Title of Invention

"MALE-SPECIFIC PROTEIN OF INDIAN MAJOR CARP, LABEO ROHITA (HAM.)-A KEY FOR SEX DIFFERENTIATION AND BROOD STOCK MANAGEMENT"

Abstract

In non-breeding seasons brood stock management is very much essential to ensure the stocking of appropriate number of males and females for effective breeding in breeding season. But, in non-breeding seasons, sex differentiation of Indian major carps (L. rohita) is impossible without sacrificing the fish, as no specific morphological or genetic markers are reported. On analysis of serum and mucus samples from all seasons, a monomeric glycoprotein of molecular weight 17.85 kDa was detected in male samples only. The male specificity nature of the protein was confirmed through western blotting and immunostaining. Thus, in the process we have discovered a protein which is specific to male Indian major carps (L. rohita) and present in all seasons thereby a key for sex differentiation in non-breeding seasons.

Full Text

a) Nature of the invention □ A male-specific protein of Indian major carp, Labeo rohita, (Ham.), that helps in sex differentiation in all season, is identified for off season brood stock management. □ This male-specific protein is a glycoprotein monomer of molecular weight 17.85 kDa and is distributed in blood serum and scale mucus of male fish. □ This male specific protein is neither detected in semen nor in gonadal extract suggesting its non-gonadal origin. □ Detection of this protein either in fish serum or mucus helps in identification of the male and their segregation. □ Sex differentiation using this innovative protein marker will be of enormous use for selecting and maintaining optimum brood stocks, that will be helpful for effective breeding and thereby higher seed production. It will also reduce the cost of keeping more males. b) Prior art: Indian major carp (L. rohita) enjoys a prime position in the Indian aquaculture scenario. Higher seed production depends mainly on off season brood stock management. In seasons other than breeding season, sex differentiation is difficult in case of Indian major carp (L. rohita). Thus, it is impossible to differentiate between males and females in seasons other than breeding season. Male and female brood stock identification is necessary for their procurement, marketing, selective breeding and other commercial and experimental purposes. Therefore, segregation of males and females in seasons other than breeding season is necessary for stocking optimum number of males and females that will be helpful for effective breeding in breeding season. Till date, no specific morphological, biological, biochemical or genetic markers for off season sex differentiation in Indian major carp {L. rohita) are reported. Thus, a sensitive and accurate sex differentiating marker of Indian major carp (L. rohita) is in high demand of the time and would help in differentiating sex of Indian major carps (L. rohita) and thereby help in proper off season brood stock management. c) Details of the invention: Blood, mucus, semen and other tissue samples have been collected from Indian major carp {L. rohita) in breeding and non-breeding seasons. Protein profiling of serum and mucus, milt and tissue extracts have been done through polyacrylamide gel electrophoresis (gel concentration ranging from 8-12.5%) under native, reducing and non-reducing conditions. Periodic acid Schiff (PAS) staining of gels have been done to confirm the glycoprotein nature of the polypeptide. The male specificity of the protein has been confirmed through western blotting and immunostaining with anti-rohu male serum antibodies raised in rabbit, sufficiently adsorbed with female serum and antibodies raised against purified male-specific serum protein component. The above study yielded a polypeptide of molecular weight 17.85 kDa in 12.5% SDS-PAGE. The protein was evident only in serum and mucus samples, collected from male fish in all seasons. The protein was neither detected in semen nor in gonadal extracts suggesting its non-gonadal origin, which is to be investigated. The protein was not reduced in 2-mercaptoethanol and was found to be a monomelic unit. Serologically the protein was found to be specific for male. So far no markers are available for sex differentiation of Indian major carp (I. rohita). Herein, after using this invention one can differentiate males from females in case of Indian major carp (L. rohita) in non-breeding seasons and stock optimum number of male and female for effective breeding. The cost of keeping more male fish can also be reduced substantially in small farm holdings. Since, the protein has been found in the mucus, it becomes easier for sex determination at any time without causing any harm to the fish. Please see Appendix-1 for further details. (Figure Removed) We claim: 1. A process for sex differentiation in L. rohita, said process comprises: - performing SDS PAGE or western blotting on a blood sample or mucus sample obtained from L. rohita; and - identifying presence or absence of the protein of molecular weight 17.85 KDa in the SDS PAGE or western blot, wherein the presence of said protein is indicative of male sex of L. rohita, wherein said protein is non-detectable in semen or gonads.

Documents:

1116-DEL-2005-Abstract-(31-05-2011).pdf

1116-del-2005-abstract.pdf

1116-DEL-2005-Claims-(29-03-2012).pdf

1116-DEL-2005-Claims-(31-05-2011).pdf

1116-del-2005-claims.pdf

1116-DEL-2005-Correspondence Others-(04-11-2011).pdf

1116-DEL-2005-Correspondence Others-(29-03-2012).pdf

1116-DEL-2005-Correspondence Others-(31-05-2011).pdf

1116-del-2005-correspondence-others.pdf

1116-del-2005-description (complete).pdf

1116-DEL-2005-Form-1-(31-05-2011).pdf

1116-DEL-2005-Form-1.pdf

1116-del-2005-form-13.pdf

1116-del-2005-form-18.pdf

1116-del-2005-form-2.pdf

1116-del-2005-form-26.pdf

1116-DEL-2005-Form-3-(31-05-2011).pdf

1116-DEL-2005-Form-5-(31-05-2011).pdf

1116-DEL-2005-GPA-(31-05-2011).pdf