There is a synergism between Assisted Reproductive Technology research and understanding basic biology of gametes and embryos. In the latter category, major efforts of ARBL faculty include study of membrane composition and capacitation of sperm. Regulation of oocyte maturation is another area of interest, particularly cytoplasmic maturation while holding nuclear maturation in abeyance. Considerable efforts are being made in studying regulation of energy metabolism of equine and bovine embryos, including flux of substrates through the pentose phosphate pathway. Gene expression in elongating bovine embryos is another area of active research. Summaries of several specific projects follow.
The fertility of cryopreserved bull spermatozoa is sufficiently high to permit its routine use in commercial settings. This is not true of spermatozoa from other species. The objectives of these projects are to develop methods to improve cryopreserving sperm from several species.
Dewit M, Marley WS, Graham JK. 2000. Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs. Cryobiology 40:36-45.
Brinsko SP, VanWagner GS, Graham JK, Squires EL. 2000. Motility, morphology and triple stain analysis of fresh, cooled and frozen-thawed stallion spermatozoa. J Reprod Fert, Suppl 56:111-120.
Crockett EC, Graham JK, Bruemmer JE, Squires EL. 2001. Effect of cooling of equine spermatozoa before freezing on post-thaw motility: Preliminary results. Theriogenology 55:793-803.
A rapid laboratory sperm assay that could predict the fertilizing capacity of a semen sample would be very beneficial to those utilizing semen for artificial insemination. Current semen assays do not adequately assess fertilization capacity. This is due in part to the fact that spermatozoa contain several cell compartments (nucleus, acrosome, plasma membrane, mitochondria etc.), all of which must be intact at the time of insemination for a sperm to fertilize an oocyte. However, commonly used laboratory assays of semen quality evaluate only one of these parameters (usually motility).
The objectives of this project are to develop an assay that evaluates multiple sperm parameters simultaneously and then determine how well this assay correlates with the fertility of semen. The figure at right shows three sperm that have been stained with three fluorescent stains. Sperm 1 has a functioning mitochondria (stained green) but acrosomal damage (stained yellow), sperm 2 is fully functional (mitochondria stained green and no cell damage), while sperm 3 is dead (stained red).
Parrish JJ, Susko-Parrish JL, Graham JK. 1999. In vitro capacitation of bovine spermatozoa: Role of intracellular calcium. Theriogenology 51:461-472.
Salazar P, Graham JK, Parrish JJ, Susko-Parrish J, Squires EL. 2000. Indirect determination of stallion sperm capacitation based on esterase release from spermatozoa challenged with lysophosphatidylcholine. J Reprod Fert, Suppl 56:407-414.
Many horse registries permit the use of cooled shipped semen to be used for artificial insemination purposes. The fertility of cooled horse semen, however, drops rapidly after 24 to 36 hours of storage. We believe that one reason for this decrease in fertility is due to lipid peroxidation that occurs to sperm membranes during incubation.
The objectives of this project are to determine if antioxidants can increase the time that stallion sperm remain motile during incubation at 5°C and if antioxidants can maintain sperm fertilizing capacity. The figure below demonstrates that at least one antioxidant maintains sperm motility for 48 hours. Additional antioxidants and the fertility of stored stallion sperm are currently being evaluated.
Denniston DJ, Squires EL, Bruemmer JE, Brinsko SP, McCue PM, Graham JK. 2000. Effect of antioxidants on the motility and viability of cooled stallion spermatozoa. J Reprod Fert, Suppl 56:121-126.