Proximal Droplets Research Articles

Effect of breed, age, season and region on sperm morphology in 11,387 bulls submitted to breeding soundness evaluation in Australia

This study reports the distribution of sperm morphology defects by breed, age, season and region of 11,387 bulls in 500 herds in Australia and near Pacific Islands during annual BBSE. Bull location was divided into 4 broad climatic regions based upon temperature, vegetation and climatic risk. Taking into account the impact of age, season, region, and breed there were differences between breeds in both percent morphologically normal sperm and in some individual categories of sperm abnormality (P < 0.001). Independent of breed, season and region, proximal droplets were significantly increased in bulls less than 20 months of age. This is the first study to comprehensively collect data from this wide geographical area and compare sperm morphology profiles among the Bos indicus and Bos taurus breeds. The findings of this study will act as a guide for veterinary practitioners and cattle breeders in the proportion of bulls that can be expected to pass the PNS test, by breed, age and region, based on a robust data set.

Identifying candidate genes associated with sperm morphology abnormalities using weighted single-step GWAS in a Duroc boar population

Artificial insemination (AI) has been used as a routine technology globally in the pig production industry since 1930. One of the preferable advantages of AI technology is that the semen of elite boars can be disseminated to the commercial sow population rapidly. Understanding the genetic background of semen traits may help in developing genetic improvement programs of boars by including these traits into the selection index. In this study, we utilized weighted single-step genome-wide association study (wssGWAS) to identify genetic regions and further candidate genes associated with sperm morphology abnormalities (proximal droplet, distal droplet, bent tail, coiled tail, and distal midpiece reflex) in a Duroc boar population.Several genomic regions explained 2.76%–9.22% of the genetic variances for sperm morphology abnormalities were identified. The first three detected QTL regions together explained about 7.65%–25.10% of the total genetic variances of the studied traits. Several genes were detected and considered as candidate genes for each of the traits under study: coiled tail, HOOK1, ARSA, SYCE3, SOD3, GMNN, RBPJ, STIL, and FGF1; bent tail, FGF1, ADIPOR1, ARPC5, FGFR3, PANX1, IZUMO1R, ANKRD49, and GAL; proximal droplet, NSF, WNT3, WNT9B, LYZL6, FGFR1OP, RNASET2, FYN, LRRC6, EPC1, DICER1, FNDC3A, and PFN1; distal droplet, ARSA, SYCE3, MOV10L1, CBR1, KDM6B, TP53, PTBP2, UBR7, KIF18A, ADAM15, FAAH, TEKT3, and SRD5A1; and distal midpiece reflex, OMA1, PFN1, PELP1, BMP2, GPR18, TM9SF2, and SPIN1. GO and KEGG enrichment analysis revealed the potential function of the identified candidate genes in spermatogenesis, testis functioning, and boar spermatozoa plasma membrane activating and maintenance.In conclusion, we detected candidate genes associated with the coiled tail, bent tail, proximal droplet, distal droplet, and distal midpiece reflex in a Duroc boar population using wssGWAS. Overall, these novel results reflect the polygenic genetic architecture of the studied sperm morphology abnormality traits, which may provide knowledge for conducting genomic selection on these traits. The detected genetic regions can be used in developing trait-specific marker assisted selection models by assigning higher genetic variances to these regions.

Dependence of the frequency of sperm defects and dimensions on sperm motility in ejaculates of Polish Landrace boars

An attempt was made to determine the dependence of the frequency of sperm defects and dimensions on sperm motility in ejaculates of Polish Landrace boars. The study was conducted on 393 ejaculates collected from 33 Polish Landrace boars. Ejaculates were grouped according to the percentage of sperm with progressive motility, distinguishing ejaculates in which the percentage of motile sperm was 70% and 80%. In each ejaculate, the frequency of morphological changes in the sperm was determined and morphometric measurements of the sperm were made. Ejaculates with a higher proportion of sperm with progressive motility were found to contain more sperm. The ejaculate volume and sperm concentration in the ejaculate were not found to be directly associated with sperm motility. The frequency of primary defects was linked to sperm motility. Ejaculates with higher sperm motility contained fewer sperm with primary defects. The frequency of minor morphological changes, however, shows no significant dependence on sperm motility in the ejaculate. The primary morphological sperm defects most often found in ejaculates are a proximal droplet and the Dag defect. Both of these morphological forms are more common in ejaculates with lower sperm motility. The most common secondary sperm defects include sperm with a simple bent tail, sperm with a free normal head, and sperm with a distal droplet. These defects were not found to depend on sperm motility in the ejaculate. Sperm cells in ejaculates with greater sperm motility had slightly larger dimensions than sperm in ejaculates with lower sperm motility. Ejaculates with higher sperm motility are preferable for use in practice, not only because more insemination portions can be prepared from them, but also due to the lower frequency of primary defects.

Genetic relationships among sperm quality traits of Duroc boars collected during the summer season

The goals of the study were to estimate the heritability and repeatability of boar sperm quality traits during the summer season, estimate phenotypic and genetic correlations between sperm quality traits, and assess the effect of these traits on total number born (TNB). Semen samples were collected and individually processed from Duroc boars (n = 363) from May through October 2017. Single sire litter information was available for 121 of the boars. Heritability, repeatability, genetic correlations, and phenotypic correlations were estimated for and among the following traits: total percentage of motile cells (MOT), total percentage of progressively motile cells (PROG), percentage of cells with a distal droplet (DIST), percentage of cells with a proximal droplet (PROX), percentage of cells with a bent tail (BENT), percentage of cells with a distal midpiece reflex (DMR), mean sperm head elongation (ELON), and total sperm cells per ejaculate (TOTSP). Heritability estimates ranged from 0.08 to 0.24 and repeatability estimates ranged from 0.21 to 0.62. The phenotypic and genetic correlations between sperm motility traits and morphological defects indicated a negative relationship. Sperm morphological defects had positive phenotypic and genetic correlations with each other. Total sperm per ejaculate showed positive genetic correlations with the motility traits. Positive phenotypic and genetic correlations existed between ELON and the motility traits. Total sperm motility (P < 0.001) positively affected TNB while DIST (P < 0.001), PROX (P < 0.001), BENT (P = 0.05) and DMR (P = 0.002) negatively affected TNB. Results indicate genetic selection could enhance sperm quality during the summer season in boars.

154 Exposure of bulls to heat stress had deleterious effects on embryo development

Concerns about global climate change reducing animal fertility are arising. Therefore, our objective was to determine the effects of increased environmental temperature on Holstein bulls and its effects on sperm quality and embryo development. Frozen semen samples were obtained from 6 bulls exposed to natural heat stress (HS) in August 2016, compared with a lower temperature (control) in March 2016 (temperature-humidity index of up to 74.5 and 40.6, respectively). We evaluated sperm morphology, embryo development, gene expression, inner cell mass/trophectoderm ratio (ICM/TE), and apoptosis cell ratio of Day-8 blastocysts. Sperm morphology was evaluated using eosin/nigrosin staining. Blastocysts were produced by routine in vitro methods (Wydooghe et al. 2014 Reprod. Fertil. Dev.). Cleavage rates were determined 48h after insemination, and blastocyst rates were determined on Days 7 and 8. Expression of NANOG, SOX2, POU5F1, DNMT (1, 3a, 3b), HSP (A1a, A2, A8, 10, 60, 90), HSF1, IFNT2, H19, SNRPN, IGF2, IGF2R, MEST, PHLDA2, MEG3, MEG9, PEG10 and PLAGL1 were analysed. Total RNA was extracted from Day-8 blastocysts for gene expression analysis using RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Reverse transcription and qPCR were performed with iScript (BioRad, Hercules, CA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (BioRad) in a CFX Connect system (BioRad). Blastocysts were differentially stained (Wydooghe et al. 2011 Anal. Biochem.) and analysed using a Leica TCS-SP8×confocal microscope (Leica Microsystems, Wetzlar, Germany). Data analyses included a GLM procedure and paired-samples Student’s t test (P ≤ 0.05). A normal distribution was verified with a UNIVARIATE procedure and Shapiro-Wilk test. A Wilcoxon signed rank test was used to analyse qPCR data. Detached heads (P=0.006) and coiled tails (P=0.018) were significantly lower in the HS group (4.9 and 0.2%, respectively) compared with the control (5.5 and 0.5%). Moreover, proximal droplets (P=0.051) were lower in the HS group (0.7%) compared with the control (1.3%). Remarkably, cleavage and blastocyst rates at Days 7 and 8 were significantly higher in control (78.4, 19.6 and 29.5%, respectively) compared with HS (75, 14.5 and 23.2%). Early and normal blastocysts were grouped as early stage, whereas expanded, hatching and hatched blastocysts were grouped as advanced stage. There was a significant reduction in the HS group of early stage blastocysts on Day 7 and of advanced stage blastocysts on Days 7 and 8. However, in Day-8 blastocysts, there was no significant difference in gene expression for any target gene. Moreover, there were no significant differences in total number of cells or apoptosis cell ratio in blastocysts. However, the ICM/TE ratio was significantly higher (P=0.021) in control (0.7) compared with HS (0.56). Sperm samples collected in August had reduced fertility compared with those obtained in March. Although fewer sperm abnormalities were present in HS, based on decreased blastocyst rates and ICM/TE ratio in embryos produced with HS semen, we inferred that molecular mechanisms for advanced blastocyst development were affected. However, those mechanisms did not involve our target genes. This work was funded by the European Union, Horizon 2020 Marie Sklodowska-Curie Action, REPBIOTECH 675526.

The Effect of Morinda citrifolia L. Fruit Extract Supplemented in to Diluent Tris-Egg Yolk Toward the Abnormality of Goat’s Spermatozoa after Freezing at -80°C

The aim of this research is to know the effect of diluent Tris-egg yolk medium added with Morinda citrifolia L. extract towards the sperms abnormality after storage at -80°C for 24 hours. The extracts added in diluent Tris-egg yolk medium were 0% (P 0 ), 10% (P 1 ), 20% (P 2 ), and 30% (P 3 ). The result showed that the percentage of sperms abnormality in P 0 , P 1 , P 2 , and P 3 were respectively 8.67%, 6.67%, 5.67%, and 10%. P 2 was the lowest percentage, while P 3 was the highest percentage of sperms abnormality. In addition, this research found twelve kinds of sperms abnormality divided into categories: major abnormality included the broke tail, absent head, detached head, dag defect, tapered head, proximal droplet, round head and abaxial; and minor abnormality included the coiled tail, shoe hook tail, bent tail, and distal droplet. It has been concluded that the allotment of 20% Morinda citrifolia L. extracts in Tris-egg yolk medium able to maintain the sperm morphology at -80°C storage for 24 hours. Keywords: goat, Morinda citrifolia L., post-thawing, spermatozoa.

Morphological defects, sperm DNA integrity, and protamination of bovine spermatozoa.

The association between sperm morphology characteristics and DNA conformation and integrity is still controversial. In bulls, major morphological sperm abnormalities have been associated with reduced fertility, and morphological assessment is used to provide an indication of potential fertility of the individual. Sperm DNA fragmentation and damage has a negative effect on embryo development and subsequently fertility, with bull spermatozoa generally displaying low levels of DNA damage and tight chromatin. However, sensitive methods for detecting chromatin damage may reveal associations with morphological defects. The objective was to determine whether morphological sperm abnormalities and variables expressing sperm DNA integrity and protamination are correlated in bulls, using the sperm chromatin structure assay (SCSA) and the sperm protamine deficiency assay (SPDA). Electroejaculated samples (n=1009) from two-year-old tropically adapted bulls were split and fixed and submitted to microscopic sperm morphology assessment, and snap-frozen for sperm nuclear integrity assessments by SPDA and SCSA. For SPDA, the variables were defective (MCB) and deprotaminated (HCB), and for SCSA, the variables were DNA fragmentation index (DFI) and high DNA stainability (HDS). HCB correlated with DFI; τKen2 =0.317 and HDS; 0.098, and MCB correlated with DFI; 0.183 (p<0.001). The percentage of morphological normal spermatozoa was correlated negatively to DFI; τKen2 =-0.168, MCB; -0.116 and HCB; -0.137 (p<0.001). HCB and DFI were both positively correlated to head defects, proximal droplets, and spermatogenic immaturity, but not to distal droplets, vacuoles, or diadems. Sperm DNA integrity and protamination, using the SCSA and SPDA, respectively, in bulls show associations with morphological parameters, particularly with head shape abnormalities and indicators of spermatogenic immaturity, including proximal droplets. The vacuoles and diadem defects were not correlated with sperm nuclear integrity, and hence, these are likely physiological features that may not directly affect sperm chromatin configuration.

In utero heat stress causes reduced testicular area at puberty, reduced total sperm production, and increased sperm abnormalities in boars

In utero stress has been shown to negatively affect intact male rats and mice, though very little research has been conducted in boars. The objectives of the present studies were to determine the effects of in utero heat stress (IUHS) on postnatal development and the response to postnatal heat stress of boars. Ten boars were selected at weaning from litters subjected to IUHS or in utero thermoneutral (IUTN) during 30–60 days of gestation. The boars were evaluated for reproductive performance from birth through 57 weeks of age (WOA). Testicular area tended to be smaller for IUHS boars compared to IUTN boars at 24 WOA (P = 0.080). Libido did not differ for IUHS or IUTN (P = 0.818). Total sperm production was reduced in IUHS boars compared to IUTN boars (P ≤ 0.038). Semen volume and semen concentration did not differ (P ≥ 0.469 and P ≥ 0.664, respectively). Total motility and progressive motility did not differ for IUHS and IUTN boars (P ≥ 0.430 and P ≥ 0.652, respectively). In utero heat stressed boars had a greater incidence of sperm with tail abnormalities than IUTN (P ≤ 0.042). In utero heat stressed boars had a lower incidence of sperm with proximal droplets following mild, postnatal heat stress compared with IUTN (P = 0.005). In utero heat stress resulted in significant reductions in sperm production and increased sperm abnormalities in boars. The IUHS boars may be slightly more tolerant to postnatal heat stress, though more research is needed.

Influence of multiple showering on quality of buffalo semen during hot-humid season

Objectives: This experiment was conducted on buffaloes to see effect of showering on maintaining good quality of buffalo semen in hot season. Materials and methods : This study was conducted on 6(six) indigenous buffalo bulls in hot summer of March and April 2017. The effect of multiple showering vs single shower alone on fresh and equilibrated semen quality was observed. The buffalos were divided into similar two groups (according to age and weight) and housed in half-walled openshed with adequate spacing and the feeding management being identical. The bulls in the control group were allowed to a single shower at 10.00 am and experimental bulls were allowed to four showers at 07.00, 10.00, 13.00 and 16.00. Temperature and humidity were recorded during this time. The average temperature was around 35°C along with 72% relative humidity. The quality of experimental bull’s semen was evaluated in terms of volume, live sperm, sperm concentration, sperm motility, motion parameters and morphology for normal and abnormal sperm. For this analysis Computer Assisted Semen Analysis (CASA) system was used. Results: Between experimental group and control group semen quality differs significantly for volume (2.04±0.13 mL; 2.53±0.27 mL) ( P <0.01); live sperm (81.38±1.22%; 90.28±1.53%) ( P <0.01), normal fraction (36.87±6.38%; 47.87±12.01%) ( P <0.05); DMR (6.89±5.86%, 5.86±1.45%) ( P <0.05) and for proximal droplet (57.86±4.30, 45.26±10.96) ( P <0.05). Motility and motion parameters for fresh semen were not significantly different. Conclusion: In short, showering showed significant effect on different parameters of fresh semen quality of buffalo but not in case of further processing. http://doi.org/10.5455/javar.2018.e239

Dip Quick Staining Modified for Morphological Evaluation to Equine Spermatozoa

Morphological analysis of the sperm cells were accomplished from 19 stallions comparing six different techniques (DIC, Karras, Eosin-Nigrosin, Dip Quick 5′, Dip Quick modified, Dip Quick) in order to verify the possibility of using a modification of Dip Quick stain technique to evaluate the morphology of equine semen. Data of sperm morphology were evaluated using Kolmogorov-Smirnov (KS) normality test. Parametric continuous data were compared for ANOVA followed by the Tukey test and non-parametric dates, for Kruskal-Wallis test followed by the Dunns test. Significant differences were considered when P < .05. Pearson product-correlation procedure was used to test the correlation between technics. High correlation were considered when r ≥ 0.7, moderate correlation when r ≥ 0.5 but r < 0.7 and poor correlation when r < 0.5. The proposed technique did not show difference (P > .05) from other techniques to morphological assessment. However, the usual Dip quick technique showed to be less efficient (P < .05) to evaluated the sperm morphology defects, especially for the display of abnormal heads, distal and proximal droplets. A High correlation was observed among techniques in the evaluation of total number of defects, but when specific sperm defects of each segment were measured the Dip Quick modified showed a high correlation compared with the usual technics, different results from the Dip Quick technic that showed a moderate correlation on the evaluation of distal and proximal droplets and poor correlation on abnormal heads evaluation. We can conclude that the modification of the Dip Quip technique improved the quality of smear for the evaluation of equine sperm morphology.

Cytoplasmic droplet acting as a mitochondrial modulator during sperm maturation in dogs

Motility acquisition during sperm maturation and passage through the epididymis is closely related to mitochondrial function and appears to occur in parallel with cytoplasmic droplet (CD) migration. However, such mechanism remains unclear in dogs. Thus, the aim of this study was to characterize the influence of sperm CD in the mitochondrial functionality during epididymal sperm maturation in dogs. Twenty-one adult dogs were submitted to elective bilateral orchiectomy. Testicles were stored for 18–24h at 5°C and epididymal sperm samples were then collected from different segments of the epididymis (caput, corpus and cauda). Samples were evaluated for computer-assisted motility analysis (CASA), presence of CD (eosin/nigrosin stain), ultrastructural CD analysis and sperm mitochondrial activity (3,3' diaminobenzidine technique) and membrane potential (JC-1 probe). Samples collected from the corpus epididymis showed higher motility and mitochondrial activity in comparison to the caput sperm. Moreover, corpus sperm had lower percentage of proximal droplets compared to caput samples, while mitochondrial membrane potential remained unchanged. Cauda samples showed higher motility, mitochondrial activity and potential, however, lower presence of sperm droplets (proximal and distal). In conclusion, the CD is essential for epididymal sperm maturation in dogs, showing important functions along the transit in the epididymis. In the corpus segment, the migration of the CD along the sperm midpiece provides a high mitochondrial activity and the onset of sperm motility. On the other hand, sperm from cauda epididymis lack CD but suffered lipid membrane changes which allow a maximum mitochondrial membrane potential and motility.

Relationship between the Corynebacterium pseudotuberculosis, phospholipase D inoculation and the fertility characteristics of crossbred Boer bucks

The aim of this study was to investigate the effects of Corynebacterium pseudotuberculosis and phospholipase D (PLD) on buck's fertility, specifically, testosterone concentration, scrotal circumference and semen quality. Crossbred Boer goats (n=13) aged 12–14 months were divided into three groups. The first group (n=3) was inoculated with one ml sterile phosphate buffer saline subcutaneously into subaxillary area as the control. The second group (n=5) was inoculated with live C. pseudotuberculosis 1×109 cfu subcutaneously into subaxillary area. The third group (n=5) was inoculated with PLD 1ml/20kg BW intravenously into jugular vein. Blood collection was done twice a week over a period of three months for testosterone analyses. Semen was collected using electro-ejaculator once every two weeks whilst scrotal circumference was measured once every three weeks. Semen volume, semen pH and sperm progressive motility, concentration, live/dead percentage and morphology were evaluated. Bucks were euthanized three months' post inoculation and histopathological examination of the testicles and epididymis was performed. The results showed seven folds' significant decrease (P<0.05) in testosterone concentration in both C. pseudotuberculosis and PLD inoculated groups compared to the control. Semen volume, percentage of dead/live and abnormal sperm morphology showed significant increase (P<0.05) in both inoculated groups compared to the control. The following sperm defects were observed; tapered sperm, decapitated and knobbed head, thickened midpiece, distal and proximal droplet, sterilizing tail and folded tail. Scrotal circumference, semen pH, semen wave pattern, sperm motility and concentration showed significant decrease (P<0.05) in both inoculated groups compared to the control. The testicles showed varied degrees of degeneration and necrosis with shrunken seminferous tubules. In conclusion, testosterone concentration, scrotal circumference and semen quality were negatively affected in both inoculated groups. Moreover, percentages of live/dead and abnormal sperm morphology were also increased. The results suggest that both C. pseudotuberculosis and PLD had detrimental effects on buck's fertility.

Morphological defects, sperm DNA integrity and protamination of bovine spermatozoa

The association between sperm morphology characteristics and DNA conformation and integrity is still controversial. In bulls, major morphological sperm abnormalities have been associated with reduced fertility, and morphological assessment is used to provide an indication of potential fertility of the individual. Sperm DNA fragmentation and damage has a negative effect on embryo development and subsequently fertility, with bull spermatozoa generally displaying low levels of DNA damage and tight chromatin. However, sensitive methods for detecting chromatin damage may reveal associations with morphological defects. The objective was to determine whether morphological sperm abnormalities and variables expressing sperm DNA integrity and protamination are correlated in bulls, using the sperm chromatin structure assay (SCSA) and the sperm protamine deficiency assay (SPDA). Electroejaculated samples (n = 1009) from two-year-old tropically adapted bulls were split and fixed and submitted to microscopic sperm morphology assessment, and snap-frozen for sperm nuclear integrity assessments by SPDA and SCSA. For SPDA, the variables were defective (MCB) and deprotaminated (HCB), and for SCSA, the variables were DNA fragmentation index (DFI) and high DNA stainability (HDS). HCB correlated with DFI; τKen2 = 0.317 and HDS; 0.098, and MCB correlated with DFI; 0.183 (p < 0.001). The percentage of morphological normal spermatozoa was correlated negatively to DFI; τKen2 = -0.168, MCB; -0.116 and HCB; -0.137 (p < 0.001). HCB and DFI were both positively correlated to head defects, proximal droplets, and spermatogenic immaturity, but not to distal droplets, vacuoles, or diadems. Sperm DNA integrity and protamination, using the SCSA and SPDA, respectively, in bulls show associations with morphological parameters, particularly with head shape abnormalities and indicators of spermatogenic immaturity, including proximal droplets. The vacuoles and diadem defects were not correlated with sperm nuclear integrity, and hence, these are likely physiological features that may not directly affect sperm chromatin configuration.

Evaluation of Cauda Epididymal Semen Quality of Crossbred Bulls in the Tropics

The objective of the study was to evaluate the quality of semen retrieved from cauda epididymis of crossbred bulls in the tropics. Testes from one hundred and twenty six recently slaughtered cross bred bulls in the tropics were used for the analyais. The total and progressive motility percentage obtained for epididymal semen were 49.17±9.26 per cent and 27.5±9.11 per cent respectively. The mean concentration obtained for epididymal semen was 37,175x106 ±7612x106 per ml. The mean percentage of live and dead sperms was 84.5±8.02 per cent and 15.5±8.02 per cent respectively. The mean percentage of normal spermatozoa, spermatozoa with abnormal heads, abnormal tails, spermatozoa with a proximal protoplasmic droplet and distal protoplasmic droplet were 35.67±2.30, 3.17±1.58, 2.33±0.61, 11.67±4.01 and 47.17±3.17 per cent respectively. Int J Appl Sci Biotechnol, Vol 4(1): 130-132

Dynamics of Sus scrofa semen quality in the western-central region of Colombia

In order to characterize the dynamics of Sus scrofa (order Artiodactyla, family Suidae) semen quality in the Western-central region of Colombia, information of 1,017 assessments from 177 males from 12 swine farms located between 700 and 2,600 meters above sea level in different inter-tropical systems was collected in the Laboratory of Reproductive Biology at Universidad de Caldas (Colombia) during seven years. The database included the seminal variables: concentration, morphology (head, midpiece and tail), membrane structural integrity (MSI), acrosome integrity (ACRI), and membrane functional integrity (MFI) proximal citoplasmic droplets (PCD), distal citoplasmic droplets (DCD), and total citoplasmic droplets (TCD). Additionally, the racial group of the male (grac): maternal cross (cm), paternal cross (cp), paternal pure (pp), maternal pure (pm) and age group (agegr): young (up to 18 months), mature (19 to 36 months) and old (over 37 months) were registered. Data was explored by estimating descriptive statistics; it was analyzed using a log-linear model consisting of a Poisson regression with a log link function using the PROC GENMOD of SAS (SAS Inst. Cary, NC). The effect of age group and racial group on semen quality variables using a mixed model for repeated measurements in the same boar over time were evaluated. The Spearman correlation coefficients between variables were calculated. The variables: macro, micro and pyriform head; double, eccentric, and thickened midpiece; tail whip; ladder bow; ball and distal reflex showed less than 1% prevalence. The highest values of FMI were found in cm and pp. Males cp, cm and pm do not differ from each other in MFI, but all had higher values than pp. The ACRI in cm was higher than cp, and these higher than pm. Presentation of TCD increased with age, with a higher percentage of PCD in older animals than in younger animals; mature animals showed intermediate values between them. The TCD exceeded 15% of presentation, being always higher for PCD than for GCP. The TCD is negatively correlated with MSI and ARCI, but positively correlated with MFI. The same trend is observed in the correlations with distal cytoplasmic droplets. However, the correlation of proximal droplets with the structural integrity loses significance, though they are negatively correlated with acrosome integrity. Values for MSI, MFI and ACRI, do not limit the quality of the samples in this population.

Effects of Experimental Trypanosoma Congolense Infection on Sperm Morphology in Yankasa Rams

Abstract The objective of the study was to determine the effect of T. congolense on the sperm morphology of Yankasa rams (YKR). Nine YKR aged 24-30 months-old were assigned into two groups of 6 infected and 3 uninfected control and were monitored for 7 weeks. The infected group of YKR was each inoculated with 1 × 106 T. congolense through the jugular vein, while the control group remained uninfected. The entire infected group developed trypanosomosis post infection (pi) characterized by sperm morphological abnormalities in the semen. There were significant (P&lt;0.001) increases in the mean percentage of acrosomal, head, middle piece and tail abnormalities. Proximal and distal droplets as well detached heads were also significantly (P&lt;0.001) increased post infection (pi). Acrosomal abnormalities, distal droplet and tail abnormalities increased from week 1 pi till the end of the study, while head abnormalities and detached heads increased from week 2 pi. Middle piece abnormalities and proximal droplets increased from week 3 and 4 pi till the end of the study respectively. The high incidence of morphological defects caused by T. congolense is capable of causing infertility from the first week pi thereby making the rams unfit for breeding at the end of the study.

Prevalence of bovine sperm-bound antisperm antibodies and their association with semen quality

The objectives of this study were to determine reference intervals (RIs) for sperm-bound immunoglobulins G and A (IgG and IgA), prevalence of antisperm antibodies (ASAs) in satisfactory and nonsatisfactory breeders, and association between ASAs and semen quality in beef bulls. It was hypothesized that ASA binding differed with breeding soundness classification and semen quality. The percentage of IgG- (IgGperc) and IgA-bound (IgAperc) spermatozoa was evaluated in satisfactory (n = 134) and nonsatisfactory (n = 71) breeder beef bulls using flow cytometry. The RI for IgGperc was 0% to 13.5%. The RIs for IgAperc were 0% to 25.8% in yearling Aberdeen Angus bulls and 0% to 12% in all other bulls. The prevalence of IgA-positive samples was higher in nonsatisfactory (14.1%) than that in satisfactory (1.5%) breeders (P = 0.0003). However, the prevalence of IgG-positive samples did not differ. Similarly, IgA binding was higher in nonsatisfactory (median; interquartile range; 2.18; 0.77%–8.57%) than that in satisfactory breeders (median; interquartile range; 1.11; 0.32%–3.16%; P = 0.0035), but IgG binding did not differ. Among ASA-positive bulls, median IgA and IgG binding was 39.7% (range, 18.8%–96.2%) and 24.8% (range, 14.2%–33.1%), respectively. Immunoglobulin A binding correlated with the percentage of total (P < 0.0001; r2 = −0.345) and progressively motile spermatozoa (P < 0.0001; r2 = −0.329), morphologically normal spermatozoa (P = 0.0004; r2 = −0.256), sperm head abnormalities (P = 0.0416; r2 = 0.149), proximal droplets (P = 0.0227; r2 = 0.167), and coiled tails (P = 0.0338; r2 = 0.156). Immunoglobulin G binding correlated with the percentage of total (P < 0.0001; r2 = −0.373) and progressively motile spermatozoa (P < 0.0001; r2 = −0.455) and sperm concentration (P = 0.0332; r2 = −0.195). Reference intervals were established for determination of cutoffs for clinically significant sperm-bound IgA and IgG with flow cytometry. Immunoglobulin A binding was both higher and more prevalent in nonsatisfactory breeder bulls. Although IgG binding did not differ with breeding soundness classification, detection of surface-bound IgG and IgA was associated with changes in semen quality.

Effects of Inclusion of Polyunsaturated Fatty Acids and Antioxidants on Spermatozoa Morphology of Potchefstroom Koekoek Cockerels

This study was conducted to determine the effects of increasing dietary polyunsaturated fatty acids (PUFA) and antioxidants (Se and vitamin E) on spermatozoa morphology. At 24 weeks of age, Potchefstroom Koekoek cockerels (n = 60) were selected from the base population. Cockerels were randomly allocated into five dietary treatments with 12 cockerels per dietary treatment. The diets consisted of commercial layer diet (CL), commercial cockerel diet (CC), modified layer diet (ML), modified layer diet +PUFA (MLP), and modified layer +PUFA+ antioxidants (MLPA) diet. At 36 weeks of age, six cockerels with ≥ 75% sperm motility rate following analysis by Computer Aided Sperm Analysis (CASA) were selected from the base population in each dietary treatment group. Semen volume, sperm cells concentration and dead cells did not differ significantly among any of the groups. However, live and normal spermatozoa were significantly higher in the MLPA group. The bent mid-piece, bent mid-piece plus droplets, proximal droplets and distal droplets were significantly lower in the MLPA group. From this study it can be concluded that supplementation of polyunsaturated fatty acids and antioxidants may be of vital importance for normal morphology characteristics of spermatozoa in Potchefstroom Koekoek cockerels.

Exploratory analysis of differences in sperm morphology in Nelore and Gir (Bos indicus) bulls

The aim of this study was to evaluate the effects of breed and season on semen quality parameters of zebu bulls. Data (1,632 registers) of semen production from Gir (n = 4) and Nelore (n = 15) bulls were collected between October 2005 and November 2009. The ejaculates were collected twice a week during various seasons (summer, fall, winter, and spring) and evaluated for the following semen parameters: ejaculate volume, sperm concentration, sperm motility, forward progressive motility (FPM), and sperm morphology. Factor analysis was used to determine the relationship among variables. The effect of breed (Gir and Nelore) and season and their cross effect on each parameter and extracted factor were tested using ANOVA. A negative correlation (P < 0.05) was observed between FPM and proximal droplet, as well as with abnormal loose head, abnormal small head, pouch formation, abnormal mid-piece, and strongly folded tail. Gir bull sperm showed more major defects, detached acrosome, and minor FPM (P < 0.01), whereas Nelore bulls showed a higher number of sperm with normally loose head.

Trypan Blue/Giemsa Staining to Assess Sperm Membrane Integrity in Salernitano Stallions and its Relationship to Pregnancy Rates

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p<0.05) and visual motility (p<0.01). A decrease in sperm motility, concentration (p<0.05) and total sperm number was found in June-July (p<0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p<0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p<0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p<0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.