Immunization with a Streptococcus bovis vaccine administered by different routes against lactic acidosis in sheep

Streptococcus bovis is an important lactic acid bacterium in the rumen, which contributes to the development of lactic acidosis. This study was designed to test the efficacy of immunization with S. bovis primed either intramuscularly (i.m.) or intraperitoneally (i.p. ) against lactic acidosis. Forty-five wethers were allocated to three treatment groups. Two groups were injected with a S. bovis vaccine by either the i.m. or i.p. route for primary immunization; both groups were further immunized by the same route(s) (oral and/or i.m.) for boosters. The third group was not immunized (control). Antibody concentrations were measured in saliva prior to and following animals being fed a grain diet, and also in the rumen fluid, before the animals were suddenly introduced to a grain diet. The average antibody concentration in the animals of the i.m. group was higher than the i.p. group (P< 0.05). The antibody concentration in the rumen fluid of immunized sheep was higher than the control animals (P< 0.01). The difference in the rumen fluid antibody concentration between the i.m. and i.p. groups was not statistically significant (P> 0.05). In the i.m. group, there was a significantly greater feed intake, higher rumen pH, lower diarrhoea scores, and less increase in blood packed cell volume following grain feeding than in the animals of the control group. The severity of diarrhoea and the increase of blood packed cell volume in the animals of the i. p. group were also less than in the animals of the control group. The results suggest that the risk of lactic acidosis can be reduced by immunization against S. bovis, and that the immunization primed i. m. is more effective than the immunization primed i.p.     

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.     

Antibody Response in Sheep Following Immunization with Streptococcus bovis in Different Adjuvants

Recent studies have shown that immunization with Streptococcus bovis using Freund's complete adjuvant (FCA) may confer protection against lactic acidosis in sheep. The major objective of this study was to compare the antibody responses to S. bovis in a practically acceptable adjuvant (Freund's incomplete adjuvant (FIA); QuilA; dextran sulphate (Dex); Imject Alum; or Gerbu) and in FCA. Thirty-five sheep were randomly allocated to 7 treatment groups. Six groups were immunized with S. bovis in an adjuvant; the other group served as the non-immunization control. The primary immunization was administered intramuscularly on day 0, followed by a booster injection on day 28. Immunization with FCA induced the highest saliva and serum antibody responses. The saliva antibody concentrations in the FIA and QuilA groups were significantly higher than those in the Alum, Dex and Gerbu groups (p<0.01). The serum antibody concentration in the FIA group was significantly higher than those in the QuilA, Alum, Dex and Gerbu groups (p<0.01). Immunization enhanced the antibody level in faeces (p<0.05), but there was no significant difference between the different adjuvant groups (p>0.05). Seven and 14 days following booster immunization, the saliva antibody levels induced by QuilA and/or FIA were comparable with the level stimulated by FCA (p>0.05). There was a strongly positive correlation (R ² = 0.770, p<0.01) between the antibody concentrations in saliva and serum. Compared with the controls, a higher faecal dry matter content was observed in the animals immunized with either FCA or QuilA. The change in faecal dry matter content was positively associated with the faecal antibody concentration (R ² = 0.441, p<0.05). These results indicate that FIA and QuilA were effective at inducing high levels of antibody responses to S. bovis, and suggest that either Freund's incomplete adjuvant or QuilA may be useful for preparing a practically acceptable vaccine against lactic acidosis.     

牛链球菌在瘤胃中产酸的代谢机制及调控

牛链球菌(S.bovis)是瘤胃主要的乳酸产生菌,在饲喂高精料饲粮导致瘤胃乳酸中毒进程中扮演重要角色。已有研究证实S.bovis利用碳水化合物代谢产酸主要受葡萄糖转运方式、酵解产酸途径中酶和中间代谢物调控。另外,研究也发现环境p H、增殖生长阶段及分解代谢控制蛋白(Ccp A)等对其产酸速率和模式也有显著影响。本文对近年来有关S.bovis利用饲料中碳水化合物发酵产酸代谢途径及影响因素研究加以综述,为从微生物代谢角度解析瘤胃乳酸中毒机制提供参考。     

分解代谢物控制蛋白A对瘤胃中牛链球菌碳水化合物代谢的调控作用

分解代谢物控制蛋白A(CcpA)是低GC含量的革兰氏阳性菌基因转录的抑制或激活因子,对瘤胃微生物代谢具有调控作用。瘤胃中主要乳酸产生菌——牛链球菌(S.bovis)中的CcpA与磷酸转移酶系统(PTS)有着密切关系,对碳源次序代谢利用及碳水化合物代谢的多种关键酶具有重要的调控作用。本文综述了有关CcpA对S.bovis碳水化合物的代谢调控的研究进展,旨在为后期进一步研究S.bovis代谢产酸机制、防控反刍动物瘤胃酸中毒提供思路和参考。     

瘤胃中乳酸的代谢及其调控机制

有关瘤胃酸中毒发生机制研究表明,瘤胃乳酸的累积可能对酸中毒诱导起重要作用,而高精料日粮下瘤胃乳酸累积主要取决于瘤胃乳酸产生菌和乳酸利用菌间的平衡程度。本文综述了瘤胃微生物对乳酸的代谢机制,包括主要乳酸产生菌[溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)、牛链球菌(Streptococcus bovis)、乳酸杆菌(Lactobacillus)]和主要乳酸利用菌[反刍兽新月单胞菌(Selenomonus ruminantium)、埃氏巨型球菌(Megasphaera elsdenii)],并简要概述了酸中毒的调控方法,旨在为反刍动物瘤胃酸中毒的乳酸中毒机制深入解析提供参考。     

Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis

In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n=3) and acidosis-resistant (AR, n=3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis. Global R analysis of bacterial profile similarity revealed that bacterial diversity was significantly different between AR and AS groups for both rumen content (P=0.001) and epithelial (P=0.002) communities. The copy number of total bacterial 16S rRNA genes in content of AS steers was 10-fold higher than that of AR steers, and the copy number of total 16S rRNA genes of epimural bacteria in AR steers was positively correlated with ruminal pH (r=0.59, P=0.04), and negatively correlated with total VFA concentration (r=-0.59, P=0.05). The expressions of host TLR2 and 4 genes were significantly higher in AR steers compared to those in AS steers. These findings enhance our understanding about the ruminal microbial ecology and host gene expression changes that may be useful in the prevention of ruminal acidosis.     

Kinetics and type of immune response following intranasal and subcutaneous immunisation of calves

Protection of animals against respiratory infections has long been known to depend on respiratory mucosal immunity. However, few studies have been reported on the immune response following intranasal (i.n.) immunisation with non-living, soluble antigens. This study determined the kinetics of the humoral and cellular immune responses in calves after i.n. immunisation with Limulus haemocyanin (LH) with cholera toxin adjuvant, or subcutaneous (s.c.) immunisation with LH in incomplete Freund's adjuvant. A proliferative response of peripheral blood mononuclear cells cultured in vitro with LH was observed in animals immunised 7-10 days after i.n. and s.c. immunisations with no significant differences between the two immunised groups. LH -specific antibody was present in the serum of animals immunised s.c. (IgM, IgG1 and IgG2) and i.n. (IgA). Although significant IgA responses were observed, i.n. immunisations in cattle with soluble protein antigens and cholera toxin as an adjuvant did not induce a strong systemic immune response.     

Humoral immunity in the ewe. 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).     

Specific antibody containing cells in the porcine respiratory tract following intraperitoneal and intratracheal immunisation

Pigs were immunised intraperitoneally with ovalbumin in Freund's complete adjuvant and subsequently challenged intratracheally with ovalbumin. The results show that the group receiving two intratracheal challenges had a significantly greater antiovalbumin-containing cell response in the lamina propria of the respiratory tract. With intraperitoneal immunisation or intratracheal challenge alone the response was negligible. The immunoglobulin isotype of the anti-ovalbumin containing cells was predominantly IgG. There was also a substantial antibody containing cell response in the lung which was also IgG. These findings are discussed in relation to immunity within the porcine respiratory tract.     

Effects of bacterial direct-fed microbials on ruminal fermentation,blood variables,and the microbial populations of feedlot cattle

A study was conducted to determine whether bacterial direct-fed microbials (DFM) could be used to minimize the risk of acidosis in feedlot cattle receiving high concentrate diets. Six ruminally cannulated steers, previously adapted to a high concentrate diet, were used in a double 3 x 3 Latin square to study the effects of DFM on feed intake, ruminal pH, and ruminal and blood characteristics. Steers were provided ad libitum access to a diet containing steam-rolled barley, barley silage, and a protein-mineral supplement at 87, 9, and 4% (DM basis), respectively. Treatments were as follows: control, Propionibacterium P15 (P15), and Propionibacterium P15 and Enterococcus faecium EF212 (PE). The bacterial treatments (10(9) cfu/g) plus whey powder carrier, or whey powder alone for control, were top-dressed once daily at the time of feeding (10 g/[steer/d]). Periods consisted of 2 wk of adaptation and 1 wk of measurements. Ruminal pH was continuously measured for 6 d using indwelling electrodes. Dry matter intake and ruminal pH (mean, minimum, hours, and area pH < 5.8 or < 5.5) were not affected by treatment (P > 0.05). However, supplementation with P15 increased protozoal numbers (P < 0.05) with a concomitant increase in ruminal NH3 concentration (P < 0.01) and a decrease in the number of amylolytic bacteria (P < 0.05) compared with the control. Streptococcus bovis, enumerated using a selective medium, was numerically reduced with supplementation of PE. Although blood pH and blood glucose were not affected by DFM supplementation, steers fed PE had numerically lower concentrations of blood CO2 than control steers, which is consistent with a reduced risk of metabolic acidosis. Although the bacterial DFM used in this study did not induce changes in DMI or ruminal and blood pH, some rumen and blood variables indicated that the bacterial DFM used in this study may decrease the risk of acidosis in feedlot cattle.     

Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation

Ewes were immunised intraperitoneally with ovalbumin and Brucella abortus in Freund's complete adjuvant, followed seven days later by intramammary immunisation in which ovalbumin was presented to one mammary gland and Brucella abortus to the other. Mammary tissue taken after a further seven days contained more antigen-specific plasma cells than ewes given intraperitoneal or intramammary immunisation alone. These cells were found predominantly in the specifically immunised gland and only a few were found in the contralateral gland. Most of these cells were of the IgG1 isotype. There was also an increase in the total number of IgG1- and IgG2-containing cells in mammary gland tissues of these ewes, indicating a non-specific response to immunisation. Following either intraperitoneal or intramammary immunisation there was also a significant increase in the number of antigen-specific IgA cells in the lamina propria of the jejunum. The gut response following intramammary immunisation alone was abrogated by chronic drainage of intestinal lymph but not mammary lymph. This suggests that antigen may relocate from the mammary gland to the intestine where an IgA response is generated from gut associated lymphoid tissue. These data provide evidence for interaction between the gut and mammary gland of sheep in response to antigen.     

Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.     

饲粮不同NFC/NDF对奶山羊瘤胃溶纤维丁酸弧菌、牛链球菌及埃氏巨型球菌含量变化的影响

试验旨在探讨在饲粮不同非纤维性碳水化合物/中性洗涤纤维条件下,奶山羊发生亚急性瘤胃酸中毒（subacute rumen acidosis,SARA）过程中瘤胃溶纤维丁酸弧菌、牛链球菌及埃氏巨型球菌数量的变化。选用6只安装有永久性瘤胃瘘管的泌乳期关中奶山羊为试验动物,采用动物自身前后对照法,试验分4期进行,每期10d,依次饲喂NFC/NDF比分别为1.02（Ⅰ期）、1.24（Ⅱ期）、1.63（Ⅲ期）、2.58（Ⅳ期）的4种饲粮,以逐渐增加饲粮精料含量的方式诱导奶山羊发生SARA,并采用实时定量PCR技术对瘤胃内溶纤维丁酸弧菌、牛链球菌及埃氏巨型球菌的数量变化进行定量分析。结果表明：①随着饲粮NFC/NDF比的逐步升高,溶纤维丁酸弧菌和埃氏巨型球菌的数量均增加,但当饲粮NFC/NDF比升至2.58时,与其他试验期相比,埃氏巨型球菌的数量极显著增加（P〈0.01）,而溶纤维丁酸弧菌的数量却极显著降低（P〈0.01）;②牛链球菌的数量随着饲粮NFC/NDF比的逐步增加呈显著下降趋势（P〈0.05）,但到第Ⅳ期又恢复到第Ⅰ期的数量。结果提示,当奶山羊发生SARA时,瘤胃牛链球菌的数量无明显变化,对低pH值敏感的溶纤维丁酸弧菌数量急剧下降,而耐酸性的埃氏巨型球菌数量表现为大幅增加。     

不同发酵类型乳酸菌对低水分粳稻秸秆青贮发酵品质及有氧稳定性的影响

为探讨不同发酵类型乳酸菌对低水分粳稻(Oryza saliva subsp keng)秸秆发酵品质和有氧稳定性的影响,本试验以稻秸(含水量50.47%)为青贮材料,设有4组,即对照组(CS),布氏乳杆菌H4001组(HS,5×10~6 cfu·g~(-1)FM),植物乳杆菌S2406组(SS,5×10~6 cfu·g~(-1) FM)及植物乳杆菌与布氏乳杆菌混合组(MS,5×10~6 cfu·g~(-1)FM),发酵时间60d,取青贮粳稻秸秆样品测定其青贮品质及有氧稳定性。结果表明:同型发酵乳酸菌植物乳杆菌S2409可显著降低青贮的pH值和氨态氮含量(P0.05),提高青贮的乳酸、粗蛋白和干物质含量(P0.05),而异型发酵乳酸菌布氏乳杆菌H4001可显著提高青贮的乙酸、水溶性碳水化合物和中性洗涤纤维的含量(P0.05)。同型发酵乳酸菌和异型发酵乳酸菌在青贮开窖后可分别延长青贮的有氧稳定性时间36h、65h。混合组发酵品质及有氧稳定性均显著优于对照组。结合不同情况单独或混合使用不同发酵类型乳酸菌可获得更加优质的青贮饲料。     

The effect of adjuvants on active and passive immunity in pregnant deer and their offspring.

Experiments were carried out to determine the effects of a range of adjuvants on immunoglobulin M (IgM) and immunoglobulin G (IgG) serum concentrations to a protein antigen administered subcutaneously to farmed female deer following mating. The antibody responses of animals immunised with keyhole limpet hemocyanin (KLH) in Freund's Incomplete Adjuvant (FIA), diethylaminoethyl dextran (DEAE-dextran) and aluminium hydroxide (alum) were compared with the response to antigen administered in the absence of adjuvants. Animals were subsequently challenged with a subcutaneous immunisation of the antigen in saline. Following parturition, the concentration of passively transferred antigen-specific antibody was measured in the serum of the offspring. The polyionic adjuvant, DEAE-dextran, produced the greatest enhancement of both primary and secondary IgG responses to KLH. Offspring suckling from mothers immunised with antigen in DEAE-dextran consequently had higher concentrations of specific antibodies in their serum than other fawns in the experiment. The adjuvants FIA and alum were approximately 20-fold less effective in enhancing antigen-specific IgG than DEAE-dextran but induced greater amounts of antigen-specific IgM. From the results presented in this paper, there is evidence that immunisation of deer during pregnancy may be an effective way of reducing morbidity in both mothers and offspring.     

Immunisation of goats against contagious caprine pleuropneumonia using sonicated antigens of F-38 strain of mycoplasma

Three groups of 15 goats each were immunised against contagious caprine pleuropneumonia (CCPP) using sonicated antigens of the F-38 strain of mycoplasma incorporated in incomplete Freund's adjuvant (IFA), emulsified in aluminium hydroxide and phosphate buffered saline respectively. Three months after immunisation, five goats from each group were challenged by the in-contact method. The goats immunised with the antigen incorporated in IFA were all solidly immune to the challenge whereas only two of five of the goats in the other two groups were protected. When the remaining 10 animals from each group were challenged six months after immunisation, those immunised with the antigen in IFA were still solidly immune while only two goats from each of the other two groups were protected. These results show that effective immunity against CCPP caused by the F-38 strain can be induced by vaccination with sonicated F-38 antigens emulsified in IFA.     

Humoral immunity in the ewe. 2. The effect of pregnancy on the primary and secondary antibody response to protein antigen

This study examines the effect of pregnancy on the quantity and isotype of antibody in ewes immunised with a novel primary antigen. The primary and secondary antibody levels to bovine serum albumin (BSA) in alum adjuvant, were compared between non-pregnant ewes and ewes immunised at different stages of pregnancy. Anti-BSA isotype specific responses were measured using an indirect ELISA. Results show the levels of immunoglobulin M (IgM) increased and persisted in response to BSA during pregnancy (P less than 0.05). Secondary immunoglobulin G1 (IgG1) titres were significantly impaired in late pregnancy and during lactation (P less than 0.05). The lower levels of immunoglobulin G2 (IgG2) were unaffected by pregnancy under these experimental conditions. Ewes were also immunised with BSA in alum adjuvant for their primary inoculum and BSA in different adjuvants for the secondary inoculation. Primary IgM persisted at higher levels during pregnancy compared with the response in non-pregnant ewes (P less than 0.05). Following the secondary injection, lower levels of anti-BSA specific IgM, IgG1 and IgG2 antibodies were produced in late pregnancy and during lactation compared with the levels in non-pregnant control ewes (P less than 0.05). Alterations in regulatory T-cell function or effector B-cell activity would most readily explain the qualitative changes in antibody titre observed following primary injection during pregnancy. The results also suggest an associated impairment of immunological memory following primary immunisation during pregnancy.     

Pediococcus acidilactici isolated from the rumen of lambs with rumen acidosis, 16S rRNA identification and sensibility to monensin and lasalocid

A lactic-acid producing bacterium was isolated from the rumen of lambs with rumen acidosis. The cells were Gram-positive, nonmotile, nonsporing, catalase negative spherical, 1.5-2.0 μm in diameter, and occur in pairs and tetrads. Analysis of 16S ribosomal RNA indicated that the rumen bacterium was a strain of Pediococcus acidilactici with 99% of nucleotide homology. This bacterium was sensible to monensin and lasalocid at the unique dose tested of 300 ppm. The concentration of lactic acid and DM degradation decreased (P < 0.05) when monensin or lasalocid were added to the culture media after 24, 48 and 72 h of incubation. In contrast, total VFA concentration and pH were higher (P < 0.05) in the culture media added with the ionophores. Up to now S. bovis is considered the main ruminal bacterium related with rumen acidosis, but the importance of P. acidilactici should be also reconsidered in experimental studies focused on the control rumen acidosis.     

Ovarian response to PMSG and GnRH in ewes immunised against oestradiol-17 beta

Forty-six adult merino ewes were immunised against oestradiol-17 beta-6 carbomethyloxime:human serum albumin and 48 comparable ewes were used as controls in an experiment to study the effects of gonadotrophin releasing hormone (GnRH) on ovulatory responses after treatment with pregnant mare's serum gonadotrophin (PMSG). All the ewes were treated with progestogen sponges for 14 days and received 1500 iu PMSG on the 12th day. Twenty-four control and 24 immunised ewes received 25 micrograms GnRH 21.5 hours and 23 hours after the sponges were withdrawn. Plasma samples were collected between 17 and 50 hours after the sponges were withdrawn and assayed for luteinising hormone (LH). Immunisation reduced the proportion of ewes which ovulated and their rate of ovulation. Injection of GnRH increased the proportion of immunised ewes ovulating (P less than 0.0005) and their rate of ovulation (P less than 0.0001). More unovulated follicles were observed in immunised ewes regardless of GnRH treatment (P less than 0.0001). The rate of recovery of eggs was reduced after immunisation. Treatment with GnRH produced a surge of LH of equal magnitude in the control and immunised ewes although not as many immunised ewes ovulated.