Onset of sexual maturity in female Göttingen minipigs & consequences for toxicity studies
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1 Onset of sexual maturity in female Göttingen minipigs & consequences for toxicity studies Birgit Peter Eveline e Rijk Helle Lorentsen*, Adrian Zeltner* *Joint project with Ellegaard Göttingen Minipigs A/S
2 Background Göttingen minipigs are increasingly used in general and reproduction toxicology studies. Typically, females are in the age of 4-5 months at study start. Females are believed to reach sexual maturity at approximately 5 months (BW ± kg). However, no supportive experimental data are available. This is critical information when studying compounds which may affect female reproductive functioning. 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
3 Study 1 Find a useful parameter to detect estrous in adult female Göttingen minipigs 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
4 Material and Methods 3 adult female Göttingen minipigs (10-11 months) Housed adjacent to adult boars Observations: Rectal body temperature daily Signs of heat (specific behavior, changes in vulvar appearance) daily Vaginal smears for estrous cycle determination daily Measurement of the reproductive hormones progesterone and 17β-estradiol in serum twice weekly 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
5 (1) Testing different Parameters for Heat in a Mature Sow P 11 P 12 E/M P/E P/E ay of Study and Stage of Estrous Cycle (Proestrus, Estrus, Metestrus, iestrus) Restlessness Ear straightening Rubbing Mounting P/E E/MM/M/ Immobility Redness/swelling of vulva Increased mucus Rectal temp. ( C) 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
6 (2) Testing different Parameters for Heat in a Mature Sow P 11 P 12 E/M P/E P/E ay of Study and Stage of Estrous Cycle (Proestrus, Estrus, Metestrus, iestrus) P/E E/MM/M/ Restlessness Ear straightening Rubbing Mounting Immobility Redness/swelling of vulva Increased mucus Rectal temp. ( C) 17β-Estradiol (pg/ml) 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
7 (3) Testing different Parameters for Heat in a Mature Sow P 11 P 12 E/M P/E P/E P/E E/MM/M/ ay of Study and Stage of Estrous Cycle (Proestrus, Estrus, Metestrus, iestrus) Restlessness Ear straightening Rubbing Mounting Immobility Redness/swelling of vulva Increased mucus Rectal temp. ( C) Progesterone (ng/ml) 17β-Estradiol (pg/ml) 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
8 Study 2 Onset of progesterone release in young female Göttingen minipigs - Pilot study 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
9 Material and Methods 2 immature female Göttingen minipigs (2.5 months) Housed in the same room as adult boars Blood sampling for progesterone analysis twice weekly 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
10 70 Onset of Progesterone Release in Young Female Minipigs 60 Progesterone (ng/ml) ,5 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 8 Age (months) Animal A Animal B (died at the age of 7 months) 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
11 Summary & Conclusions (Studies 1 + 2) Progesterone analysis seems to be an useful method for detection of estrous cycle. First onset of progesterone release at approximately 5.5 and 6.5 months of age (BW: 12.3 and 17.3 kg) Other parameters, like external signs of heat, cytology of vaginal smears, rectal body temperature and release of 17β-estradiol were not useful. 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
12 Study 3 Onset of progesterone release in young female Göttingen minipigs - Main study 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
13 Material and Methods 14 young female Göttingen minipigs (3-4 months) Housed in the same room as adult boars etermination of progesterone levels in serum twice weekly Necropsy after completion of 1-2 cycles of progesterone release or, if negative, at 7 months of age Histopathology of ovaries, uterus and vagina 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
14 Age and body weight at first progesterone peak Body weight (kg) Age (months) 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
15 Summary In 7/14 sows the first progesterone peak was measured at months of age ( kg) In 6/14 sows, progesterone release started much later, i.e. at months of age ( kg) In one sow no progesterone was detected until necropsy at the age of 7 months. 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
16 Progesterone peaks of 12 different females 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
17 Progesterone figures show: A cycle length of days. Since the cycles were very regular, the next peak(s) could be predicted based on the former peak(s). Therefore, necropsy on different time points within the cycle could well be established based on the information of the previous peak(s). To confirm that the first progesterone peak is directly correlated to the onset of sexual maturity a morphologic examination of the reproductive organs is needed. 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
18 Morphology: sections of ovary-uterus-vagina Myometrium Glands Lumen Lumen 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
19 Best morphologic proof for sexual maturity 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
20 Is first progesterone peak is directly correlated to the onset of sexual maturity? Corpora lutea - degenerating Corpora lutea mature Corpora lutea degenerating (previous cycle) Corpora lutea mature Corpora lutea degenerating (previous cycle) Corpora albicans (old corpora lutea from two cycles ago) YES 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
21 Composition of the Estrous Cycle in the Minipig..ProE E MetE ie ProE.. 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
22 Ovaries: from follicular to late luteal phase Low magnification GF: Graafian follicle; dc: degeneration CL; df: disruption Follicle; nc: newly formed CL CL: mature corpus luteum; tf: tertiary follicle 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
23 Ovaries: from follicular to late luteal phase High magnification Follicular Ovulation early Luteal mid Luteal late Luteal First cycle Second cycle 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
24 Uterus: from proliferative to late secretory phase Proliferation Ovulation early Secretory mid Secretory late Secretory Low magnification S L L G G S M M 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
25 Uterus: from proliferative to late secretory phase High magnification Proliferation Ovulation early Secretory mid Secretory late Secretory 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
26 Vagina: from proliferative to late secretory phase Proliferation Ovulation early Secretory mid Secretory late Secretory 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
27 Animal without progesterone peak (7 months) Immature Mature 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
28 Conclusions Progesterone measurements are useful to determine the onset of ovulation (sexual maturity) in female minipigs. The variation in the age of sexual maturity is much higher than expected ( months); a better chance for sexual maturity will be at the age of 6.5 months. The stage of female reproductive cycle in the minipig can be determined by the morphologic characteristics of mainly the ovary and the uterus. Addition of estrous cycle staging by uterine/ovarian histopathology further enhances reliable evaluation of reproductive system function in toxicity studies. 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
29 References Tortereau A., Howroyd P., Lorentsen H. (2013) Onset of puberty and normal histological appearances of the reproductive organs in peripubertal female Göttingen minipigs. Toxicol Pathol.41(8): E. de Rijk, H. v.d. Brink, J. Lensen, A. Lambregts, H. Lorentsen, B. Peter (in press) Estrous cycle-dependent morphology in the reproductive organs of the female Göttingen Minipig. Toxicol Pathol. THANK YOU FOR YOUR ATTENTION 14 November BSTP/ACCP/MRF WIL Research Europe en Bosch
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