Alarelin active immunization influences expression levels of GnRHR,FSHR and LHR proteins in the ovary and enhances follicular development in ewes

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle‐stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty‐two pre‐pubertal ewes were assigned to experimental groups 1 to 5 (EG‐I to EG‐V) and control group (CG). Ewes in EG‐I, EG‐II and EG‐III were subcutaneously injected with 200, 300 or 400 μg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG‐IV and EG‐V were subcutaneously injected with 200 μg and 300 μg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P < 0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG‐IV and EG‐V were higher than that in EG‐I, EG‐II and EG‐III from days 35 to 45. Expressions of GnRHR protein in EG‐IV and EG‐V were lower than that in CG (P < 0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG‐IV and EG‐V (P < 0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG‐III and EG‐V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin‐immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P < 0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.     

Nasal immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis for improving ovarian responses and fertility in cross‐bred buffaloes

This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross‐bred buffaloes. A total of 134 cross‐bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 10¹⁰ CFU/ml of the C501 (pVAX‐asd‐IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF_2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX‐asd‐IS) elicited significant immune responses, and anti‐inhibin IgG antibody titres in group T1 were significantly higher (p < .01) than groups T3 and C. After the second GnRH treatment, the growth speed of the dominant follicles in group T1 was significantly faster (p < .05) than groups T3 and C. The number and diameter of large follicles (≥10 mm) as well as ovulatory follicles in group T1 were the greatest in all groups, resulting in a greater conception rate in buffaloes with positive anti‐inhibin antibodies. These results demonstrate that immunization with the C501 (pVAX‐asd‐IS) vaccine, coupled with the Ovsynch protocol, could be used as an alternative approach to improve reproductive performance in cross‐bred buffaloes.     

Ovarian response to pregnant mare serum gonadotropin and porcine pituitary extract in gilts actively immunized against gonadotropin releasing hormone

Two experiments were conducted to determine the effect of exogenous gonadotropins on follicular development in gilts actively immunized against gonadotropin releasing hormone (GnRH). Four gilts, which had become acyclic after immunization against GnRH, and four control gilts were given 1,000 IU pregnant mare serum gonadotropin (PMSG), while four additional control gilts were given saline. Control animals were prepuberal crossbred gilts averaging 100 kg body weight. Control gilts given saline had ovaries containing antral follicles (4 to 6 mm in diameter). Control gilts given PMSG exhibited estrus and their ovaries contained corpora hemorrhagica and corpora lutea. PMSG failed to stimulate follicular growth in gilts immunized against GnRH, and ovaries contained regressed corpora albicantia and small antral follicles (less than 1 mm in diameter). Concentrations of luteinizing hormone (LH) and estradiol-17 beta (E2) were non-detectable in gilts immunized against GnRH and given PMSG. In the second experiment, five gilts actively immunized against GnRH were given increasing doses of PMSG every third day until unilateral ovariectomy on d 50. PMSG failed to stimulate follicular growth, and concentrations of follicle stimulating hormone (FSH), E2 and LH were not detectable. Six weeks later, gilts were given a booster immunization and then were given 112 micrograms LH and 15 micrograms FSH intravenously every 6 h for 9 d. The remaining ovary was removed on d 10. Although LH and FSH concentrations were elevated, administration of gonadotropins did not stimulate follicular growth or increase E2 concentrations. These results indicate that neither PMSG or exogenous LH and FSH can induce E2 synthesis or sustain follicular development in gilts actively immunized against GnRH.     

Immunization of Sheep Against GNRH Early in Life: Effects on Gonadotropins,Follicular Growth and Responsiveness of Granulosa Cells To FSH and IGF-I in Two Breeds of Sheep With Different Prolificacy (Romanov and ILE-DE-France)

The prolific Romanov (R, ovulation rate = 3) and non-prolific Ile-de-France (IF, ovulation rate = 1) breeds were compared for their ovarian sensitivity to gonadotropins and IGF-I before puberty. For this purpose, the effects of in vivo immunization against GnRH on populations of ovarian follicles and in vitro sensitivity of granulosa cells to FSH and IGF-I were studied in prepuberal lambs from both breeds. Seventeen prepuberal lambs of each breed were actively immunized against GnRH between 3 wk and 6 mo of age. Relative to untreated lambs, FSH levels at 4, 5, and 6 mo of age were (respectively) 41%, 25%, and 29% for IF, and 43%, 24%, and 36% for R lambs. In a first experiment, histological analysis of ovaries was performed. Immunization treatment decreased the number of small (100–390 μm in diameter) and large size follicles (<1500 μm) in both breeds at 6 mo of age. In both breeds, gonadotropin (FSH - LH -hCG) treatment increased the number of large size follicles (<1500 μm in diameter) and induced the formation of preovulatory follicles in immunized as well as untreated lambs. The ovulation rate was less in immunized animals, but it was not different between breeds. In a second experiment, the effects of FSH and IGF-I were studied on granulosa cells from follicles between 1000 and 2000 μm in diameter. In both breeds, IGF-I increased granulosa cell proliferation, but enhanced progesterone secretion was observed only in R lambs after FSH and IGF-I stimulation. Granulosa cell response to FSH treatment was lost by immunization, whereas response to IGF-I remained unchanged in both breeds. These results indicate that long-term immunization of prepuberal lambs against GnRH reduced systemic concentrations of FSH, follicular development, and response to gonadotropins in vivo, similarly in the prolific R and the non-prolific IF breed. However, granulosa cells from R lambs had higher steroidogenic capacities and were more responsive to FSH. In addition, these results suggest that IGF-I could play an important role in regulating growth of small follicles both in immunized and non-immunized lambs.     

Hepatitis B Surface Antigen S Gene is an Effective Carrier Molecule for Developing GnRH DNA Immunocastration Vaccine in Mice

Relatively molecular mass of GnRH antigens is small and hence needs to couple to a large carrier molecule to enhance its immunogenicity. This study investigated whether hepatitis B surface antigen S (HBsAg‐S) gene can be used as an effective carrier molecule for developing GnRH DNA immunocastration vaccine. Two copies of human GnRH gene were fused with HBsAg‐S gene for constructing a recombinant plasmid pVAX‐HBsAg‐S‐2GnRH that coded for 27 kDa target fusion protein. Ten male mice were divided into two equal groups, treatment and control. The vaccine (50 μg/mice) prepared in saline solution was injected into male mice at weeks 0, 1, 2, 4 and 7 of the experiment. Vaccine's efficacy was evaluated in terms of GnRH‐specific IgG antibody response, plasma testosterone levels, testicular weight and extent of the testicular tissue damage. The specific anti‐GnRH antibody titre in vaccinated animals was significantly higher than in controls in only 4th week of immunization (p < 0.05). In addition, vaccinated animals showed lower testicular weight than those of the controls (p < 0.05). Spermatogenesis in seminiferous tubules in vaccinated animals was suppressed. In conclusion, in this study, the engineered plasmid to be used as a GnRH DNA vaccine induced antibody response and suppressed spermatogenesis in mice. This suggests that HBsAg‐S gene can be an effective carrier molecule for developing GnRH DNA immunocastration vaccine when relatively molecular mass of the aimed antigens is small.     

Histological Characteristics and Steroid Concentration of Ovarian Follicles at Different Stages of Development in Pregnant and Non-pregnant Dairy Cows

The aim of the study was to investigate the histological characteristics and steroid concentrations in follicular fluid of different populations of follicles at different stages of development, during pregnancy and the oestrous cycle in cows. Follicles from ovaries collected at a slaughterhouse were allocated into three size categories (small, 2–5.9 mm; medium, 6–13.9 mm; and large, 14–20 mm) in pregnant and non-pregnant cows. Slices were stained with HE and PAS for histological analysis. Follicular fluid was pooled according to size and pregnancy status and estradiol, testosterone and progesterone concentrations in follicular fluid were determined by RIA. Characteristics of healthy follicles did not differ, regardless of follicle size or pregnancy status. Total histological atresia was significantly higher in pregnant cows than in non-pregnant cows (p < 0.05). Estradiol increased and testosterone decreased significantly, while follicles increased in size, in both non-pregnant and pregnant cows (p < 0.05). Nonpregnant cows had the highest estradiol values in follicles of all sizes. Medium and large follicles from pregnant cows showed the lowest testosterone concentration (p < 0.05). Progesterone levels increased with follicle size only in non-pregnant animals. In large follicles, progesterone concentration was significantly higher in non-pregnant cows than in pregnant cows (p < 0.05). Considering steroid concentration and histological findings, most large follicles might be atretic during pregnancy in cattle.     

Use of the gonadotropin‐releasing hormone (GnRH) stimulation test to monitor gonadal function in intact adult male cats

The gonadotropin‐releasing hormone (GnRH) stimulation test is a common procedure used to investigate normality of the pituitary‐gonadal axis in mammals. There is very little information on the technique, its efficacy and side effects in small animals and in particular no information for male cats. In dogs, such test is performed by intravenous (IV) administration. With cats, the number of times the animal needs to be restrained for blood sampling should be the least possible. The purpose of this study was to assess efficacy and side effects of the GnRH stimulation test in tomcats comparing the IV with the intramuscular (IM) route of administration. A GnRH stimulation test was performed in eight adult tomcats through IM or IV administration of 50 μg gonadorelin. The response of the pituitary‐gonadal axis was assessed by measuring serum testosterone on blood samples collected prior to and 1 hr following treatment. When considering each single group of cats, the post‐stimulation serum testosterone values were significantly higher than the pre‐treatment ones (p < .05). When comparing the two groups of cats, basal testosterone concentrations did not differ, and also post‐GnRH testosterone concentrations did not differ. In conclusion, in the cats of our study, the GnRH stimulation test produced the same results following the IM or the IV route of administration. Therefore, in tomcats, the IM route can be considered as effective as the IV one and should be preferred when doing a GnRH test.     

X monosomy in a Virilized Female Cat

An infertile Siamese female cat was subjected for clinical, histological, cytogenetic and molecular studies due to ambiguous external genitalia (vulva, vagina, rudimentary penis and scrotum‐like structure) and masculine behaviour. An elevated oestrogen activity and a detectable level of testosterone were found. The cat underwent laparotomy. The gonads and the uterus were removed and subjected for histological studies, which showed ovaries with corpora lutea and a some primordial follicles. Chromosome studies of lymphocyte and fibroblast cultures, with the use of Giemsa staining, G‐banding and whole X chromosome painting by fluorescence in situ hybridization, revealed pure X monosomy. Molecular analysis showed the absence of the SRY gene. Our study revealed for the first time that X monosomy in cats may be associated with virilization, in spite of the lack of the SRY gene.     

Effect of immunization with bovine luteinizing hormone receptor on ovarian function in cats

OBJECTIVE: To determine the effect of immunization with bovine luteinizing hormone receptor (LH-R) on ovarian function of cats. ANIMALS: 9 adult female domestic cats. PROCEDURE: 7 cats were immunized with 0.5 mg of LH-R encapsulated in a silastic subdermal implant (3 x 10 mm); 2 served as control cats. Receptors had 80% specific binding to 125I-human chorionic gonadotropin with a binding capacity of 2,682 pM/mg. Cats received booster injections of LH-R. Cats were induced to ovulate with luteinizing hormone (LH) releasing hormone on day 345. Samples of venous blood and vaginal cells were collected through day 395. Observation of estrus behavior continued until day 516. Serum concentrations of estradiol, progesterone, thyroid gland hormones, LH, and LH-R antibody were determined. RESULTS: LH-R antibody was detected in the sera of immunized cats within 21 days after implantation. Detection of LH-R antibody was associated with suppression of serum progesterone to < or = 0.5 ng/mL during the study period, compared with concentrations of 5 to 10 ng/mL in control cats. Immunized cats did not display signs of estrus. Release of LH after administration of LH-releasing hormone indicated an intact hypothalamic-pituitary axis but poor corpus luteum function. Serum estradiol concentrations remained between 30 to 40 pg/mL in immunized and control cats. With the decrease antibody titers, hormone concentrations returned to a pattern consistent with that during fertility. CONCLUSIONS AND CLINICAL RELEVANCE: Active immunization with LH-R suppressed corpus luteum function in cats. The effect was reversible. An LH-R-based antifertility vaccine may have clinical application in other vertebrates.     

Evaluation of Follicular Development and Oocyte Quality in Pre‐pubertal Cats

Our study was conducted to assess the follicular development and availability of sound ovarian oocytes for in vitro production (IVP) of embryos in pre‐pubertal cats. The relationship between body and ovarian weight was examined in 93 cats. The results revealed that ovarian weight rapidly increased until 100 days of estimated age. By histological evaluation of ovaries obtained from 11 pre‐pubertal cats with estimated age of <20, 20–40 and 100–120 days, it was clarified that the increase in ovarian weight during kitten growth accompanied the increase in the number and size of antral follicles. The follicular diameter and percentage of normal oocytes in secondary/antral follicles also increased as estimated age (body weight) increased. The oocytes obtained from pre‐pubertal cats with 100–120 days of estimated age were used for IVP of embryos. The results showed that the success rates of in vitro maturation, in vitro fertilization and development to blastocysts after in vitro culture in pre‐pubertal cats were lower than in sexually mature cats. However, the percentage of blastocysts based on the cleaved embryos and cell number of blastocysts in pre‐pubertal cats were comparable to those in mature cats. In conclusion, these results suggest that the ovaries of pre‐pubertal cats with ≥100 days of age contain oocytes with in vitro developmental competence to blastocysts.     

Immunization against gnRH in mature mares: antibody titres,ovarian function,hormonal levels and oestrous behaviour

The aim of the present study was to investigate the effect of active immunization against GnRH in mature Standardbred mares (three experimental and one control mare) on antibody titres, ovarian function, hormonal levels and oestrous behaviour. The mares were individually teased with a stallion once each day. During the first part of the experiment (period I: late April until November), blood was sampled every third day during the first 3 months, thereafter once per week. In the second part of the experiment (period II: December until August), sampling was carried out every second week. Progesterone, oestradiol-17beta and LH were analysed. Rectal gynaecological examination was made with the same intervals as the blood samplings and included palpation of the genital organs and ultrasonography. The experimental mares were immunized against GnRH with a GnRH-BSA conjugate. Equimune (Vetrepharm, Bracetown, Business Park, Clonee, Co. Meath, Republic of Ireland) was used as adjuvant. The mares were immunized on four occasions (20-30 day intervals) and GnRH antibody titre was determined. All immunized mares produced antibodies against GnRH but the maximum titres as well as the duration of a greater than 10% binding capacity varied between the mares (1 : 1600 to 1 : 50 000; 5 to 12 months, respectively). After the first injection, all mares showed one oestrus and ovulated at the regular time. In two of the mares, the immunization resulted in ovarian atrophy. Their hormone levels of progesterone, oestradiol- 17beta and progesterone decreased to basal levels and the cyclical hormone pattern was interrupted from approximately 30 days onwards. They continued to show oestrous signs but with irregular durations and intervals. The third mare showed ovarian suppression only for short periods and not in both ovaries at the same time; the hormone levels were basal for only about 20 days (days 50-70) and the mare ovulated on day 75 after start of immunization. The other mares ovulated after 13.5 and 15 months, respectively. It is concluded that the effect of immunization against GnRH in mature mares was individual concerning antibody titre response and the suppression of ovarian activity and hormone levels. Mares with totally inactive ovaries continued to show oestrous signs but with irregular intervals and durations.     

Dominant follicles development and estradiol‐17β concentrations in non‐ovulating and ovulating post‐partum Bulgarian Murrah buffaloes

This study was designed to evaluate the dominant follicles development and the estradiol‐17β concentrations in non‐ovulating and ovulating post‐partum buffaloes. Sixteen Bulgarian Murrah buffaloes were submitted to transrectal ultrasonographic examination from the 1st post‐partum day until day 50, 3 days apart. The follicular diameter of the different categories of follicles and the ovulations was recorded. The animals were allocated into two groups: I (n = 6) non‐ovulating and II (n = 10) ovulating buffaloes. Serum estradiol‐17β concentrations on the days for dominant follicle registration were measured by enzyme‐linked immunosorbent assay. The results were statistically processed by analysis of variance, non‐parametric and correlation analysis. The mean intervals between calving and first dominant follicle detection differed significantly (p < .05) among the groups (19.5 ± 6.2 vs. 13.8 ± 5.1 days), while the mean intervals between registered dominant follicles from two successive waves were comparable. The mean follicular diameters for the same category follicles in both groups were similar. Different estradiol‐17β concentrations (p < .05) for the first dominant follicle between non‐ovulating (23.5 ± 7.0 pg/ml) and ovulating (33.3 ± 8.4 pg/ml) buffaloes were determined. The cumulative percentages of buffaloes with firstly detected dominant follicle and ovulating animals correlated positively (r ≥ .84; p < .05) to post‐partum days. In conclusion, non‐ovulating and ovulating post‐partum Bulgarian Murrah buffaloes showed differences in the development of the first dominant follicle and estradiol‐17β concentrations during the time of dominant follicles detection.     

Feedlot performance of steers and bulls actively immunized against gonadotropin-releasing hormone.

Feedlot performance and testicular and pituitary function were assessed in cattle actively immunized against GnRH. In Trial 1, 50 steers were either unimmunized (n = 10), actively immunized against keyhole limpet hemocyanin (KLH; n = 10), or immunized against a GnRH-KLH conjugate (n = 30). Fifteen of 30 steers immunized against GnRH-KLH received a secondary immunization 8 wk after primary immunization. Antibodies against GnRH were not evident in unimmunized steers or steers actively immunized against KLH. Antibodies against GnRH were noted in all immunized animals (n = 30) within 6 wk of primary immunization and anti-GnRH antibody concentrations became maximal 20 to 24 wk after immunization. The increasing anti-GnRH titer in immunized steers was associated with decreasing serum concentrations of LH. Serum concentrations of LH were depressed (P less than .05) within 8 wk of primary immunization and reached a nadir by wk 20. The patterns of increase in GnRH titer and decrease in serum concentrations of LH did not differ (P greater than .05) in animals receiving primary immunization alone or primary and secondary immunization. Feedlot performance and carcass quality were not affected (P greater than .05) by immunization against KLH or the GnRH-KLH conjugate. In Trial 2, 60 bull calves (mean weight = 325.2 +/- 2.8 kg) were randomly assigned to a 2 x 3 factorial experiment. The two classes (n = 30) were 1) unimplanted and 2) implanted with Synovex-S. The three treatments (n = 20) were 1) intact control, 2) actively immunized against GnRH, and 3) castrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active Immunization against Gonadotrophin-releasing Hormone in Chinese Male Pigs

We have investigated, under the normal conditions of local Chinese pig farming, castration of young male pigs by vaccination with a newly developed vaccine against gonadotrophin releasing hormone (GnRH). Because of the very early onset of puberty, long fattening period and relatively harsh circumstances in Chinese pig production, an investigation of the endocrine response of Chinese breeds to this type of vaccination was of particular interest. Fifteen crossbred boars (Yorkshire × Yanan) from three different litters were randomly assigned to three groups of five animals each. The first group was immunized at 13 weeks of age with a GnRH tandem dimer OVA‐conjugate in Specol and received a booster immunization 8 weeks later. The second group was injected with Specol alone and served as untreated controls. The remaining group was surgically castrated at the time of weaning (at 6 weeks of age). Pigs were fed ad libitum from weaning onwards. All animals were slaughtered at 31 weeks of age. Immunized boars had undetectable or low serum testosterone (0.09 ± 0.12 ng/ml), low fat androstenone (0.05 ± 0.01 μg/g) levels and very low testes weights (19.1 ± 4.3 g). Intact controls had much higher serum levels of testosterone (9.76 ± 4.81 ng/ml), fat androstenone levels (2.26 ± 0.87 μg/g) and testes weights (114.3 ± 29.41 g) at slaughter. Both the immunized and castrated group grew significantly faster than intact boars (p < 0.01). Average daily gains in immunized, castrated and intact animals were 0.69 ± 0.08, 0.63 ± 0.05 and 0.42 ± 0.07 kg (mean ± SD), respectively. The present data demonstrate for the first time that the newly developed anti‐GnRH vaccine works very well under practical Chinese pig farming conditions, and can be an attractive alternative to surgical castration.     

人抑制素主动免疫对母鸡产蛋和内分泌的影响

本文报道了主动免疫抑制素对母鸡卵泡发育、排卵率和外周E2 和P的影响。用人工合成人抑制素α亚基 ( 1 -2 6Tyr.Gly)片断与KLH连接作免疫原 ,免疫 8只母鸡 ,1 2只作对照 ,观察其产蛋率的变化、卵巢卵泡的发育 ;用放免法测定排卵周期中外周E2 和P。免疫组产蛋率比对照组高 (P <0 0 5) ,卵巢上≥ 1 0mm的卵泡数无显著差异 ,但白泡、小黄泡数差异较大。第 3、4、5次免疫后产蛋率显著提高 ,但在实验期抑制素免疫对蛋重没有影响。用放免法测定发现处理组外周基础水平的雌二醇比对照组上升     

Pulsatile administration of gonadotropin-releasing hormone agonist to gilts actively immunized against gonadotropin-releasing hormone

Sexually mature gilts (n = 20) were actively immunized against GnRH. Primary and booster immunizations of GnRH conjugated to bovine serum albumin induced production of antibodies in all gilts. Nineteen of the gilts became acyclic with suppressed concentrations of gonadotropins and estradiol. Intravenous challenges with 100 micrograms GnRH and 5 micrograms D-(Ala6, des-Gly-NH2(10)) ethylamide GnRH (a GnRH agonist that did not cross-react with antibodies produced by the gilts) caused release of LH and FSH, indicating maintenance of secretory capacity of pituitary gonadotropes in the immunized animals. Gilts were given 100 ng GnRH agonist at 2-h intervals for 72 h (n = 4) or 144 h (n = 10) or did not receive agonist (n = 5). Blood samples were taken every 6 h, and detectable concentrations of LH were observed in 42% and 52% of samples taken from gilts treated with or without agonist. In contrast, serum concentrations of FSH and estradiol were undetectable. Reproductive tracts and anterior pituitaries were taken from gilts at the conclusion of pulsatile administration of GnRH agonist or at 144 h for controls. Pituitary concentration of LH and FSH, uterine wet and dry weight, and size of the uterus were similar among groups. Paired ovarian weights for treated gilts pulsed with GnRH agonist for 72 h were heavier (P less than .05); however, ovaries from all immunized gilts were atrophied without follicular structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization of pigs against chicken gonadotropin-releasing hormone-II and lamprey gonadotropin-releasing hormone-III: effects on gonadotropin secretion and testicular function

The objective of this experiment was to evaluate the effects of active immunization against 2 GnRH isoforms on gonadotropin secretion and testicular function in pigs. Synthetic chicken (c) GnRH-II and lamprey (l) GnRH-III peptides, with the common pGlu-His-Trp-Ser sequence at the N-terminal omitted, were conjugated to BSA. Forty-eight male piglets were randomly assigned to 1 of 4 treatments. Pigs on treatment 1 were actively immunized against cGnRH-II, whereas pigs on treatment 2 were actively immunized against lGnRH-III. Control pigs on treatment 3 were actively immunized against the carrier protein (BSA), and pigs on treatment 4 were castrated and actively immunized against BSA. The BSA conjugate was emulsified in Freund's Incomplete Adjuvant and diethylaminoethyldextran. Primary immunization was given at 13 wk of age (WOA) with booster immunizations given at 16 and 19 WOA. Body weight and plasma samples were collected weekly beginning at 11 WOA. Treatments did not affect BW during the experimental period. Antibody titers were increased in animals immunized against cGnRH-II and lGnRH-III (P < 0.001). Cross-reactivity of the antibodies to mammalian GnRH or between cGnRH-II and lGnRH-III was minimal. Concentrations of testosterone were maximal in control boars (treatment 3) and minimal in control barrows (treatment 4) and immunized pigs (treatment x week; P < 0.01). Immunized animals had concentrations of LH (P < 0.001) and FSH (treatment x week; P < 0.03) that were less than control barrows and similar to control boars. At the end of the experiment, intact (noncastrated) pigs were exsanguinated. Testes were removed immediately; Leydig cells were isolated and treated with 0, 1, or 10 ng/mL of LH. There was an LH x GnRH treatment effect on testosterone concentrations (P < 0.03), indicating that Leydig cells were sensitive to the immunization protocol and doses of LH. Taken together, these data suggest that immunization against GnRH isoforms decreased gonadotropin secretion compared with control barrows. Additionally, immunization against cGnRH-II and lGnRH-III reduced the ability of Leydig cells to respond to LH challenges.     

Reproductive function and feedlot performance of beef heifers actively immunized against GnRH1

Two trials were conducted to examine reproductive function and feedlot performance by heifers after active immunization against GnRH. In trial 1, heifers were not immunized or were immunized with one of three doses of a GnRH-KLH (keyhole limpet hemocyanin) conjugate in Freund's complete adjuvant. Antibodies against GnRH were not detectable in non-immunized heifers (n = 9). However, antibodies against GnRH were noted in all immunized animals (n = 30) within 8 wk of primary immunization; anti-GnRH antibody concentrations were at a maximum 16 to 20 wk after immunization. This increased anti-GnRH titer was associated with a decreased serum concentration of progesterone. Ovarian and uterine weight and tissue concentrations of LH and GnRH receptor were reduced (P less than .05) by immunoneutralization of GnRH. Similarly, immunization against GnRH reduced (P less than .05) weight gain during feedlot confinement. In trial 2, feedlot performance after insertion of anabolic steroid implants (Synovex H) was evaluated in non-immunized heifers (n = 15), heifers actively immunized against GnRH-KLH (n = 15) or KLH alone (n = 15), or non-immunized heifers treated with melengestrol acetate (MGA; n = 15). Serum concentrations of progesterone were depressed in anti-GnRH and MGA-fed groups, but ovarian and uterine weights were depressed (P less than .05) only in heifers immunized against GnRH. Total weight gain and gain during the final 4 wk of confinement did not differ (P greater than .05) among groups with steroid implants. The GnRH-KLH conjugate is an effective immunogen in heifers, leading to suppression of reproductive activity. The depression of weight gain that attends development of anti-GnRH titers may be reversed by use of implants that contain anabolic steroids.     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Effect of a Gonadotropin-releasing Hormone Vaccine (ImprovacTM) on Steroid Hormones, Boar Taint Compounds and Performance in Entire Male Pigs

The objective of this study, comprising two trials, was to evaluate the effect of a gonadotropin‐releasing hormone (GnRH‐vaccine Improvac^TM; Pfizer Ltd) in a sample of the Swedish pig population. The pigs (n = 120) were assigned to three groups: control (entire male pigs), surgical castration and immunization against GnRH. Surgically castrated pigs did not express detectable levels of either testosterone or estrone sulphate (E1S) in plasma, or androstenone in fat and had lower skatole and indole levels in fat than entire male pigs. Immunization significantly reduced testes weight and bulbourethral gland length, plasma levels of the testicular hormones testosterone and E1S, and fat levels of androstenone, skatole and indole. Skatole levels in plasma were significantly lower than in entire male pigs in the second trial, but not in the first due to overall low skatole levels. All immunized pigs and surgically castrated pigs expressed skatole concentrations in fat below the level of 0.2 μg/g, above which meat is regarded as tainted. In contrast, eight entire male pigs exceeded this level. Indole levels in plasma from immunized pigs were lower than those from entire male pigs. Surgical castration caused lower daily weight gain in the suckling period compared with piglets raised intact, whereas in the post‐weaning period no difference was observed. Immunization resulted in higher feed intake and daily weight gain after the second injection. The estimated lean meat content was improved in comparison with the castrated pigs, but was lower than for entire male pigs. Dressing percentage was lower in immunized pigs than in surgically castrated and entire male pigs. The frequency of skin damage did not differ between immunized and entire male pigs or between immunized and surgically castrated pigs.