Cloning method using oocytes matured in vivo

Abstract

A method is described of selecting a porcine oocyte or a population of oocytes with improved developmental competence following either in vitro or in vivo maturation.

1 . A method of producing a porcine oocyte optimised for developmental competence, the method comprising maturing a harvested oocyte in vitro for between about 36 to about 48 hours.  
     
     
         2 . A method as claimed in  claim 1 , in which the in vitro maturation is for between about 42 to about 46 hours  
     
     
         3 . A method as claimed in  claim 1  or  claim 2 , in which the in vitro maturation is for between about 43 to about 45 hours.  
     
     
         4 . A method of producing a porcine oocyte optimised for developmental competence, the method comprising harvesting an oocyte from a pig from about 42 to about 50 hours post-injection with a gonadotropin hormone, wherein the oocyte was ovulated from about 42 to about 50 hours post-gonadotropin injection.  
     
     
         5 . A method as claimed in  claim 4 , in which the oocyte is harvested from the pig from about 44 to about 48 hours post-injection with a gonadotropin hormone, wherein the oocyte was ovulated from about 44 to about 48 hours post-gonadotropin injection.  
     
     
         6 . A method as claimed in  claim 4  or  claim 5 , in which the oocyte is harvested from the pig from about 45 to about 47 hours post-injection with a gonadotropin hormone, wherein the oocyte was ovulated from about 45 to about 47 hours post-gonadotropin injection.  
     
     
         7 . A method as claimed in any one of  claims 4  to  6 , in which ovulation of the oocyte is detected using transcutaneous ultrasonography.  
     
     
         8 . A method of harvesting an oocyte from a porcine mammal in response to the administration of a gonadotropin hormone, the method comprising administration of the gonadotropin hormone towards the end of the diurnal rhythm of the porcine mammal, and subsequently harvesting the ovulated oocyte.  
     
     
         9 . A method of enhancing the developmental competence of a harvested porcine oocyte, the method comprising administration of a gonadotropin hormone to a porcine mammal towards the end of the diurnal rhythm of the animal prior to harvesting the oocyte such that ovulation is delayed.  
     
     
         10 . A method of nuclear transfer, the method comprising the introduction of a nucleus from a donor cell into a porcine oocyte produced by a method as defined in any one of  claims 1  to  7 .  
     
     
         11 . A method of in vitro production of a porcine embryo, the method comprising the step of in vitro fertilization (IVF) of an oocyte produced by a method as defined in any one of  claims 1  to  7 .  
     
     
         12 . A composition of porcine oocytes selected for optimal developmental competence, wherein the oocytes substantially comprise oocytes matured in vitro for between about 36 to about 48 hours.  
     
     
         13 . A composition as claimed in  claim 12 , in which the oocytes substantially comprise oocytes matured in vitro for between about 42 to about 46 hours.  
     
     
         14 . A composition as claimed in  claim 12  or  claim 13 , in which the oocytes substantially comprise oocytes matured in vitro between about 43 to about 45 hours.  
     
     
         15 . A composition as claimed in  claim 12 , in which at least about 50% of the oocytes were matured in vitro for about 36 to about 48 hours.  
     
     
         16 . A composition of porcine oocytes selected for optimal developmental competence, wherein the oocytes substantially comprise oocytes harvested from at least one pig from about 42 to about 50 hours post-injection with a gonadotropin hormone, wherein the oocyte was ovulated from about 42 to 50 hours post-gonadotropin injection.  
     
     
         17 . A composition as claimed in  claim 16 , in which the oocytes substantially comprise oocytes harvested from the at least one pig from about 44 to about 48 hours post-injection with a gonadotropin hormone, wherein the oocytes were ovulated from about 44 to about 48 hours post-gonadotropin injection.  
     
     
         18 . A composition as claimed in  claim 16  or  claim 17 , in which the oocytes substantially comprise oocytes harvested from the at least one pig from about 45 to about 47 hours post-injection with a gonadotropin hormone, wherein the oocytes were ovulated from about 45 to about 47 hours post-gonadotropin injection.  
     
     
         19 . A composition as claimed in  claim 16 , in which at least about 50% of the oocytes were harvested from the at least one pig from about 42 to about 50 hours, post-injection with a gonadotropin hormone, wherein the oocytes were ovulated from about 42 to about 50 hours post-gonadotropin injection.  
     
     
         20 . A composition as claimed in any one of  claims 16  to  19 , in which ovulation of the oocyte is detected using transcutaneous ultrasonography.  
     
     
         21  A method of producing a porcine oocyte optimised for developmental competence, the method comprising: 
 (i) harvesting a cumulus oocyte complex (COC) from a follicle of a pig ovary; and  
 (ii) maturing said COC in a physiologically acceptable culture medium comprising a gonadotropin hormone for between about 36 hours to about 48 hours.  
 
     
     
         22 . A method of producing a porcine oocyte optimised for developmental competence, the method comprising: 
 (i) administering progesterone or an analogue thereof to a pig for 18 days to establish a reference heat in the animal;    (ii) thirteen to sixteen days subsequently administering to the animal a first injection of Pregnant Mares Serum Gonadotropin (PMSG);    (iii) after a time period of about 88 hours administering to the animal a second injection of human chorionic gonadotropin (hCG); and    (iv) harvesting an oocyte from a pig from about 42 hours to about 50 hours post-injection with hCG, wherein the oocyte was ovulated from about 42 hours to 50 hours post-hCG injection as determined by transcutaneous ultrasonography.    
     
     
         23 . A method of producing a porcine oocyte optimised for developmental competence, the method comprising: 
 (i) administering to the animal progesterone or an analogue thereof for four or five days at days 11 to 15 of the estrous cycle;    (ii) subsequently administering to the animal PMSG one day later and hCG four days later to cause ovulation of an oocyte; and    (iii) harvesting an oocyte from a pig from about 42 to about 50 hours post-injection with hCG, wherein the oocyte was ovulated from about 42 to 50 hours post-hCG injection as determined by transcutaneous ultrasonography.    
     
     
         24 . The use of separate first and second administrations of gonadotropin hormone to harvest an oocyte from a porcine mammal in which the first administration of gonadotropin hormone is towards the end of the diurnal rhythm of the animal.  
     
     
         25 . The use of a gonadotropin hormone in the manufacture of an agent for the harvesting of an oocyte from a porcine mammal whereby the gonadotropin hormone is administered towards the end of the diurnal rhythm of the animal.  
     
     
         26 . A kit comprising a gonadotropin hormone wherein the kit contains instructions for administration of the gonadotropin hormone to a porcine mammal for harvesting an oocyte from the animal in which administration of the hormone is towards the end of the diurnal rhythm of the animal.  
     
     
         27 . A kit comprising a first gonadotropin hormone and a second gonadotropin hormone wherein the kit contains instructions for administration of the gonadotropin hormones to a porcine mammal for harvesting an oocyte from the animal in which administration of the first hormone is towards the end of the diurnal rhythm of the animal and administration of the second hormone is separated from the first administration by about three days.