Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection

Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, 505 185th Avenue, Beaverton, Oregon, OR 97006, USA.
Theriogenology (Impact Factor: 1.8). 06/2005; 63(9):2356-71. DOI: 10.1016/j.theriogenology.2004.05.033
Source: PubMed


The objective was to develop a sperm freezing procedure suitable for use in the propagation of valuable founder animals by assisted reproductive technologies. Here, we report a comparison of processing methods by measuring the motility of fresh and frozen-thawed rhesus monkey spermatozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed post-thaw with or without a density gradient centrifugation step. Among the four study series, motility post-thaw was improved with density gradient centrifugation (17-24% versus 75%, P<0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total n=377) were co-cultured on BRL cells and observed for fertilization and development. With spermatozoa frozen in straws in liquid nitrogen vapors, the fertilization rate after ICSI was lower than with fresh spermatozoa (40-44% versus 77-86%, P<0.05), even with the Percoll-enriched fraction that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date. Additionally, a 3-4 h incubation after thawing improved the fertilization rate with spermatozoa from a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate after ICSI with motile, frozen-thawed primate spermatozoa was observed comparable to that obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used with ICSI to facilitate propagation of desirable primate genotypes.

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Available from: Shoukhrat Mitalipov, Mar 26, 2015
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    • "In rhesus monkeys, the use of cryopreserved sperm in standard, intravaginal artificial insemination (AI) has not been successful and live births have been reported only through intrauterine insemination (Sá nchez-Partida et al, 2000) or intracytoplasmic sperm injection (ICSI; Yeoman et al, 2005). These latter techniques are not readily available at most primate colonies around the world; therefore, propagation by using frozenthawed sperm with standard AI would be very valuable for practical use. "
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    ABSTRACT: Sperm cryopreservation in rhesus monkeys has not been successful when applied in standard, intravaginal artificial insemination; thus, there is a need for substantial improvement in current cryopreservation protocols. The present study was part of our systematic approach to optimize the cryopreservation procedure. Specifically, we tested whether modification of the concentration of egg yolk, the dilution method, and the time delay between ejaculation and adding egg yolk would result in significant improvement of postthaw motility for ejaculated sperm. We also tested the effects of presence and absence of egg yolk on cryopreservation of ejaculated and epididymal sperm. Our findings indicated that the concentration of egg yolk (2%-50%, vol/vol), the dilution method, and the delay (1-5 hours) in addition of egg yolk had no significant effect on postthaw motility of ejaculated rhesus monkey sperm. The presence of egg yolk yielded significantly higher motility after thawing than samples without egg yolk for ejaculated and epididymal sperm. The present study suggests that as long as egg yolk is present in the extender, details such as the amount of egg yolk, as well as when and how to add the egg yolk, have little impact on the ultimate freezing outcomes for ejaculated rhesus monkey sperm. We also discuss the possible mechanism of the protective role of egg yolk in sperm cryopreservation.
    Preview · Article · Feb 2009 · Journal of Andrology
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    • "In fact, a recent study with rhesus monkey sperm has shown that there were no significant differences in chromosome damage between fresh sperm and frozen-thawed sperm when motile sperm were selected for ICSI (Li et al, 2007). In addition to percentage motility, recording the forward progression scale (Yeoman et al, 2005) and the sperm longevity may provide a more accurate estimation of sperm quality after thawing. Future studies should evaluate the possibility of developing a composite index that incorporates the progressive scale into the motility estimation as well as the sperm longevity after thawing. "
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    ABSTRACT: Recently, there has been an increased interest in preservation of epididymal sperm as a potential source of material for genetic resource banking; however, cryopreservation of epididymal sperm from the rhesus monkey has not been explored. This study evaluated the effect of prolonged refrigerated storage of the intact cauda epididymides at various conditions on the postthaw motility of rhesus monkey epididymal spermatozoa, and also tested whether altering cryoprotectants and cooling methods could improve post-thaw motility for epididymal sperm after refrigerated storage. Motility before freezing decreased significantly after refrigerated storage (0 degrees C) for a period of 24 or 48 hours. Although postthaw motility was not significantly different after 24 hours of refrigerated storage, epididymides stored at a higher temperature (4 degrees C-10 degrees C) yielded better results, but postthaw motility still decreased significantly after 48 hours of refrigerated storage at 4 degrees C. Comparisons of glycerol and ethylene glycol at 3% and 6% revealed similar postthaw motility. However, consistently high postthaw motility was obtained with 3% glycerol throughout all freezing trials regardless of whether samples were collected fresh or after refrigerated storage for 24 or 48 hours. Cooling at a higher rate of 220 degrees C/min was found to yield better postthaw motility than the slower rate of 29 degrees C/min. Thawing time duration was evaluated, and a minimum of 30 seconds was required for thawing 0.25-mL straws containing 50-microL semen samples. An overall average of 42% postthaw motility was obtained for rhesus monkey epididymal sperm packed in 3% glycerol and cooled after 24 or 48 hours refrigerated storage. These postthaw motility results for epididymal sperm indicate that this method should be practical for use in preserving epididymal sperm, even if tissue must be shipped from sites remote from the cryopreservation laboratory.
    Preview · Article · Jan 2008 · Journal of Andrology
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    ABSTRACT: Fresh and frozen-thawed rhesus monkey sperm were analyzed for DNA damage using the comet assay and for chromosome damage by cytogenetic analysis after intracytoplasmic sperm injection (ICSI) into mouse oocytes. The percentage of fresh sperm with damaged DNA in ejaculated semen was 0 to 2.7% (n = 5). Conventional cryopreservation and storage in liquid nitrogen caused DNA damage in 25.3% to 43.7% of sperm; when sperm were frozen without cryoprotectants, 52.7% to 92.0% of thawed sperm had DNA damage. However, no significant difference in chromosome damage was found between fresh sperm and frozen-thawed sperm when motile sperm were selected for ICSI. The percentage of sperm with abnormal karyotypes ranged from 0 to 8.3%. The most common structural chromosomal abnormalities in fresh motile sperm and frozen-thawed motile sperm were chromosome breaks or fragments. Our findings suggest that genetically competent frozen-thawed macaque sperm can be selected for fertilization by using only motile sperm for ICSI.
    Full-text · Article · Feb 2007 · Journal of Andrology
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