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United States Department of Agriculture

Agricultural Research Service

Research Project: National Animal Germplasm Program

Location: Plant And Animal Genetic Resources Preservation Research Unit

Title: A preliminary report on the impact of cryopreservation diluent on ram sperm physiology and cervical artificial insemination

Authors
item Purdy, Phil
item Spiller, Scott
item Rice, S -
item Koeoke, K -
item Mcguire, E -
item Mcguire, K -
item Stobart, S -
item Blackburn, Harvey

Submitted to: Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: June 25, 2014
Publication Date: June 25, 2014
Citation: Purdy, P.H., Spiller, S.F., Rice, S.D., Koeoke, K.L., Mcguire, E.M., Mcguire, K.E., Stobart, S., Blackburn, H.D. 2014. A preliminary report on the impact of cryopreservation diluent on ram sperm physiology and cervical artificial insemination. Symposium Proceedings. Western Section American Society of Animal Science, June 25-27, 2014, San Angelo, Texas.

Interpretive Summary: A meta-analysis of our fertility trials using Chi-square demonstrated that use of the skim-milk-egg yolk (SMEY) diluent resulted in greater fertility (42%) compared with the TRIS diluent (15%). Therefore, we hypothesized that the cryopreservation diluent utilized in these trials may have a strong impact on ram sperm function. To test this, we collected ram semen samples, diluted and cooled them, and analyzed them to determine the impact of the diluents on sperm motility and physiology. Samples diluted in SMEY had greater proportions of greater proportions of motile and progressively motile sperm and were considered of a higher quality based on physiology assays compared with the samples diluted in TRIS. These results demonstrate that diluent influences the physiology of ram sperm and in particular that TRIS may modulate cellular function. However, additional analyses are required in order to fully understand the impact of these diluents on sperm physiology and fertility.

Technical Abstract: A meta-analysis of our fertility trials using Chi-square demonstrated that use of the skim-milk-egg yolk (SMEY) diluent resulted in greater fertility (42%) compared with the TRIS diluent (15%; P < 0.05). Therefore, we hypothesized that cryopreservation diluent (SMEY or TRIS) utilized in these trials may have a strong impact on ram sperm physiology. To test this, ram semen samples (n = 3) were collected and aliquots diluted to 600 x 106 sperm/mL in either SMEY or TRIS diluents. The samples were cooled to 5 °C over 2 h. Aliquots (200 x 106 sperm) were removed and diluted in 39 °C capacitation media and maintained at this temperature for the duration of the capacitation analyses. Aliquots of the capacitating samples were removed after 180 min of incubation for computer automated semen analysis (CASA) and for flow cytometry (analysis of membrane integrities, intracellular calcium, mitochondrial activity, and phospholipid organization). The analyses were repeated after incubation of the samples in the cryopreservation diluents for 24 h at 5 °C. Samples diluted in SMEY had a greater proportions of sperm with functioning mitochondria (58% vs 41%) and were better able to accumulate intracellular calcium compared with TRIS samples (12% vs 5%), respectively (P = 0.01). Diluent did not affect plasma membrane integrity or apoptotic characteristics (P > 0.05). Diluent and holding time did not affect the proportion acrosome reacted sperm or the membrane phospholipid organization but SMEY had greater proportions of motile and progressively motile sperm (31 and 20%, respectively) compared with TRIS (21 and 10%, respectively; P < 0.001). These results demonstrate that diluent influences the physiology of ram sperm and in particular that TRIS may modulate accumulation of intracellular calcium and mitochondrial activity; both of which are necessary for capacitation to occur. However, additional analyses are required in order to fully understand the impact of diluent on sperm physiology and fertility.

Last Modified: 10/21/2014
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