Progress 10/01/99 to 09/30/04
Outputs
OBJECTIVES: To use biomolecular techniques such as polymerase chain reaction (PCR) to select ejaculates of varying X-Y chromosomal ratio for use in the sex ratio manipulation in producers' calf crops. To determine how spermatozoal separation technologies impact spermatozoal mitochondrial function. To determine what is the relationship between sperm mitochondrial function and parental energy utilization. To improve the efficacy of semen separation by the SEPDEVICE both in terms of sperm numbers, yield and sperm quality. APPROACH: Duplex gel (DG-PCR) and real time (RT-PCR) PCR technologies were compared for evaluating the effect of ejaculation frequency on percent Y-chromosome DNA bearing sperm (%YCDBS). For the DG-PCR and RT-PCR, primers were designed for single copy genes specific to the X and Y chromosomes. Freezing technologies were used as a harbinger for freezing semen in cochettes. The relationship of mitochondrial point mutations to motility (MOT) and ATP use
rate (ATPUSE) as well as an altered semen collection schedule was used to compare the bull behavior, collection efficiency and semen quality across breeds. DG-PCR and RT-PCR techniques were used for assessing of X to Y chromosome-bearing spermatozoal ratios in bull and boar ejaculates. Spermatozoal ratios were compared to sex ratios in piglet. ACCOMPLISHMENTS: The correlation between %YCDBS per ejaculate as determined by DG-PCR and that determined by RT-PCR was nonsignificant. The correlation between RT-PCR and %male piglets was moderate yet significant, while the correlation between DG-PCR and %male piglets was nonsignificant. The quality of the semen frozen in 0.5mL straws was no different from that frozen in cochettes. There were no differences between bull behavior, collection efficiency and semen quality parameters when compared between Holstein and Brahman bulls. The occurrence of point mutations in the NADH-complex 1 gene sequence was expressed as percent of disagreement
(%DISAGREE) and number of disagreements (NDISAGREE) with bovine heart mtDNA. There was a significant breed difference in both %DISAGREE and NDISAGREE. For Brahman bulls there were significant positive correlations between %DISAGREE and NDISAGREE and MOT and between these and ATPUSE were positive but negligible. For Holstein bulls, there were no significant correlations between %DISAGREE and NDISAGREE and MOT, while correlations between these and ATPUSE were negative but not significant. SIGNIFICANCE: The %YCDBS variation and PCR identification would allow ejaculate selection to alter the sex ratio of calf crops. Cochettes could be used for freezing semen for species that require a large inseminate volume. A different collection schedule was not warranted for Brahman bulls. There were fewer mtDNA mutations in Brahman and Holstein mitochondrial populations. The number of mtDNA point mutations related to MOT and metabolism. Since mitochondria are maternally inherited, use of dam pedigree
information in selection schemes could help maintain or improve the bull's fertility.
Impacts
Both PCR techniques were effective in quantifying the variation in percent Y-CBS in bull and boar ejaculates. Manipulation of percent Y-CBS via collection regime may prove valuable for altering the secondary sex ratio in animal agriculture.
Publications
- Yates,J.H., Paul,J.B., Canal,A.L. and Chandler,J.E. 2001. The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species. J. Animal Sc. 80, Suppl 2: Abst#35.
- Chandler,J.E., Harrison, C.M. and Canal, A.M. 2001. Spermatozoal Methylene Blue Reduction: an Indicator of Mitochondrial Function and its Correlation with Motility. Theriogenology.54:261-271.
- Harrison, C.M. 2000. Methylene Blue Reduction Rate:an Indicator of Mitochondrial Function in Dam/Son Pairs. MS Thesis, Louisiana State University.
- Chandler, J.E. and Canal, A.M. 2002. Collection Frequency Affects Percent Y-Chromosome Bearing Sperm, Sperm Head Area and Quality of Bovine Ejaculates. Theriogenology. 57:1327-1346.
- Yates,J.H. 2002. The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species. MS Thesis, Louisiana State University.
- Chandler, J.E., Canal, A.L., Stanley, C.C, Williams, C.C, Anderson, A.N. and Blouin, D.C. 2004. Comparisons of mutations in the NADH complex 1 gene sequence between tissues from Holstein bulls and their dams and correlations with spermatozoal motility. J. Animal Sc. 83, Suppl 2: Abst# 7753.
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Progress 01/01/03 to 12/31/03
Outputs
OBJECTIVE: Proportional assessment of X and Y chromosome-bearing spermatozoa in bull and boar ejaculates using conventional and real-time PCR techniques. ACCOMPLISHMENTS: Experiments were designed to evaluate the effects of two collection regimes on %Y-CBS. Conventional PCR combined with gel electrophoresis and image analysis was employed to determine %Y-CBS in ejaculates from two bulls collected on 7-day intervals and two bulls on 21-day intervals. Real-time PCR technology was used to quantify %Y-CBS in the same ejaculates. Boar ejaculates were also analyzed with both techniques and compared to the percent male piglets in litters resulting from the assayed ejaculates. Collection day (P<0.0001) significantly affected %Y-CBS as determined by both PCR methods. Ejaculate nested within bull (P<0.07) was significant in the conventional PCR study and collection frequency (P<0.0001) in the realtime PCR trial. Ejaculate nested within boar was highly significant
(P<0.0001) for both technologies. Boar was significant (P<0.002) in the conventional PCR study. Predicted %Y-CBS determined by real-time PCR was significantly correlated (0.52, P=0.004) to percent male piglets.
Impacts
SIGNIFICANCE: Both PCR techniques were effective in quantifying the variation in percent Y-CBS in bull and boar ejaculates. Manipulation of %Y-CBS via collection regime may prove valuable for altering the secondary sex ratio in animal agriculture.
Publications
- Yates, J.H., Chandler, J.E., Canal, A.L. and Paul. J.B. 2003. The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species. Theriogenology 60,(9):1665-1677.
- Paul, J.B. 2003. Proportional assessment of x and y chromosome-bearing spermatozoa in bull and boar ejaculates using conventional and real-time PCR techniques. A Dissertation. Louisiana State University and Agricultural and Mechanical College.
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Progress 01/01/02 to 12/31/02
Outputs
OBJECTIVE: Mitochondrial DNA (mtDNA) was extracted from semen to compare the relationship of point mutations to motility and ATP use. ACCOMPLISHMENTS: Ejaculates (n=16) were obtained via an artificial vagina from two Holstein and two Brahman bulls. Semen was processed and stored in 0.5 ml French straws. Polymerase chain reaction was used to amplify a 955 bp sequence of the NADH1complex I gene. The sequence was divided into two segments: segment 1 (SEG1) (range 3131 to 3588bp); segment 2 (SEG2) (range 3567 to 4055bp). The DNA sequence of each segment was compared to the appropriate segment of mtDNA sequence of the Bos taurus heart. The occurrence of point mutations was expressed as percent of disagreements (%DISAGREE) with heart mtDNA and percent of ejaculates with more than 1 mutation at the same position (%GT1MUT@P) in the DNA sequence. Data were analyzed by least squares and correlation methods. In the first study in the Holstein ejaculates, SEG1 had 33.0 %DISAGREE
and 60%GT1MUT@P while SEG2 had 26.6 %DISAGREE and 83.9%GT1MUT@P. Sequence analysis is currently being done on the Brahman ejaculates. The second experiment was done to detect the effects of mtDNA point mutations on sperm motility and ATP use in Holstein and Brahman bulls. Percent progressive motility was measured immediately post thaw (MOT0) and again after a 3h-37C incubation (MOT3). ATP concentration was measured by the luciferin/luciferase reaction and detected in a scintillation counter before (PMOLES0) and after the incubation (PMOLES3). The average motility of the thawed semen across all ejaculates was 34.4 %. Strong positive associations existed between motility and ATP concentrations. Correlation between MOT0 and MOT3, PMOLES3, %DISAGREE in SEG2 and %GT1MUT@P in SEG2 were significant. Significant negative correlations existed between the metabolic parameters (MOT0 and PMOLES3) and the SEG2 sequence information (%DISAGREE and %GT1MUT@P). These correlations were all greater than
70%. SIGNIFICANCE: Based on these results, there could be a relationship between the number of point mutations in mitochondrial DNA and sperm motility and metabolism. As the number of mutations increased, sperm motility and metabolism decreased. Since it is believed that the mitochondrial population in mammalian species is largely derived from the maternal cytoplasm, this could imply that dam information should be included in pedigree designs of future bulls. This would help maintain or improve the bull's fertility.
Impacts
Currently, when the pedigree of a new dairy bull is constructed, information from the dam is not directly included. Since sperm motility is a function of the mitochondria and is involved in the fertilization process, and if mitochrondria are derived from the mother, it would seem necessary to include her information in the pedigree in order to influence her son's sperm motility and thus his fertility.
Publications
- R. Paul Lang, Kenneth L. Riley, John E. Chandler and Terrence R. Tiersch. 2003. The use of dairy protocols for sperm cryopreservation of Blue Catfish, Ictalurus fucatus. Journal of the World Aquaculture Society. 34, (1):66-75.
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Progress 01/01/01 to 12/31/01
Outputs
OBJECTIVE: The objectives were to evaluate an altered semen collection schedule using the supposed behavioral differences between bovine species and to compare the relationship of point mutations to motility and ATP use. ACCOMPLISHMENTS:In the first experiment, bull behavior and semen quality parameters measured the efficiency. Holstein and Brahman bulls were collected during a morning and a night collection weekly. Ejaculates were obtained via artificial vagina over four-weeks. The first collection of the week was rotated between night and day. Sampling order and collection teams were randomized throughout the study. Bull behavior parameters included reaction time to first mount, time to ejaculation, a refractory period test, and a thrust intensity test. As a managerial factor, the numbers of handler interruptions were counted. Semen parameters measured included total volume, initial motility, concentration, post-thaw motility, percent intact acrosomes, 3-hour post
thaw motility, primary and secondary abnormalities. The second experiment was performed to detect the effects of mtDNA point mutations on sperm motility (MOT) and ATP utilization rate (ATPUSE)in Bos taurus and Bos indicus. Mitochondrial DNA (mtDNA)was extracted from semen. Polymerase chain reaction was used to amplify a 955 bp segment of the NADH1complex I gene. The segment was divided into two sequences; sequence 1 (SEQ1)(range 3131 to 3588bp); sequence 2 (SEQ2)(range 3567 to 4055bp). Each sequence was compared to the known mtDNA sequence of the Bos taurus heart. The occurrence of point mutations was expressed as percent of disagreement (%DISAGREE) with heart mtDNA and number of disagreements (NDISAGREE). Data were analyzed by least squares and correlation methods. In the first experiment, the bull within breed interaction was significant for all bull behavior parameters and the managerial parameter. The bull within breed interaction for total motile sperm harvested was not
significant, but differed between breeds. There was a mixed response for bull within breeds for the post freeze semen viability parameters. Bull within breeds was not significant for the semen morphology parameters. The night versus day treatment was significant for the managerial parameter. In the second study, there was significant bull within breed differences in the %DISAGREE and NDISAGREE for both SEQ1 and SEQ2. Breed differences were significant for NDISAGREE in SEQ2. For the Brahman bulls there were significant positive correlations between %DISAGREE and NDISAGREE for both SEQ1 and SEQ2 and sperm motility. Correlations between %DISAGREE and NDISAGREE for both SEQ1 and SEQ2 with ATPUSE were positive but negligible. For the Holstein bulls, there were no significant correlations between %DISAGREE and NDISAGREE for both SEQ1 and SEQ2 and sperm motility. Correlations between %DISAGREE and NDISAGREE for both SEQ1 and SEQ2 with ATPUSE were negative but not significant.
SIGNIFICANCE:Consideration of a different collection schedule for Bos indicus cattle was not warranted. The difference in mtDNA mutations could be an indication of genetic drift between the Brahman and Holstein mitochondrial populations.
Impacts
Consideration of a different collection schedule for Bos indicus cattle was not warranted. This study has also shown that mtDNA from sperm cells is not significantly different from the mtDNA of heart, kidney, and other organs, and may be an easily obtained source of mtDNA for other research.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Objectives:1)To evaluate the percent Y-chromosome DNA bearing sperm (%YCDBS)in bovine ejaculates by duplex gel and real time polymerase chain reaction(PCR). 2)To evaluate the use of cochettes (a flat plastic bag)as semen freezing containers. Experiment 1: Ejaculates were processed separately, packaged in .5 ml straws, frozen and stored in liquid nitrogen(LN). Individual ejaculate DNA was extracted and quantified by spectroscopy. For the duplex gel technology(DG-PCR), primers were designed for single copy genes specific to the X-(factor IX, F9)and Y-(SRYB)chromosomes. Amplified X and Y products were separated by gel electrophoresis, extracted and quantified at 260 nm. PCR product was electrophoresed on agarose gels containing ethidium bromide. Gels were imaged on a UV transilluminator with a SIT camera. Image analysis software was used to determine the integrated density of the bands. Standards were made to contain the following ratio of X to Y product (0:100, 20:80,
40:60, 60:40, 80:20, and 100:0). Unknowns were analyzed with the same primers and the images were quantified by comparison to the standard curve. Real time PCR(RT-PCR) was done on the same DNA, using primers for the same gene as duplex gel technology. The reporter flouroprobes were FAM for F9 and VIC for SRYB. Correlation analysis was done on DG-PCR and RT-PCR ejaculate means. The mean RT-PCR was 53.51% YCDBS with 0.45 standard error, ranging from 44.46 to 60.83% YCDBS across ejaculates. The mean DG-PCR was 55.00% YCDBS with 0.69 standard error, ranging from 42.55 to 73.51% YCDBS across ejaculates. The correlation between %YCDBS per ejaculate as determined by DG-PCR and RT-PCR was 0.15 (P>r=28, H0:Rho=0). Experiment 2. A rack for suspending cochettes about two inches above the LN level was constructed to accommodate twelve cochettes in two rows of six. Bovine semen collected by artificial vagina was evaluated for concentration and percent progressive motility (PPM). Semen was extended
to 15 million motile cells/straw in a classical two part, egg yolk-sodium citrate-glycerol extender containing antibiotics. The second extender part was added to cooled semen by drip method. The semen was packaged into .5 ml French straws or cochettes (5 ml each). Air was removed from the cochettes and they were heat-sealed. The cochettes and straws were frozen via a standard straw protocol. Freezing was achieved by reducing the temperature from 5C to -135C in 8 minutes. Both straws and cochettes were then plunged into LN. Straws were thawed in a 37C water bath for 20 seconds. Semen was incubated in a 37C water bath. Cochettes were thawed in a circulating 37C water bath for 20 seconds. They remained in the water bath for 1 more minute before opening. Samples were evaluated on a warm coverslipped slide for PPM with phase microscopy. An aliquot was fixed with 0.2% gluteraldehyde for acrosomal integrity (PIA)evaluation. Samples were incubated for 3 hours and re-evaluated for PPM and PIA.
PIA was determined by apical ridge presence using DIC microscopy. The quality of the semen frozen in straws was no different from that in cochettes(P>0.05).
Impacts
Significance:Both DG-PCR and RT-PCR techniques showed variation in the %YCDBS from ejaculate to ejaculate. This variation and PCR identification could allow producers to select ejaculates for artificial insemination that would alter the sex ratio of the calf crop. Cochettes could be used as a semen freezing container for species (fish, swine and equine) that require large inseminate volumes.
Publications
- Paul, J.B., Canal, A.M. and Chandler, J.E. 2000. X and Y chromosome specific duplex PCR standardized to quantify sex ratio variation in bulls and boars. J. Dairy Sc. 83, Suppl 1:198.
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