progesterone (P4) were studied in Nili-Ravi buffaloes. For this purpose, 23 animals within 1 week after calving were randomly divided into three groups: without oxytocin (CON; n = 7), 10 i.u. oxytocin (LOW; n = 8), 30 i.u. oxytocin – (HIGH; n = 8) and used to record the PEI for the study period of 154 days. At subsequent estrus, three buffaloes from each group (not served) were selected randomly to monitor two cycles for 6 weeks. Transrectal ultrasonography was performed to evaluate follicular and corpus luteum (CL) development, and blood sampling was done for progesterone (P4) analysis. These results revealed that postpartum oestrous interval (PEI) decreased significantly in oxytocin-treated groups. The number of small, medium and total follicles on the left ovary was significantly higher in the HIGH group. However, an overall number of small and total follicles on both right and left ovaries was significantly higher in CON and HIGH groups. On the other hand, there was no difference in the number of follicles on the right ovary among all treatment groups. The same was true for the size of pre-ovulatory follicles, CL, P4 concentrations and oestrous cycle length. The intervals from onset of estrus to ovulation and from standing estrus to ovulation were increased considerably in the HIGH group. It is concluded that exogenous oxytocin administration resulted in the shortening of PEI but triggered a delay in ovulation. Moreover, a higher dose of oxytocin could stimulate the growth of small, medium, and total follicles in postpartum Nili-Ravi buffaloes.     

The effect of active immunization against inhibin on gonadotropin secretions and follicular dynamics during the estrous cycle in cows

The hypothesis of the present study is that active immunization of cows against inhibin would neutralize endogenous inhibin, increase circulating levels of follicle stimulating hormone, and subsequently affect follicular dynamics and the ovulation rate during the estrous cycle. Thirteen cows were immunized against inhibin alpha-subunit and, 6 cows were immunized with a placebo. Both groups were given 4 booster immunizations 7, 14, 21, and 34 weeks after the primary injection. Ovaries were examined daily after the 2nd, 3rd, and 4th booster immunizations by transrectal ultrasonography for 25 days. After the 4th booster immunization, blood samples were collected daily for one complete estrous cycle to measure FSH and LH. The results showed that the immunized cows generated antibodies against inhibin, and that they had higher FSH levels compared with the controls. The number of follicular waves during the estrous cycle was higher in the immunized cows (3 or 4 waves) than in the controls (2 or 3 waves). Moreover, the immunized cows had a greater number of follicles during the estrous cycle compared with the control cows. The maximum number of follicles was 14.8 +/- 1.7 vs 5.4 +/- 0.2 in inhibin-immunized and control cows, respectively, during the first follicular wave and 13.9 +/- 1.9 vs 5.6 +/- 0.7, respectively, during the ovulatory wave. Multiple ovulations were increased in the immunized cows. However, the ovulation rate varied greatly in the immunized animals. In conclusion, immunization against inhibin increased FSH secretions during the estrous cycle in the cows. Moreover, the immunized cows had a greater number of follicular waves during the estrous cycle and a greater number of follicles, and this could be used as a potential source of oocytes for use in IVF/embryo transfer programs.