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Analysis of the occurrence of freemartinism among single female calves born from multiple embryo transfer
Theriogenolog
y 39:239,
1993
ANALYSIS OF THE OCCURRENCE OF FREEYARTINISM AMONG SINGLE FEMALE CALVES BORN FROM MULTIPLE EMBRYO TRANSFER H. Kadokawa, M. Minezawa, H. Takahashi, 0. Sasaki and T. Kariya Department of Animal Industry Hokkaido National Agricultural Experiment Station Sapporo 062, Japan Induction of twin-birth by embryo transfer is an efficient method to obtain a higher number of calves for beef cattle production, but it is expected to increase freemartinism. Owing to strong demand for females with normal reproductive function, especially in the Japanese Black breed cattle, an investigation of the occurrence of freemarttnism among single females produced by multiple embryo transfer was carried out. Chromosome typing and PCR (Polymerase Chain Reaction) amplification of male-specific DNA were chosen to detect freemartins. Leukocyte DNA was prepared for PCR by Higuchi’s rapid method from whole blood (Higuchi, 1969, Perkin EimerlCetus Newsletter 21) and the primer pair was synthesized from the sequence of MDYZ (Perret et at., 1990, Genomics, 6M2-490). PCR was carried out for 1 min at 95°C 1.5 min at 60°C and for 1.5 min at 72°C in 30 cycies in lOOt.11of reaction mixture containing 600 ng of sampfe DNA. The presence of the expected size of PCR products was confirmed by ethidium bromide staining on a 2.0% agarose gel. With this method, the male-specific DNA couM even be detected from a mixture having 1 part mate in 10000 parts of femaie+male DNA. The greater sensitivity of this method than chromosome typing (in 100 ceils) was confirmed in another experiment using 17 females from male-female twins. Twenty-two single females born by non-surgical twin embryo transfer were later investigated for the occurrence of freemartinism by both chromosome typing and PCR methods. Df these, 21 single females (95.5%) were classified as normal (i.e., no male cell among 100 metaphase spreads in chromosome typing, and mate-spedfic DNA negative in PCR) by both methods. One female (4.5%) was, however, classified as a freemattin by PCR (male-specific DNA positive), but it was dassified as normal cytogeneticaily. This meant that the individual had a low degree of heterosexual chimerlsm and that the low degree of freemartinism derived from multiple embryo transfer was difficult to diagnose by ordinary chromosome typing, while it was detectable by PCR. The genital tracts of all females were found to be normal by rectal examination. f3ased on these results, it could be conduded that most single females born from multiple embryo transfer are expected to have normal reproductive function.
Copyright (Q 1993 Butterworth-Heinemann
239
y 39:239,
1993
ANALYSIS OF THE OCCURRENCE OF FREEYARTINISM AMONG SINGLE FEMALE CALVES BORN FROM MULTIPLE EMBRYO TRANSFER H. Kadokawa, M. Minezawa, H. Takahashi, 0. Sasaki and T. Kariya Department of Animal Industry Hokkaido National Agricultural Experiment Station Sapporo 062, Japan Induction of twin-birth by embryo transfer is an efficient method to obtain a higher number of calves for beef cattle production, but it is expected to increase freemartinism. Owing to strong demand for females with normal reproductive function, especially in the Japanese Black breed cattle, an investigation of the occurrence of freemarttnism among single females produced by multiple embryo transfer was carried out. Chromosome typing and PCR (Polymerase Chain Reaction) amplification of male-specific DNA were chosen to detect freemartins. Leukocyte DNA was prepared for PCR by Higuchi’s rapid method from whole blood (Higuchi, 1969, Perkin EimerlCetus Newsletter 21) and the primer pair was synthesized from the sequence of MDYZ (Perret et at., 1990, Genomics, 6M2-490). PCR was carried out for 1 min at 95°C 1.5 min at 60°C and for 1.5 min at 72°C in 30 cycies in lOOt.11of reaction mixture containing 600 ng of sampfe DNA. The presence of the expected size of PCR products was confirmed by ethidium bromide staining on a 2.0% agarose gel. With this method, the male-specific DNA couM even be detected from a mixture having 1 part mate in 10000 parts of femaie+male DNA. The greater sensitivity of this method than chromosome typing (in 100 ceils) was confirmed in another experiment using 17 females from male-female twins. Twenty-two single females born by non-surgical twin embryo transfer were later investigated for the occurrence of freemartinism by both chromosome typing and PCR methods. Df these, 21 single females (95.5%) were classified as normal (i.e., no male cell among 100 metaphase spreads in chromosome typing, and mate-spedfic DNA negative in PCR) by both methods. One female (4.5%) was, however, classified as a freemattin by PCR (male-specific DNA positive), but it was dassified as normal cytogeneticaily. This meant that the individual had a low degree of heterosexual chimerlsm and that the low degree of freemartinism derived from multiple embryo transfer was difficult to diagnose by ordinary chromosome typing, while it was detectable by PCR. The genital tracts of all females were found to be normal by rectal examination. f3ased on these results, it could be conduded that most single females born from multiple embryo transfer are expected to have normal reproductive function.
Copyright (Q 1993 Butterworth-Heinemann
239