Mehrdad POURJAFAR, Khalil BADIEI, Hassan SHARIFIYAZDI, Seyed NAGHIB, Aliasghar CHALMEH, Mohammad DIVAR

Application of Hormonal and Single Multiplex PCR Assays for Detection of Freemartinism in a Horned Goat

ABSTRACT The present study describes a rare case of freemartinism of the 60XX/60XY chimera type in an adult 7-year-old horned, mixed Lori breed phenotypically doe. Signs of masculinisation were evident in both the behavioral patterns and body conformation, but there was no structure similar to penis and hypospadias was observed in this goat. External genital organs were similar to that of a female one. In this study, a single multiplex PCR assay was used to determine the freemartinism status (XX-XY blood chimerism) based on semi-quantitatively co amplify a sex-based polymorphism in the amelogenin locus (AMX and AMY). In addition, serum concentrations of anti-Müllerian hormone (AMH), testosterone and progesterone hormones of this horned intersex goat were evaluated. Serum AMH levels in the present case (0.2 ng/ml) were similar to male (0.2 ng/ml) and lower than female (0.6 ng/ml) goat. The serum concentrations of testosterone (0.2 ng/ml) were remarkably high in comparison to normal control male and female ones (0.02 ng/ml). Progesterone level in the intersex goat (0.4 ng/ml) was close to control buck (0.7 ng/ml) and remarkably lower than control doe (10 ng/ml). The results of the present study showed that the use of hormonal pattern, especially AMH level, and single multiplex PCR technique provide an easy and alternative approaches for indication of XX/XY chimerism or mosaicism in intersex freemartin goats. Key Words: Freemartin, XX/XY chimerism, intersex, horned goat, single multiplex PCR, anti-müllerian hormone ÖZET BOYNUZLU BİR KEÇİDE FREEMARTİNİZMİN HORMONAL VE TEKLİ MULTİPLEKS PCR ANALİZ UYGULAMALARI İLE TESPİTİ Bu çalışmada, nadir bir olgu olan freemartinizmin dişi fenotipine sahip Lori ırkı melezi 7 yaşındaki boynuzlu ergin bir keçide 60XX/XY kimera tipi bildirilmektedir. Davranış biçimlerinde ve vücut yapısında maskülinizasyon belirtileri bulunur iken, penis veya hypospadias gözlenmemiştir. Dış genital organlar dişi keçi ile benzerlik göstermektedir. Bu çalışmada amelogonin lokusundaki (AMX ve AMY) cinsiyete bağlı polimorfizmin semi-kantitatif olarak çoğaltılması ile freemartinizm durumunun (XX-XY kan kimerizmi) belirlenmesinde tekli multipleks PCR yöntemi kullanılmıştır. Ayrıca anti-Müllerian hormon (AMH), testesteron ve progesteron hormonlarının serum konsantrasyonları da değerlendirmeye alınmıştır. Mevcut vakadaki serum AMH düzeyleri (0,2 ng/ml) tekeye benzer (0,2 ng/ml) olarak, dişi keçiden (0,6 ng/ml) ise düşük olarak saptanmıştır. Serum testesteron konsantrasyonu (0,2 ng/ml) kontrol tekeye ve dişi keçiye (0,02 ng/ml) göre önemli düzeyde yüksek bulunmuştur. İnterseks keçide progesteron düzeyi (0,4 ng/ml) kontrol tekedeki düzeye (0,7 ng/ml) yakın, kontrol dişi keçideki düzeyden (10 ng/ml) ise belirgin olarak düşüktür. Mevcut çalışmanın sonuçları interseks freemartin keçilerde XX/XY kimerizmi veya mozaizmini belirlemede AMY düzeyi ve tekli multipleks PCR tekniğinin kolay ve alternatif bir yöntem olduğunu ortaya koymaktadır. Anahtar Kelimeler: Freemartin, XX/XY kimerizmi, interseks, boynuzlu keçi, tekli mutlipleks PCR, anti-müllerian hormon

Anahtar Kelimeler:

Freemartin, XX/XY kimerizmi, interseks, boynuzlu keçi, tekli mutlipleks PCR, antimüllerian hormon

PDF

___

Basrur, P.K., Kochhar, H.S., 2007. Inherited Sex Abnormalities in Goats. In: Youngquist, R.S., Threlfall, W.R. (Eds.), Current Therapy in Large Animal Theriogenology, 2nd Ed. Saunders, USA, pp. 590-594.
Batista, M., Gonzalez, F., Cabrera, F., Palomino, E. , Castellano, E. , Calero, P., Gracia, A., 2000. True hermaphroditism in a horned goat with 6OXX/6OXY chimerism. Canadian Veterinary Journal 41, 562-564.
Behringer, R.R., 1994. The in vivo roles of müllerianinhibiting substance. Current Topics in Developmental Biology 29, 171-187.
Bongso, T.A., Thavalingam, M., Murkherjee, T.K., 1982. Intersexuality associated with XX/XY mosaicism in horned goat. Cytogenetics and Cell Genetics 34, 315-319.
Bosu, W.T.K., Basrur, P.K., 1984. Morphological and hormonal features of an ovine and a caprine intersex. Canadian Journal of Comparative Medicine 48, 402-409.
Cate, R.L., Mattaliano, R.J., Hession, C., Tizard, R., Farber, N.M., Cheung, A., Ninfa, E.G., Frey, A.Z., Gash, D.J., Chow, E.P., 1986. Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal cells. Cell 45, 685-698.
Christensen, B.W., Drost, M., Troedsson, M.T., 2009. Diseases of the Reproductive System. In: Smith, B.P. (Ed.), Large Animal Internal Medicine, 4th Ed. Elsevier, pp. 1427-1470.
Cole, J. , Broadwell, M., Rogers, G., 1997. Intersexuality in a Charolais heifer. Veterinary Record 141, 656-657.
Cribiu, E.P., Chaffaux, S., 1990. Intersexuality in domestic mammals. Reproduction Nutrition Development Supplement 1, 51-61.
Dunn, H.O., Johnson, R.H., 1981. Sample size for detection of Y chromosomes in lymphocytes of possible freemartins. Cornell Veterinary Medicine 71, 297-304.
Ennis, S., Gallagher, T.F., 1994. A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus. Animal Genetics 25, 425-427.
Ennis, S., Vaughan, L. , Gallagher, T.F., 1999. The diagnosis of freemartinism in cattle using sex specific DNA sequences. Research in Veterinary Science 67, 111-112.
Fujishiro, A., Kawakura, K., Miyake, Y.I., Kaneda, Y., 1995. A fast, convenient diagnosis of the bovine freemartin syndrome using polymerase chain reaction. Theriogenology 43, 883-891.
Hafez, S.A., Huckle, W.R., Caceci, T., 2005. Anatomical, histological and genetic investigations of a sexually anomalous goat. Veterinary Record 157, 513-516.
Kevenaar, M.E., Meerasahib, M.F., Kramer, P., van de Lang-Born, B.M., de Jong, F.H., Groome, N.P., Themmen, A.P., Visser, J.A., 2006. Serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice. Endocrinology 147, 3228-3234.
McNiel, E.A., Madrill, N.J., Treeful, A.E., Buoen, L.C. , Weber, A.F., 2006. Comparison of cytogenetics and polymerase chain reaction based detection of the amelogenin gene polymorphism for the diagnosis of freemartinism in cattle. Journal of Veterinary Diagnostic Investigation 18, 469-472.
Monniaux, D., Baril, G.R., Laine, A.L., Jarrier, P., Poulin, N., Cognié, J. , Fabre, S., 2011. AntiMüllerian hormone as a predictive endocrine marker for embryo production in the goat. Reproduction 142, 845-854.
Padula, A.M., 2005. The freemartin syndrome: an update. Animal Reproduction Science 87, 93-109.
Rico, C., Fabre, S., Médigue, C., di Clemente, N., Clément, F., Bontoux, M., Touzé, J.L., Dupont, M., Briant, E. , Rémy, B., Beckers, J.F., Monniaux, D., 2009. Anti-mullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow. Biology of Reproduction 80, 50-59.
Ricordeau, G., 1981. Genetics: Breeding Plans. In: Gall, C. (Ed.), Goat Production, New York, Academic Press.
Rota, A., Ballarin, C., Vigier, B., Cozzi, B., Rey, R., 2002. Age dependent changes in plasma antiMüllerian hormone concentrations in the bovine male, female, and freemartin from birth to puberty. General and Comparative Endocrinology 129, 39-44.
Saba, N., Cunningham, N.F., Millar, P.G., 1975. Plasma progesterone, androstenedione and testosterone concentrations in freemartin heifers. Journal of Reproduction and Fertility 45, 37-45.
Seguin, B.E., Zhang, T., Buoen, L.C. , Weber, A.F., Ruth, G.R., 2000. Cytogenetic survey of Holstein bulls at a commercial artificial insemination company to determine the prevalence of bulls with centric fusion and chimeric anomalies. Journal of the American Veterinary Medical Association 216, 65-67.
Smith, M.C., Dunn, H.O., 1981. Freemartin condition in a goat. Journal of the American Veterinary Medical Association 178, 735-737.
Szatkowska, I., Zych, S., Udala, J. , Dybus, A., Aszczyk, P.B., Sysa, P., Dabrowski, T., 2004. Freemartinism: Three cases in goats. Acta Veterinaria Brno 73, 375-378.
van Rooij, I.A., Broekmans, F.J., te Velde, E.R., Fauser, B.C., Bancsi, L.F. , de Jong, F.H., Themmen, A.P., 2002. Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve. Human Reproduction 17, 3065-3071.
Visser, J.A., de Jong, F.H., Laven, J.S., Themmen, A.P., 2006. Anti-Mullerian hormone: a new marker for ovarian function. Reproduction 131, 1-9.
Yadav, B.R., 1988. Freemartinism in goat. Indian Journal of Animal Science 58, 123-126.