A fine structure genetic analysis evaluating ecoregional adaptability of a Bos taurus breed (Hereford).

Ecoregional differences contribute to genetic environmental interactions and impact animal performance. These differences may become more important under climate change scenarios. Utilizing genetic diversity within a species to address such problems has not been fully explored. In this study Hereford cattle were genotyped with 50K Bead Chip or 770K Bovine Bead Chip to test the existence of genetic structure in five U.S. ecoregions characterized by precipitation, temperature and humidity and designated: cool arid (CA), cool humid (CH), transition zone (TZ), warm arid (WA), and warm humid (WH). SNP data were analyzed in three sequential analyses. Broad genetic structure was evaluated with STRUCTURE, and ADMIXTURE software using 14,312 SNPs after passing quality control variables. The second analysis was performed using principal coordinate analysis with 66 Tag SNPs associated in the literature with various aspects of environmental stressors (e.g., heat tolerance) or production (e.g., milk production). In the third analysis TreeSelect was used with the 66 SNPs to evaluate if ecoregional allelic frequencies deviated from a central frequency and by so doing are indicative of directional selection. The three analyses suggested subpopulation structures associated with ecoregions from where animals were derived. ADMIXTURE and PCA results illustrated the importance of temperature and humidity and confirm subpopulation assignments. Comparisons of allele frequencies with TreeSelect showed ecoregion differences, in particular the divergence between arid and humid regions. Patterns of genetic variability obtained by medium and high density SNP chips can be used to acclimatize a temperately derived breed to various ecoregions. As climate change becomes an important factor in cattle production, this study should be used as a proof of concept to review future breeding and conservation schemes aimed at adaptation to climatic events.

Female sex selection using clomiphene citrate and albumin separation of human sperm.

BACKGROUND: This study was undertaken to assess whether the use of clomiphene citrate in conjunction with albumin-separated sperm would alter the sex ratio (expressed as the proportion of males) towards females and, if so, whether this skewing was due solely to the induction of ovulation. METHODS: The sex ratios of 184 single and 42 twin births at five assisted reproduction biology clinics were determined. The normal approximation to the binomial distribution was used to determine significant differences between these sex ratios and the established sex ratios for single, twin and combined (single and twin) non- and ovulation-induced births. RESULTS: The non-ovulation-induced sex ratios for singletons (51.4%) and twins (50.2%) were greater than the treatment singleton (27.7%; P < 0.001) and twin (33.3%; P < 0.01) sex ratios respectively. Correspondingly, the non-induced sex ratio for combined births (51.4%) was greater than the treatment sex ratio (28.8%; P < 0.001). The previously established induced singleton and twin sex ratios (48.1%) were lower than the non-induced sex ratio (51.4%), but higher than the treatment singleton (27.7%; P < 0.001) or twin (33.3%; P < 0.03) sex ratios. The ovulation-induced combined ratio (48.1%) was less than the non-induced combined (51.4%) sex ratio, although greater than the treatment combined sex ratio (28.8%; P < 0.001). CONCLUSION: Clomiphene citrate in conjunction with albumin-separated sperm decreased the sex ratio; a reduction that was not exclusively due to induction of ovulation.

The use of buck and ram extenders and two packaging systems to cryopreserve aoudad (Ammotragus lervia) spermatozoa.

Because the aoudad has been hunted to near extinction, cryopreservation of their semen would be useful for DNA conservation and for the possible re-establishment of captive bred animals to their former ranges. This study was conducted to investigate the effectiveness of cryopreserving aoudad spermatozoa. Semen samples from four post-pubertal animals were collected using electro-ejaculation. Microscopic analysis was performed to assess the percentages of progressively and non-progressively motile spermatozoa as well as intact acrosomes in samples prior to freezing and post-thaw. Extended samples (0.2 mL) were frozen using 2 different extenders and packaging systems and stored in LN2 Post-thaw data were arcsine-transformed and analyzed using ANOVA, 2 x 2 factorial. Samples that were processed using the ram/straw method had a significantly higher percentage (P < 0.05) of spermatozoa with intact acrosomes than did any other system. In addition, samples that were processed with the buck/pellet system had significantly greater percentages (P < 0.05) of progressive and non-progressively motile spermatozoa than the samples processed using either extender and packaged in straws. This study illustrates that some aoudad spermatozoa may be cryopreserved using the extender/processing systems developed for the domestic buck and ram.

Assessing the fertility potential of equine semen samples using the reducible dyes methylene green and resazurin.

The objective of this study was to determine if spermatozoal reduction of the dyes methylene green to colorless and resazurin to pink or colorless was associated with the fertility potential of an equine semen sample. Fifty samples from 38 stallions were evaluated for the number of spermatozoa per milliliter and number of motile sperm per milliliter. Methylene green (20 micrograms/mL of semen) or resazurin (85 micrograms/mL of semen) was added to 3-mL aliquots of semen. Semen samples were identified as having low fertility potential (< 200 x 10(6) total cells/mL and < 140 x 10(6) motile cells/mL) or high fertility potential (> or = 200 x 10(6) total cells/mL and > or = 140 x 10(6) motile cells/mL). The sensitivities were 80% for the methylene green, 68% for the resazurin to pink, and 60% for the resazurin to colorless tests. The specificities were 80% for the methylene green, 79% for the resazurin to pink, and 92% for the resazurin to colorless tests. The overall accuracies were 80% for the methylene green test, 74% for the resazurin to pink test, and 76% for the resazurin to colorless tests. The methylene green and resazurin reduction tests can provide valuable information on the fertility potential of an equine semen sample.

Modified resazurin reduction test for determining the fertility potential of bovine spermatozoa.

The objective of this study was to determine if reduction of the dye resazurin by bovine sperm is associated with the concentration of motile and progressively motile sperm. Metabolically active sperm reduce resazurin (blue color) to resorufin (pink color) and upon further reduction to dihydroresorufin (white color). A total of 68 semen samples from 20 Limousin bulls were collected using electroejaculation. The concentration of motile and progressively motile sperm was determined. Resazurin was added to each semen sample (8.8 microgram/mL semen), samples were then incubated at 37 degrees C for a maximum of 15 min, and visual color changes indicative of dye reduction were noted. Samples were identified as having either low fertility potential (< 200 x 10(6)/mL motile sperm and < 100 x 10(6)/mL progressively motile sperm) or high fertility potential (> or = 200 x 10(6)/mL motile sperm and > or = 100 x 10(6)/mL progressively motile sperm). Assessment of the reduction of resazurin from blue to pink (< or = 3.5 min) allowed identification of 88% of the low and 94% of the high fertility potential samples. In addition, the reduction of resazurin from blue to white (< 15 min) resulted in identification of 82% of the low and 76% of the high fertility potential samples. The modified resazurin reduction test is useful for determining the fertility potential of bovine spermatozoa.

Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human spermatozoa.

Functional differences among fresh 24-hour extended and cryopreserved human spermatozoa were assessed using both computer-assisted semen analysis (CASA) and flow cytometry. The objective was to determine if there were interrelationships among various qualitative parameters of the fresh and treated samples when assessed by these two automated methods. Fertile donor specimens (n = 15) were split and examined for sperm motility and curvilinear velocity using CASA within 1 hour postejaculation, after 24 hours in TEST-yolk buffer at 5 degrees C and after cryopreservation in TEST-yolk-glycerol medium. Flow cytometric analyses were performed on 24-hour extended and cryopreserved (CP) samples after fluorescent staining with rhodamine 123 to quantify mitochondrial function and carboxydimethyl fluorescein diacetate and propidium iodide to assess plasma membrane integrity. The percentages of spermatozoa with functional mitochondria and intact membranes along with the proportion of dead cells were identified and quantified by flow cytometry. Quadrant analyses of these data were used to determine the relative red and green fluorescent intensities. The initial sperm motility was correlated to the motility observed for the 24-hour stored and the CP samples. The sperm velocity of both the initial and the 24-hour extended samples was correlated to the velocity of CP samples. As for the comparison of the two automated methods for assessing seminal quality, the only sperm motion parameter that was correlated with a sperm population identified by flow cytometry (quadrant 4) was the curvilinear velocity of the sperm after 24 hours storage (r = 0.69) and after cryopreservation (r = 0.74). The present findings indicate that additional research is needed to determine if prefreeze analyses of donor sperm could be useful in predicting the post-thaw integrity of CP samples and, thereby, be useful in screening potential semen donors.

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa.

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.

Sex ratio of male sex preselected children born to couples with exclusively female offspring.

The present study was undertaken to determine if couples with exclusively female children had a decreased probability of a male child after using male sex preselection. Selection criteria for subjects necessitated that couples have had only female children previously and had produced a child at one of 14 centers after using protocol 3 (n = 70) and modified 3 (n = 28) male sex preselection. Prior to sex selection, protocol 3 couples produced a combined total of 135 female children for an average of 2.01 (range 1-4) females per couple; modified 3 couples produced a combined total of 62 female children for an average of 2.21 (range 1-4) females per couple. The normal approximation to the binomial distribution was used to determine significant differences between the sex ratios prior to and after male sex preselection. For couples using protocol 3 there were significant differences in the sex ratio prior to sex preselection (0%) and after sex preselection (73.0%) (p less than .00003). There were also significant differences between the sex ratio prior to modified 3 (0%) and after sex preselection (86%) (p less than .00003). Couples using male sex preselection do not have a decreased probability of a male child if they have had exclusively female children.

Couples with exclusively female offspring have an increased probability of a male child after using male sex preselection.

The present study was undertaken to determine whether couples with one or more daughters and no sons had an increased probability of having a male child after using male sex preselection. The sex ratio of children born to couples after using 'protocol 3' (n = 70) or 'modified protocol 3' (n = 28) male sex preselection at one of 14 centres was determined. The normal approximation to the binomial distribution was used to determine significant differences between these sex ratios and the established sex ratio for children born to couples with one previous daughter and no sons. The sex ratios of both protocol 3 sex-preselected children (73.0%; P less than 0.0001) and modified protocol 3 sex-preselected children (86%; P less than 0.0001) were significantly different to the established sex ratio (control) for a current child born to parents with one previous daughter and no sons (50.1%). Couples with one or more daughters and no sons will have an increased probability of a male child after using male sex preselection.

The resazurin reduction test provides an assessment of sperm activity.

OBJECTIVE: The objective of the study was to determine if reduction of the dye resazurin by semen could be correlated with the concentration of motile sperm. DESIGN: After assessment of sperm count and motility, specimens were incubated for 1 hour with resazurin (25 micrograms/mL of semen) and visual color changes indicative of dye reduction noted. SETTING: Specimens were obtained from men seeking care for infertility at one of four sites: (1) University of California, San Francisco (UCSF) In Vitro Fertilization Program; (2) UCSF Andrology Laboratory; (3) a gynecological practice in Maine; and (4) a private andrology laboratory in Southern California. PATIENTS: Individuals were self-selected by their desire to have a semen analysis in conjunction with the diagnosis or treatment of infertility. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The reduction of the dye resazurin and its correlation with motile sperm density. RESULTS: When the motile sperm concentration was greater than or equal to 20 X 10(6)/mL, 86% of specimens produced a positive color change. Conversely, 86% of specimens with a motile sperm concentration of less than 20 X 10(6)/mL either did not change color or changed only over a narrow range. CONCLUSION: Reduction of resazurin offers an assessment of the active sperm in a specimen without the need to do a sperm count or evaluation of motility.

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

The effect of cottonseed components on testicular development in pubescent rams.

The effect of gossypol on testicular development in rams was examined using 2 experimental approaches. Trial I utilized direct ruminal deposition of gossypol acetic acid (GAA; 95% purity), whereas Trial II compared 2 types of dietary cottonseed, regular and glandless. For Trial I, 18 Polypay or Polypay x Dorset ram lambs, aged 4 to 6 mo, were surgically fitted with rumen cannulae. These 18 ram lambs were divided into groups of 6 which were given either 0, 2.2 or 6.6 mg of GAA/kg body weight daily via gelatin capsules directly into the rumen. All rams in Trial I were slaughtered after 4 or 8 w of treatment to allow recovery of their scrotal contents. The mean testicular weight for the group of rams given 6.6 mg GAA/kg/d was 8% (p less than .05) less than for the rams given 0 mg GAA/kg/d. The caudal epididymal sperm were examined microscopically for percentage of progressively motile sperm and flow cytometrically for mitochondrial function using rhodamine 123 (R123). Differences in sperm motility were not evident among the treatment groups, nor were differences detectable in the 2 major sperm populations, B and C, that were identified by flow cytometry. In Trial II, 21 Polypay and Polypay-Dorset ram lambs, aged 10 mo, were randomly assigned into 3 groups (n = 7) and fed either glandless fuzzy cottonseed (GFC), glandless flake cottonseed (GFL) or regular fuzzy cottonseed (RFC). All rams were fed for 5 w a total daily ration (TDR) equivalent to 3.5% of their body weight daily with 25% being cottonseed. After 8 w the scrotal contents were recovered by castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two-flow cytometric systems.

A dual fluorescent staining system utilizing 5 (and-6)-carboxy-4',5'-dimethyl fluorescein diacetate (CDMFDA) and Hydroethidine (HED) was developed to provide quantifiable information reflective of spermatozoal viability and fertilizing potential. Cryopreserved spermatozoa from ten bulls on which there was fertilizing capacity information were incubated for 1.5, and 3 hr at 39 degrees C prior to fluorogenic staining. Spermatozoa were analyzed using both a FACS Analyzer and an EPICS V flow cytometer to determine if a particular fluorescence pattern was due to an instrumental artifact or cellular processes. Five fluorescent cellular populations were identified by the FACS Analyzer and three populations by the EPICS V. Spermatozoa were quantified after each incubation time for red (HED) and green (CDMFDA) fluorescence. Viable spermatozoa retained the greatest amount of both green and red fluorescence. Dead or moribund spermatozoa had a decrease in over-all fluorescence. The number of viable cells at 0 hr plus the number of dead or morbid cells at any time period were identified by the FACS Analyzer as important in estimating the potential fertility of a bull. The EPICS V identified the number of dead or moribund cells as being related to nonreturn rates. Incubation of samples decreased cellular viability, which resulted in reduced levels of both green and red fluorescence. Similarities between data obtained with both flow cytometers illustrated that cellular processes, not instrumental artifacts, were responsible for the decrease in over-all fluorescence when viability declined, the relationship between the number of cells with specific fluorescence levels and nonreturn rates, and the incubative-induced changes in fluorescence patterns.