Comparison of computer-assisted sperm analysis and smartphone-applied sperm analysis for evaluation of frozen-thawed bull semen.

This study aims to compare the efficacy of computer-assisted sperm analysis (CASA) and smartphone-applied sperm analysis (SASA) in assessing the quality of frozen-thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA-37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA-25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA-37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen-thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p > .05). However, significant differences among the groups were observed for total motile spermatozoa values (p < .001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p < .001). The immotile spermatozoa values were significant between the groups (p < .001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen-thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis.

Evaluation of predictive ability of linear type gonadal traits on reproductive capacity of breeding dairy bulls.

Dimensions of linear type traits facilitate selection of livestock for breeding and rearing. To date, use of linear type traits for selection of breeding bulls is highly concentric to scrotal circumference (SC), with probable overlook to other important traits. Present study reported the importance of various gonadal linear type traits on spermatozoa production, age-related changes in gonadal linear type traits of bulls and predictive ability of these traits on bulls' reproductive potentials. Among all gonadal traits, testicular density (TD), scrotal volume (SV), paired testicular weight (PWT) and SC were found most important predictor variables in order, which can discriminate between good/poor breeding bulls, that is, produced frozen semen doses (FSD) or not. Dimensions of gonadal traits increased significantly up to 36 months age and thereafter, development became slow and negligible. In contrast, TD decreased by 30%, 51%, 64%, 68% and 71% at 12, 24, 36, 48 and >49 months age, respectively, from its base value at 6 months. Bulls of lower TD (≤0.88 g/cm3) had significantly higher ejaculate volume (+9%), sperm motility, sperm concentration (+100 million/mL) and sperm output (+26%)/ejaculate as compared to bulls of higher TD (>0.88 g/cm3). Discriminant function was developed using TD, SV, PWT and SC to identify bulls of superior reproductive potentials. It was concluded that among the investigated traits, TD was the strongest to discriminate between FSD and Non-FSD bulls. Therefore, our findings suggested that TD could be more potential trait than SC for dairy bulls' breeding soundness evaluation and assessment of reproductive ability.

Optimising Laparoscopic Artificial Insemination in Lohi Sheep: Effects of Timing, Sperm Concentration and Body Condition on Fertility Outcome in Subtropical Climates.

The objective of this study was to determine the optimal timing, sperm concentration, and body condition score (BCS) for laparoscopic artificial insemination (LAI) in the subtropical Lohi sheep breed. In Experiment 1, Lohi ewes (n = 80) were synchronised through progestin-sponges (day 0-day 11), administering PGF2α (d-cloprostenol 75 µg/mL; i.m. @ 75 µg /ewe) on day 09 and eCG (i.m. @ 300 IU/ewe) on day 11. Ewes were divided equally into four groups (n = 20 each) and then LAI was performed at 48 (T48), 60 (T60), 72 (T72) and 96 (T96) hours post-sponge removal using 200 million sperm/insemination. In Experiment 2, ewes (n = 81) were synchronised as in Experiment I. Following synchronisation ewes were divided into four groups, each subjected to LAI employing varying sperm concentrations: 10 (S10; n = 21), 20 (S20; n = 20), 50 (S50; n = 20), or 100 (S100; n = 20) million per insemination. Inseminations were performed within a time window of 48-60 h post-sponge removal, based on the findings from Experiment 1. In both experiments ewes were categorised according to BCS, that is, medium 3 and high > 3. Results of Experiment 1 revealed a quadratic response that ewes inseminated at 60-h post-sponge removal exhibited significantly higher pregnancy and twinning rates (p = 0.02). The results of Experiment 2 revealed that sperm concentration had a quadratic effect, 50 million sperm per insemination resulted in maximal pregnancy rates (p = 0.01). Additionally, ewes with medium BCS (≤ 3) had higher pregnancy rates than high BCS (> 3) ewes. In conclusion, ewes with medium BCS (≤ 3) are ideal candidate for LAI and can be inseminated at 60 h with minimal sperm 50 × 106/dose post-sponge removal to achieve a maximum pregnancy rate.

Contraceptive effect of a gonadotropin-releasing hormone vaccine on captive male brown bears (Ursus arctos).

Fertility control has traditionally been applied in zoos to control captive populations, and reversible contraception is important. However, contraceptive methods for male bears have not been reported. We aimed to establish a reversible contraceptive for male brown bears by investigating the effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac®) that was developed for the immune castration of pigs. We vaccinated six bears with two sequential doses of 400 (n = 2) or 600 µg Improvac® (n = 4) with a 1-month interval during the pre-breeding season (February to April). We compared the reproductive parameters (testosterone levels and semen parameters) of the six vaccinated and four non-vaccinated (control) bears once during the breeding season (May or June). To investigate whether the reproductive performance could be restored in the following year of contraception, we also compared the reproductive parameters once during the breeding season in two bears between the year with GnRH vaccination and the following year without vaccination. Vaccination treatments suppressed reproductive parameters in 5 bears, although vaccination with 400 µg of Improvac® was not effective in one bear. Testosterone levels and the rate of progressive sperm motility were significantly lower, and total sperm count and testis size tended to be lower in vaccinated bears, compared with the controls. Blood biochemical findings and direct observations after Improvac® vaccination did not reveal side effects. Moreover, testosterone levels and spermatogenic scores of two bears were restored in the following year. We confirmed that the Improvac® vaccine elicited a reversible contraceptive effect in male brown bears.

Age-dependent genetic and environmental variance of semen quality in Nordic Holstein bulls.

The aim of this study was to estimate genetic and environmental parameters, across bull's age, for semen quality traits including pre- and postcryopreservation semen concentration, sperm motility, and sperm viability as well as ejaculate volume and number of doses per ejaculate. A data set on 96,595 ejaculates from 2,831 Nordic Holstein bulls collected between 2006 and 2019 was used. Genetic and environmental parameters were estimated using a random regression model and applying the average-information REML approach. Spline functions were chosen to fit the additive genetic and permanent environmental effects across bull's age, and the optimal number of knots was chosen using cross validation. Residual variance heterogeneity was assumed in different bull age classes. The estimated repeatabilities of semen quality traits ranged from 0.16 to 0.85 across different ages of bulls. The estimated heritabilities of semen quality traits ranged from 0.02 to 0.56 across different ages of bulls. The results indicate possibilities for genetic improvement of semen quality traits through selective breeding.

Evaluation of sperm and hormonal assessments in Wagyu, Nellore, and Angus bulls.

Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature bulls have a small scrotal circumference. This study aimed to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Frozen-thawed semen from Wagyu, Nellore, and Angus sires were analyzed for sperm kinetics (computer-assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity, lipid peroxidation and hormone [luteinizing hormone (LH) and testosterone] serum concentration. The results showed that Wagyu had lower total motility and an increased number of sperm with no motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations. We concluded that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from these breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation, or hormone (LH and testosterone) concentrations.

Exploring the metabolome of seminal plasma in two different horse types: Light versus draft stallions.

The application of the 'omics' studies in the field of animal reproduction has been aimed at identifying novel biomarkers of fertility since the last few years. When assessing reproductive efficiency in horses, breed should also be taken into account as it can influence semen quality and fertility. Considering the growing interest in metabolomic analysis to evaluate male fertility, we aimed to investigate the metabolomic profile of seminal plasma in two different horse breeds. Twelve healthy stallions, n.6 American Quarter Horse (AQH) and n.6 Italian Draft Horse (IDH) stallions, regularly used for artificial insemination, were included in the study. Two semen collections, performed 30-day apart, were considered for the assessment of semen parameters including gel-free volume, spermatozoa (spz) concentration, spz progressive motility and seminal plasma analysis by 1 H-NMR.Semen characteristics differed between IDH and AQH (p < .05) as well as the first cycle conception rate that was higher in AQH than IDH (p = .001). Metabolomic analysis quantified 56 molecules in equine seminal plasma, with 11 metabolites showing different concentrations in IDH compared to AQH (p < .05).This study provided evidence of differences in seminal plasma metabolites' concentrations between studied horse types, highlighting specific metabolomic fingerprints characterizing AQH and IDH sperm.

The benefits of Maca (Lepidium meyenii) extract administration for male rabbits affected by environmental heat stress.

Heat stress (HS) is the most substantial environmental issue in rabbit health status impairment, which can lower productivity. Maca is a common medicinal plant with important biological activities including antioxidant, anti-inflammatory, anti-fatigue, and neuroprotective effects. The present research explored the alleged protective role of Maca extract in alleviating the adverse impact of HS on rabbits. Growing V-line 6-week-old buck rabbits (N = 48) were orally administered Maca extract at levels of 0 (MAC0), 200 (MAC2), 400 (MAC4), or 600 (MAC6) mg/head twice per week. Compared to the control groups, rabbits provided Maca had better growth performance, feed intake and feed conversion ratio. Liver and tests weights were significantly higher (p < 0.05) in Maca treated groups compared to the control. hemoglobin, white blood cell, red blood corpuscles, platelet count, hematocrit, and lymphocytes were significantly increased in the MAC4 group compared with the control group. MAC4 treatment significantly reduced aspartate aminotransferase and malondialdehyde levels compared with other groups. Moreover, the concentrations of total bilirubin, creatinine, glucose, total cholesterol, and triglycerides were lower (p < 0.05) in the MAC4 group than in the controls. Oral administration of Maca improved cortisol and testosterone values, total antioxidant capacity, and superoxide dismutase. The MAC4 group exhibited significant improvement (p < 0.05) in sperm motility, survival, membrane functionality, concentration, and libido, with a significant decrease in abnormal sperm compared with the control group. Also, aforementioned group showed sperm cells with a normal structure and intact plasma membranes, acrosomes, and well-organised axonemal components. Histopathological screening of liver sections showed moderate to severe degenerative and necrotic changes in rabbits exposed to HS, which were alleviated with Maca oral administration. In conclusion, oral administration of Maca extract at 400 or 600 mg/head-weekly enhanced growth performance, hemato-biochemical attributes, antioxidant status, and semen quality in rabbits during the hot season.

Genetic analysis for semen quality traits in buffalo bulls.

This study was attempted to estimate the genetic parameters of semen quality traits in buffalo bulls. The study data consisted of 10975 ejaculates from 45 Murrah buffalo bulls (aged 24-72 months) used for breeding program during year 2010 to 2020. Semen quality traits (ejaculate volume, concentration of sperm, mass activity, initial and post-thaw motility, number of sperms per ejaculate, motile sperm number and discard rates) were studied. It was observed that average ejaculate volume was 2.82 ± 1.45 mL with mean concentration of 1040.12 ± 523.26 million/mL. Higher heritability was observed for number of sperms per ejaculate, number of motile sperm and sperm concentration. Significant phenotypic correlation was obtained between volume and number of sperms per ejaculate as well as volume and number of motile sperms. Likewise, significant phenotypic correlation was evident between sperm concentration with sperm number per ejaculate. Highest phenotypic correlation was obtained between sperm count per ejaculate and motile sperm count. Estimated genetic trends showed significant change in volume and motile sperm number. In conclusion, this study ascertains that genetic parameters of semen traits can be considered during the selection of buffalo bulls in breeding program.

Semen characteristics of ganders kept as genetic resources conservation programme.

The creation of genetic reserves of domesticated animal species and breeds almost become a necessity in the recent years, but there is a question what is the value of semen of males kept ex situ in vivo as gene conservation flocks. Presented studies assessed the response to semen collection by dorso-abdominal massage and the quantitative and qualitative semen characteristics of six goose breeds (Pomorska, Garbonosa, Kuban, Landes, Roman and Slovakia) covered by the genetic resources' protection programme. Fourteen semen collection attempts were performed per male. In each breed there were ganders with low and high sensitivity to massage. The most positive reactions were stated in Pomorska ganders (67.9%) and the least in Kuban breed (52.60%). Individual male evaluation showed that only in three breeds (Pomorska, Garbonosa and Kuban) there were individuals showing 100% susceptibility to semen collection, in some breeds only one to four positive reactions (ending with ejaculation) were noted. Results obtained indicated breed and male effect on analysed semen traits, with the exception of sperm motility. The highest number of live normal sperm (44.2% on average), sperm concentration (530 × 106 ml-1 ), the highest Semen Quality Factor (92.9) and sperm motility (50.30%) were found in semen of Kuban ganders, while the lowest values of these traits (28.7%; 230 × 106 ml-1 ; 11.4 respectively) in Slovakia ganders. The lowest sperm motility (38.3%) was observed in ganders of Roman breed, but comparing to the other breeds existing differences were not significant. Significant differences in sperm morphology between individual ganders were also observed.

Association of linear type pelvic traits with testicular characteristics and reproductive capability of breeding dairy bulls.

Associations of pelvic linear type traits (PLT) on production and reproduction of cows were widely documented; in contrast, importance of PLT on bulls' reproductive ability, semen quality, semen cryo-preservability, frozen semen doses (FSD) production etc. was inadequately reported. The present study was conducted on Frieswal bulls (N = 378, age: 6-91 months, m) to assess age-wise growth dynamics of pelvic dimensions, classifying age into 6 groups (6 m, 7-12 m, 13-24 m, 25-36 m, 37-48 m, >48 m). Iso-age group bulls' (N = 100; 25-36 m) records were analysed after classifying seasons (summer/rainy/winter) of semen collection, if pelvic morphometric traits had any practical associations with testicular traits, semen quality and FSD production or not. A discriminant function was developed based on PLT, which showed poor predictive ability to distinguish FSD/non-FSD category bulls. Age significantly influenced growth and dimensions of gonadal and external pelvic morphometry traits. The dimensions of PLT increased at higher rate up to 36 m age and thereafter enhancement became insignificant. Pelvic linear type traits were positively (p < .01) associated with testicular/scrotal traits. Among PLT studied, pelvic triangle area (PTA) was the strongest discriminating variable to distinguish between good and poor breeding bulls. Bulls of larger PTA (≥1000 cm2 ), at average 30 m age, produced relatively inferior-quality semen, viz. lower volume (-3%), sperm concentration (-48 × 106 /mL), motility, semen quality index, total sperm counts/ejaculate (-601 × 106 ), motile sperm counts/ejaculate (-474 × 106 ) and total post-thaw motile sperm counts (-226 106 ), than bulls of lesser PTA (<1000 cm2 ). It was concluded that external PTA could be a useful addition to the bulls' breeding soundness evaluation.

Comparison of two staining techniques on the manual and automated canine sperm morphology analysis.

Detailed and direct analysis of semen, including sperm morphology, enables a diagnosis of male fertility. This study aimed to describe an economical and verified protocol for canine spermiograms and compare the effectiveness of Sperm Stain® and Sperm Blue® (Microptic, Spain) in veterinary practice. Sperm assessment was conducted manually, using a standard optical microscope, and via computerized semen analysis using the SCA® CASA (Sperm Class Analyzer® CASA System-MICROPTIC, Spain). This study showed that Sperm Blue® is a better solution for computerized sperm quality analysis of healthy dogs. At the same time, Sperm Stain® turned out to be more helpful in identifying specific morphological defects of sperm. Automated canine sperm morphology analysis worked better with Sperm Blue stain, but Sperm Stain simplified manual evaluation of various organelles' defects. Standard, manual examination is more error-prone for an inexperienced andrology technician, but it seems to be still a gold standard technique for canine sperm assessment.

Genetic evaluation of semen production potential in Indigenous bulls using random regression models in a Bayesian framework.

Genetic potential of indigenous bulls with respect to semen production traits over the age of the bulls at semen collection was analyzed using random regression models (RRMs). Data pertaining to 59,641 ejaculates from 189 bulls of 18 indigenous breeds collected from BAIF (Bharatiya Agro Industries Foundation) was utilized for this study. Six semen production traits, viz., ejaculate volume (EV, ml), sperm concentration (SC, 109/ml), initial sperm motility (ISM, %), post-thaw motility (PTM, %), the total number of spermatozoa per ejaculate (TNS, 109/ejaculate), and the theoretical number of semen doses (TNSD) were studied. Univariate and RRM were used to obtain variance components and genetic parameter estimates. Two hundred thousand Gibbs samples were generated for each trait with a burn-in of 20,000 and thinning interval of 50 in a Bayesian framework. Legendre polynomials with orders of fit up to 5 for additive and permanent environmental effects were used. RRM modeled the heritability and repeatability for all ages between 3 and 10 years (productive lifespan). Heritability estimates ranged from 0.18 to 0.36, 0.18 to 0.45, 0.02 to 0.06, 0 to 0.001, 0.09 to 0.32, and 0.14 to 0.42 while the repeatability estimates ranged from 0.41 to 0.72, 0.36 to 0.79, 0.04 to 0.10, 0 to 0.001, 0.37 to 0.56, and 0.32 to 0.57 for EV, SC, ISM, PTM, TNS, and TNSD, respectively. Variability of estimates over the age of the bulls obtained through RRM could be useful to further refine the breeding program for age at selection, deciding the production period and age at culling.

Sperm production and quality in European eel (Anguilla anguilla) in relation to hormonal treatment.

Aquaculture production relies on controlled management of gametogenesis, especially in species where assisted reproduction is needed for obtaining gametes in captivity. The present study used human chorionic gonadotropin (hCG) treatments to induce and sustain spermatogenesis in European eel (Anguilla anguilla). The aim was to evaluate effects of strip-spawning timing (12 vs. 24 hr) after weekly administration of hCG and the necessity of a primer dose (in addition to weekly hormonal treatment) prior to strip spawning (primer vs. no-primer) on sperm quality parameters. Sperm parameters included milt production (weight), density and sperm kinematics at Week 9, 11 and 13 after onset of treatment. Spermiation commenced in 11.5% of males in Week 5 and by Week 9, and all males produced milt. Male weight, milt production, sperm density and spermatocrit did not differ among hormonal treatments during the experimental period. Overall, male weight decreased from 106.3 to 93.0 g, milt weight increased from 3.5 to 5.4 g, sperm density counts decreased from 11.7 × 109 to 10.5 × 109  cells/ml, and spermatocrit decreased from 46.5% to 40.5%. Furthermore, spermatocrit was positively related to haemocytometer counts (R2  = .86, p < .001), providing a reliable indicator of sperm density. Differences in sperm kinematics were observed depending on strip-spawning timing after hormonal injection (12 vs. 24 hr) but with no consistent pattern. These sperm quality parameters also did not consistently differ between the no-primer and primer treatments. Considering that each male may be stripped 4-5 times over the 2-3 months spawning season, omitting the primer would reduce animal handling, material costs and labour intensity, while sustaining high-quality sperm production.

Mobility of Japanese quail spermatozoa and its relationship to egg fertility.

Sperm mobility (SM) appears to be primary determinant of fertility in chicken and turkey. The aims of this study were to extend the concept to the Japanese quail by developing an assay to quantify SM, explaining the basis of SM using motility properties measured by CASA, and exploring the relationship between SM and egg fertility. The study was carried out in three stages: i) males (n = 20) and females (n = 20) were mated individually; ii) ejaculates were collected from 20 males, and SM was measured; iii) males (n = 20) and females (n = 20) were mated individually. In Stages I and III, data were collected for egg fertility, SpermOPVL and HolesIPVL . In Stage II, SM assay was developed and assay conditions were defined: effect of sperm numbers on absorbance in Accudenz solution; effect of Accudenz concentration on sperm motility and mobility; effect of quail proctodeal gland foam extract and incubation temperature on SM at 37 and 41°C. The recorded absorbance of sperm movement was dependent on sperm numbers in the sperm suspension overlaying the Accudenz (p < .001). At 41°C, SM, progressively motile sperm, VCL, VSL and VAP were negatively affected by Accudenz concentration (p < .05). The effect of foam on SM and motility depended on an interaction between the concentration of foam extract and incubating temperature. Males were categorized into low, average and high SM phenotypes. These categories differed significantly (p < .001), but sperm motility and SM were not related to egg fertility. In conclusion, SM assay can be used to identify mobility phenotypes, but the poor relationship between SM and egg fertility indicates a need for further studies on interaction between the concentration of foam extract, incubating temperature, and in vivo sperm movement and egg fertilization success.

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics.

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.

Development of a RAPD marker-based classification criterion for quality semen production in Holstein crossbred bulls.

In cattle production systems, an intense selection pressure for production traits has resulted in the decline of fertility traits. To optimize an efficient reproduction system, the inclusion of both male and female fertility traits in the selection process is very much essential. RAPD (Random Amplified Polymorphic DNA) was developed as a molecular biology tool and has been extensively used, to study intra- and interspecific genetic diversity. The present study was undertaken to utilize RAPD primers to investigate the association between DNA markers and semen quality traits viz. Sperm concentration, total sperm count ejaculate and initial sperm motility and thereby to identify good/poor semen producers. DNA isolated from the blood samples of healthy bulls was subjected to RAPD-PCR. The multiple regression analysis followed by independent t test was carried out to identify suitable markers. Based on the results, only 12 bands were identified as marker suitable for any of the quality trait. This includes, OPA2 ~ 760, OPA2 ~ 700, OPA6 ~ 1,200, OPA9 ~ 400, OPA9 ~ 380, OPA12 ~ 970, OPA14 ~ 715, OPA14 ~ 605, OPA16 ~ 485, OPA17 ~ 860 and OPA18 ~ 480. Multiple regression analysis selected, OPA2 ~ 760 and OPA2 ~ 1,750 for sperm concentration and OPA2 ~ 760, OPA2 ~ 700, OPA9 ~ 620, OPA4 ~ 670 and OPA18 ~ 1,015 for total sperm count/ejaculate. But the t test revealed a significant association between OPA2 ~ 760 and total sperm count. Further, discriminant function analysis also identified this marker in the first step itself. The results of the present study can be exploited as a low-cost alternative strategy for identification of good /poor semen producers in crossbred bulls at an early age.

Multivariate models for estimating jackass semen production and quality.

The purpose of this study was to analyse the effects of season, age, gonad and accessory sex glands on semen characteristics of jackass and to construct multivariate regression models to predict semen quality. In autumn, spring and summer, semen characteristics of 30 sexually mature donkeys (1,014 ejaculations) were analysed to investigate the effect of seasons on semen quality, and gonad and accessory sex gland parameters of 12 jackasses were measured immediately after ejaculation by ultrasonography to investigate the effect of seasons on reproductive organ size. Semen (598 ejaculates), gonad and accessory sex gland parameters of 40 jackasses aged between 3 and 7 years were analysed in autumn to investigate the effects of age and reproductive organ size on semen quality and to construct multivariate models. To verify the accuracy of the models, semen (476 ejaculates), gonad and accessory sex gland parameters of 20 jackasses were measured from March to June. Results revealed that semen, gonad and accessory sex gland parameters were not affected by season and age. Progressive motility (PM) was positively correlated with long axis of the spermatic cord (LASC) and negatively correlated with percentages of sperm abnormality (PSA). Total sperm count (TSC) was positively correlated with testis circumferences (TC) and cross-sectional area of cauda epididymis (CSACE). TC, CSACE, LASC and PSA were included into multivariate models to predict PM, TSC and functional sperm count (FSC) in 20 jackasses (PM = 72.332 + 0.428 LASC - 0.441 PSA; TSC = -169.929 + 8.728 TC + 0.253 CSACE; FSC = -206.645 + 8.788 TC + 0.258 CSACE). The predicted and observed values corresponded well. In conclusion, the tested models can be used for predicting semen quality of donkey.

Evaluation of the use of SYBR-14 and propidium iodide stain in a fluorescent computer-assisted spermatozoal quantification method in dogs.

The aim of this study was to evaluate the use of SYBR-14/propidium iodide (PI) stain in a computer-assisted spermatozoal quantification (CASQ) method of determining spermatozoal concentration in canine semen. In Experiment A, the spermatozoal concentration was measured (n = 52) with a haemocytometer and by CASQ under fluorescent illumination using green long-pass (G-LP) and red long-pass filters at measurement concentrations of <25 million/ml. For the red filter, the limits of agreement between the haemocytometer and CASQ were -6.3% to 6.8% and -7.5% to 6.2% between the haemocytometer and CASQ for the G-LP filter. For the red filter, the mean precision CVs were 2.21% ± 4.33% (mean ± 95% CI) for the haemocytometer, 2.19% ± 4.29% for CASQ and using the G-LP filter 2.13% ± 4.18% for the haemocytometer and 2.66% ± 5.21% for CASQ. In Experiment B, spermatozoa were also examined with a green spectrum short-pass (G-SP) filter (n = 50) at measurement concentrations of <12.5 million/ml. The limits of agreement between the haemocytometer and CASQ were -5.4% to 7.8% using the red filter, -15.8% to 14.3% using the G-LP filter and -13.1% to 11.3% using the G-SP filter. The mean precision CVs for the haemocytometer and CASQ, respectively, were 2.68% ± 5.26% (mean ± 95% CI) and 1.93% ± 3.72% using the red filter and 2.01% ± 3.95% and 3.55% ± 6.95% using the G-LP filter, and 3.96% ± 7.76% for CASQ using the G-SP filter. Using the red filter, the agreement between the haemocytometer and CASQ and the precision of both haemocytometer methods and CASQ were better than when using green filters. The CASQ method performed using green filters produced acceptable results; however, CASQ using a red filter with PI staining alone was superior to that using green filters and SYBR-14/PI staining.

Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles.

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 106 sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1 mg/ml papain or 5 U/ml bromelain were used (n = 10 straws per treatment). Immediately after thawing (38°C for 40 s), sperm concentration of each straw within treatment was readjusted to 15 x 106 sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p < .05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p < .05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p < .05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.