Ethics statement
The pre-clinical study was conducted in the facilities of Zoetis Manufacturing & Research Spain S.L. (Olot, Spain), whereas the two clinical studies were conducted at a commercial farm representative of the pig industry in Belgium (ILVO, Melle, Belgium). All experimental procedures were approved by the Zoetis Animal Welfare Committee and the competent authorities.
Animals and experimental design
All studies were performed double blinded. The pre-clinical study was performed earlier in time with a reduced number of animals and in a control environment, where safety and efficacy parameters were evaluated. Clinical studies were performed later, in farm conditions, with a larger number of animals evaluating only efficacy parameters. For the purpose of this article, only the efficacy parameters were discussed, in order to compare the three studies.
Pre-clinical study (Fig. 1A1, 1A2): fifty-seven clinically healthy, cross-bred gilts were enrolled at 56 days of age (8 weeks of age) and randomly allocated to one of three treatments (control, early priming [Fig. 1A] or late priming [Fig. 1B]). All animals were vaccinated at 8, 14 and 18 weeks of age either with saline or with the anti-GnRF analog (Improvac®, Zoetis Belgium SA, Belgium; 306 μg/dose). Control animals always received saline. Early priming animals were vaccinated with anti-GnRF analog at 8 and 18 weeks of age and with saline at 14 weeks of age; and late priming gilts with saline at 8 weeks of age and with anti-GnRF analog at 14 and 18 weeks of age. All treatment groups were commingled throughout the study. Vaccination was performed with 2 mL of either saline or anti-GnRF analog (Improvac®, Zoetis Belgium SA, Belgium) subcutaneously with a 16 G ¾ needle and a Simcro Sekurus safety device (Simcro, UK). They were administered in the right upper side of the neck, left side of the neck, and right lower side of the neck for the 1st, 2nd and 3rd administration respectively.
Clinical studies 1 and 2 (Fig. 1B C): clinical study 1 enrolled eighty fattening cross-bred gilts, and clinical study 2 enrolled forty fattening cross-bred gilts. In both studies, gilts were randomly allocated into two treatment groups (control or anti-GnRF analog). Two doses of anti-GnRF analog or saline were administered at two different inter-dose intervals (IDI): 14 and 18 weeks of age (clinical study 1: four-week IDI), or 10 and 18 weeks of age (clinical study 2: eight-week IDI). Both treatment groups were commingled throughout the studies. Vaccination was performed with 2 mL of either saline or anti-GnRF analog subcutaneously with a 16 G ¾ needle and a Simcro Sekurus safety device (Simcro, UK). They were administered in the right side of the neck, and left side of the neck for the 1st and 2nd administration respectively.
All animals were fed with an age-appropriate feed, and both feed and water were provided ad libitum.
Experimental design of the studies including timing of vaccination (safety gun), blood sampling (blood tube), oestrus detection, and slaughter (pig icon) and: (A) pre-clinical study (n = 57) with early (A1) and late priming (A2), (B) the 1st clinical study with late priming (n = 80), and (C) 2nd clinical study with early priming (n = 40)
Measurements and sample collection
Oestrus detection: gilts were observed three times per week for signals of oestrus from two weeks after the last administration until the end of each study (26 weeks of age for the pre-clinical study, and 27 weeks of age for the clinical studies). Animals from each pen were moved to a central area, where 1 out of 3 adult boars was present. Boars were alternated per oestrus check. After a 5-minute accustoming period, signs of standing oestrus were checked with every gilt. Oestrus detection was assessed by rubbing the flank and pressing the back of the gilt in the presence of the boar. Gilts were deemed to be in oestrus when they remained in standing response, immobile with arched back, and cocked ears.
Serum collection: Blood samples were collected before each vaccination and then approximately every two weeks post 2nd vaccination until the end of the study for the pre-clinical and clinical study 2. For clinical study 1, blood samples were taken before each vaccination and then every 28 days until the end of the study (included). Blood samples were kept for at least two hours at room temperature and then centrifuged for ten minutes at 2500 g ± 100 g at room temperature. The serum was stored at -80 ± 10ºC until further analysis.
Reproductive tract examination: at the end of each study, at slaughter, the uteri and ovaries were removed and weighed, and the length of the uterine horns was recorded. Each ovary was scored in relation with the size of the developing follicles and corpus luteum. The scoring system used: 0 (no follicles); 1 (immature follicles, 3–4 mm); 2 (mature follicles, 5–11 mm); and 3 (mature follicles and luteal tissue).
Laboratory methods
Anti-GnRF antibodies in serum: measured with a noncompetitive ELISA test [9], which uses GnRF peptide as the capture phase. Dilutions of pig sera were added to plates coated with GnRF peptide. After several washes, the plates were incubated with horseradish peroxidase-labeled anti-swine immunoglobulin. Finally, 3,3’,5,5’-tetramethylbenzidine substrate was added. After a short incubation time, the enzyme reaction was stopped, and the color generated was measured spectrophotometrically. The amount of color produced was directly proportional to the level of antibodies present in the sample. The limit of quantification (18.56 U/mL) had been previously determined in a validation study. The analysis was performed in the facilities of Zoetis Inc. (Kalamazoo, USA).
Progesterone and estradiol levels in serum: analyzed by High Performance Liquid Chromatography (HPLC) – Mass Spectrometry (MS/MS) method. Progesterone-D9 and Estradiol-D3 were used as internal standards and swine serum as the matrix. The analytical range was from 10.0 to 10,000 pg/mL for both. The analyses were performed in the facilities of Zoetis Inc (Kalamazoo, USA).
Statistical analysis
Data summaries and analyses were performed with a centralized data management system (SAS/STAT User’s Guide V. 9.4, SAS Institute, Cary, NC, USA). The individual animal was the experimental unit for all the statistical analyses.
The weight of the uterus, the sum of the weight of both ovaries and mean uterine horn length were analysed using a general linear mixed model. The fixed effect in the model was the treatment. The random effects were batch (for the clinical studies), block within batch (for the clinical studies) and residual. Treatment least squares mean, standard errors, 95% confidence interval, minimums and maximums were calculated. Contrasts were used to compare treatments within each study. Frequency distributions of follicle sizes (scores) were calculated for each treatment group within each study.
The presence or absence of at least one standing oestrus post-last vaccination (2nd for the clinical studies and last administration for the pre-clinical study) was analysed using the Cochran-Armitage test adjusting for block. Frequency distributions of the variable were calculated for each treatment group. Treatments within study were compared using a contrast.
At each time point, the proportions of pigs with anti-GnRF titers below the level of quantification in each treatment group were calculated. The data was logarithm transformed prior to analysis and then analyzed using a general linear mixed model for repeated measures, with treatment, time point and their interaction as fixed effects, and random effects of batch (for the clinical studies), block within batch (for clinical studies), animal within batch (for the clinical studies), block and treatment, and residual. Treatment comparisons within study were made at each time point using contrasts. For animals with GnRF antibody titers below the level of quantification, these were defined as half the level of quantification and included in the analyses. The treatment least squares means, standard error and 95% confidence intervals were back-transformed for each time point. Treatment minimums and maximums were also calculated for each time point.
The number and percentage of pigs with levels below the limit of quantification for progesterone and estradiol were calculated for each treatment and each time point. The progesterone and estradiol levels were analysed the same way as the anti-GnRF values except that they were not transformed.
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