A new reciprocal translocation in a subfertile bull

Three bulls of the Montbéliarde breed that exhibited fertility rates lower than 30% following more than 400 artificial inseminations were examined. Semen quality (sperm motility and morphology) from these bulls was normal. Fertilizing ability estimated from in vitro embryo production results was studied for two of them. In vitro production rate was very low for one bull (A) and normal for the other (B). Cytogenetic analyses were carried out on the three bulls using chromosome banding techniques. These analyses revealed a reciprocal translocation (12;17)(q22;q14) in bull B. Based on family analyses, the hypothesis of a de novo origin of this rearrangement is proposed.     

Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp following short-term storage of semen

A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely 'linear', 'circular' and 'haphazard', the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.     

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Determination of adenosine triphosphate in human semen to estimate the fertilizing potential and to quantify sperm antibodies

The measurement of the ATP content of fresh semen is as accurate as the estimation of sperm motility by conventional methods in discriminating between semen of fertile versus subfertile men. The ATP content of frozen thawed donor semen is correlated with the probability of conception per cycle of insemination. Exact quantification of cytotoxic sperm antibodies in serum is possible with the adenosine-triphosphate-release-cytotoxicity test, since measurement is free of the bias of microscopic examination. The procedure has been simplified by testing only one serum dilution and calculating the ‘sperm toxicity index’.     

Quality of semen stored at +15 degrees/16 degrees C as related to fertility of artificially inseminated swine

Linear correlations between swine semen quality and fertility (birth rate and number live born pigs) were studied. Doses of swine semen with 4 × 10⁹ motile spermatozoa were used after being stored at +15°/16°C. However, mean or high correlations could not be found. Low correlations occurred between head-acrosome anomalies, total anomalies and birth rate. It is likely that the doses of semen with 4 × 10⁹ motile spermatozoa were too concentrated for the fertility level in the swine breed employed.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: II. A fertility assay for frozen-thawed semen

Frozen-thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona-free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ?.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ? ?.85). Similar correlations were found between fertility and the penetration rates of zona-free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen-thawed in egg yolk extender. Frozen-thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona-free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.     

Selection of buffalo bulls: Sexual behavior and its relationship to semen production and fertility

Forty-four buffalo bulls, used for artificial insemination, were studied to develop libido, mating ability and sexual behavior indices for selection purposes. For each index, 5 categories (i.e., excellent, very good, good, fair and poor) were established. The sexual behavior index was found to be more reliable than the libido and mating ability indices. Buffalo bulls in good to excellent categories were considered acceptable sires.

Reaction time, sexual aggressiveness, and scores of libido, mating ability and sexual behavior differed significantly among the various categories of the 3 indices. Libido significantly correlated with mating ability (r=0.89; P<0.001). Sexual behavior expressed significant relationship with age (r=0.41; P<0.01) and body weight (r=0.48; P<0.01), but was nonsignificant with the scrotal circumference (r=0.28; P>0.05) of buffalo bulls. However, these relationships were absent (P>0.05) in the acceptable sires. Semen production was correlated with sexual behavior in only the fair and poor categories of buffalo bulls (r=0.84; P<0.005). Sexual behavior had no relationship with the fertility rate of buffalo bulls (r=0.44; P>0.05). It is concluded that the sexual behavior index can be used successfully for the selection of buffalo bulls. Excellent- to good bulls should be used in an artificial breeding program if they qualify in the other selection indices.     

The advantages of low-density lipoproteins in the cryopreservation of bull semen

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen–thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P < 0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P > 0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P < 0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.     

Polymorphisms in candidate genes as markers for sperm quality and boar fertility

Alpha-actinins (ACTN1 and ACTN4) and gamma-actin (ACTG2), were investigated as candidate genes on the basis of their known functions, for their possible association with sperm concentration, motility, semen volume per ejaculate, plasma droplets rate, abnormal sperm rate and the fertility traits, non-return rate and number of piglets born alive. Polymorphisms were identified in intron 18 (G>A) of the porcine ACTN1 gene and in exon 22 (A>C) of the porcine ACTN4 gene by comparative sequencing of animals from the Pietrain (PI) and Hampshire (HA) breeds. Pietrain (n = 244) and crossbreed PI x HA (n = 112) boars from an artificial insemination boar station were genotyped for the single nucleotide polymorphisms (SNPs) within ACTN1 and ACTN4 as well as for a previously described microsatellite within ACTG2. The study provides evidence for effects of ACTN1 on fertility and of ACTG2 on sperm quality traits, while no indication of impact of ACTN4 on any of these traits was found.     

In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations

The acrosome reaction (AR) is a prerequisite for normal sperm fertilizing capability and can be studied in vitro after induction by various agents. The efficacy of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential. During the past two decades, a number of attempts have been made to relate the in vitro-induced AR to field fertility in several species. However, to our knowledge, no studies have combined in vitro induction of the AR with the simultaneous detection of sperm viability and acrosomal status using a high-precision flow cytometric technique. Furthermore, large-scale fertility trials using low-dose inseminations are pending. In the current study, the relationship between field fertility and the in vitro-induced AR was investigated using three ejaculates from each of 195 bulls, 156 Holstein and 39 Jersey bulls (Bos taurus), participating in a progeny test program including low-dose inseminations. A range of insemination doses, varying from 2.0 × 10⁶ to 15 × 10⁶ sperm/dose, was obtained by a controlled dilution process applied to each ejaculate. Different insemination doses were distributed at random among 75,610 experimental first inseminations in 4721 herds and 208 artificial insemination (AI) technicians. Simultaneous detection of sperm viability and acrosomal status was achieved using a triple color flow cytometric technique. Sperm samples from the bulls displayed a wide range of ability to acrosome react in response to calcium ionophore A23187. Both reproducibility of the AR response after induction and relationship between ability to acrosome react and field fertility was highly dependent on the definition of AR inducibility. Six basic and six combined AR indices were assessed. The AR index expressing the fraction of acrosome reacted sperm in the live sperm population after induction by ionophore had the highest repeatability, best described the biological variation in the studied population, and yielded the best significant predictive values on field fertility among the 12 indices considered. Moreover, the ability of sperm to acrosome react appeared to be a noncompensable trait that affects fertility regardless of the number of sperm per insemination dose. The current results therefore indicate that this sperm parameter is important in the field and also may play a role in the IVF laboratory.     

The 1999 and 2010 WHO reference values for human semen analysis to predict sperm DNA damage: A comparative study

Standard semen parameters are often used to predict male fertility, but whether the 2010 World Health Organization (WHO 2010) thresholds are better predictors than the 1999 thresholds has not been investigated. In this study, we addressed this issue using sperm DNA fragmentation (SDF) as a marker of male fertility in 134 subfertile male individuals. To compare the predictive value of the 1999 thresholds with the 2010 cutoffs, the Youden indices (YIs) of all possible thresholds were calculated using receiver operating characteristic (ROC) curves and compared to each other and to the respective YIs of optimal thresholds. We found that the area under the ROC curves of progressive motility and vitality was the highest among standard semen parameters, and that the YI of both parameters from the 2010 manual was comparable to the respective optimal YIs. In contrast, the threshold of sperm concentration and total sperm number from both WHO recommendations demonstrated low YIs, with substantial differences to the respective optimal YIs. The YIs of normal morphology cutoffs from both WHO manuals were slightly different from each other and from the respective optimal YIs. In conclusion, the 2010 thresholds for progressive motility and vitality are superior to the 1999 thresholds in predicting SDF, whereas the cutoff value of sperm concentration, total sperm number and normal morphology may need further revisions to increase their accuracy.     

The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation

Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell^® extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2 mM), cysteine (2 mM), and control, were designed to analyze the antioxidants in Bioxcell^®. Insemination doses were processed so that each 0.25-ml straw contained 15 × 10⁶ sperm.The addition of cysteine led to higher motility, compared to the other groups (P < 0.001). Cysteine showed a greater protective effect on the percentages of acrosome damage and total abnormalities in comparison to the other groups (P < 0.001). No significant differences were observed in hypo-osmotic swelling test (HOST), following supplementation with antioxidants during the freeze-thawing process. No significant difference was observed in non-return rates among groups. In biochemical assays, the additives did not show effectiveness on the elimination of malondialdehyde (MDA) formation and maintenance of GSH and GSH-Px activities, when compared to controls. CAT activity (35.1 ± 8.1 kU/g) was demonstrated to be significantly higher upon the addition of 2 mM taurine (P < 0.001), while the level of MDA increased, indicating oxidative stress in this group. SOD activity (21.4 ± 2.9 U/g protein) was significantly elevated in the group with cysteine, compared to the other groups (P < 0.001).     

Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis

The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.     

“Decoding the Dots”: The ImageStream system (ISS) as a novel and powerful tool for flow cytometric analysis

The aim of this article is to provide a brief review of the ImageStream system (ISS). The ISS technology was developed as a novel method for multiparameter cell analysis and subsequently as a supportive tool for flow cytometry (FC). ISS integrates the features of FC and fluorescent microscopy collecting images of acquired cells for offline digital image analysis. The article presents an overview of the main characteristics of ISS and a comparison between ISS, FC and the laser scanning cytometer (LSC). We reviewed ISS applications focusing on those involved in cellular phenotyping and provide our own experience with using ISS as a supportive tool to classical FC and demonstrate the compatibility between FC and ISS photometric analysis as well as the advantages of using ISS to confirm FC results.     

Flow cytometric sexing of spider sperm reveals an equal sperm production ratio in a female-biased species

Producing equal amounts of male and female offspring has long been considered an evolutionarily stable strategy. Nevertheless, exceptions to this general rule (i.e. male and female biases) are documented in many taxa, making sex allocation an important domain in current evolutionary biology research. Pinpointing the underlying mechanism of sex ratio bias is challenging owing to the multitude of potential sex ratio-biasing factors. In the dwarf spider, Oedothorax gibbosus, infection with the bacterial endosymbiont Wolbachia results in a female bias. However, pedigree analysis reveals that other factors influence sex ratio variation. In this paper, we investigate whether this additional variation can be explained by the unequal production of male- and female-determining sperm cells during sperm production. Using flow cytometry, we show that males produce equal amounts of male- and female-determining sperm cells; thus bias in sperm production does not contribute to the sex ratio bias observed in this species. This demonstrates that other factors such as parental genes suppressing endosymbiont effects and cryptic female choice might play a role in sex allocation in this species.     

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric,microscopic and oxidative parameters in ram semen

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l⁻¹ pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l⁻¹) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.     

Impact of genomic selection of AI dairy sires on their likely utilization and methods to estimate fertility: a paradigm shift

Breeding of dairy cattle is undergoing a paradigm shift to genomic selection of potential sires and dams. This undoubtedly will affect how bulls are managed in an artificial insemination (AI) center and impact methods to estimate their ‘fertility'. Our goal is to help decision-makers understand the contents of a straw of semen, current estimates of sire fertility, and how estimates might evolve in a genomic era. Sire fertility is estimated from outcome (pregnant or not) after 300 to > 2,000 inseminations and reported in units (U) as a sire's deviation from a population (> 500 bulls) average pregnancy rate (PR). Too often users do not recognize that imprecision of an estimate encompasses a 3-U range, or more. ‘True fertility' of the sire whose semen is inseminated influences outcome far less than ‘true fertility' of each female and a myriad of microenvironment and management factors. Further, AI centers discard substandard collections and intentionally adjust number of sperm per straw so that differences in pregnancy rates achieved by different sires are minimized! For > 80% of Holstein bulls, estimated ‘sire conception rates' are within a 5.4-U range. In the future, most sires will be 15 to 40 mo old and services will accumulate at > 1,000/mo. Estimated sire conception rates still will be a deviation from the population mean, but should be based on records for the most recent 6 or 12 mo, rather than 48 or 60 mo. Repeated ‘snap shots' every 2 mo would allow AI centers to adjust number of sperm per AI straw from genomic-sires in a timely manner, to maintain high pregnancy rates, and to meet market demands with sires producing ∼40% as many sperm as mature ‘proven sires' of yesteryear.     

Male facial attractiveness and masculinity may provide sex‐ and culture‐independent cues to semen quality

Phenotype‐linked fertility hypothesis (PLFH) predicts that male secondary sexual traits reveal honest information about male fertilization ability. However, PLFH has rarely been studied in humans. The aim of the present study was to test PLFH in humans and to investigate whether potential ability to select fertile partners is independent of sex or cultural background. We found that on the contrary to the hypothesis, facial masculinity was negatively associated with semen quality. As increased levels of testosterone have been demonstrated to impair sperm production, this finding may indicate a trade‐off between investments in secondary sexual signalling (i.e. facial masculinity) and fertility or status‐dependent differences in investments in semen quality. In both sexes and nationalities (Spanish and Colombian), ranked male facial attractiveness predicted male semen quality. However, Spanish males and females estimated facial images generally more attractive (gave higher ranks) than Colombian raters, and in both nationalities, males gave higher ranks than females. This suggests that male facial cues may provide culture‐ and sex‐independent information about male fertility. However, our results also indicate that humans may be more sensitive to facial attractiveness cues within their own populations and also that males may generally overestimate the attractiveness of other men to females.