The Predictive Value of Porcine Seminal Parameters on Fertility Outcome under Commercial Conditions

The aim of this study was to address the question of whether differences in farrowing rate and litter size after the use of different ejaculates could be predicted using the standard semen parameters under commercial conditions. In this study, a total of 1818 sows were used to evaluate the fertility predictive value of different sperm parameters. Logistic regression analysis (univariate and multivariate) was used to correlate the dichotomous farrowing rate data to the sperm parameters. Linear regression was also used to determine the relationship between litter size and semen parameters (Pearson correlation and multiple regression). Receiver-operating curves (ROC) were used to determine the overall performance characteristics of each semen variable in the logistic regression model. Semen analysis, under commercial conditions, allows to identify ejaculates with very low fertility potential but the pre-selection of the samples, the high number of sperm per doses and the high quality of the semen used in artificial insemination (AI) programmes reduces the variability. Therefore, it is unlikely to detect fertility differences associated with seminal parameters.     

Genetic Parameters for Semen Production Traits in Austrian Dual-Purpose Simmental Bulls

Genetic parameters were estimated for semen production traits collected in an Austrian AI centre in the years 2000-2004. In total, 12,746 ejaculates from 301 Austrian dual-purpose Simmental (Fleckvieh) AI bulls were examined considering different effects on ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. The model for genetic parameter estimation included the fixed effects age of bull, collection interval, number of collections on collection day, bull handler, semen collector, year and month of collection, a random additive genetic component and a permanent environmental effect. Correlations between estimated breeding values for semen traits and male fertility from the routine evaluation were calculated. The fertility trait considered in the routine evaluation is non-return rate 90 for the first insemination. All semen production traits were moderately heritable. Heritabilities for volume, concentration, percentage of viable spermatozoa, total number of spermatozoa and motility were 0.18, 0.14, 0.10, 0.22 and 0.04, respectively. Correlations between breeding values for semen quality traits and routinely estimated breeding values for male fertility were low and ranged from 0.08 to 0.17 indicating that semen production traits are rather poor predictors of male fertility.     

Multivariate Analyses for Determining the Association of Field Porcine Fertility With Sperm Motion Traits Analysed by Computer‐Assisted Semen Analysis and With Sperm Morphology

This study investigates the association of semen traits with boar fertility. The fertility outcome (farrowing rate – FR and total piglets born – TB) of 14 boars was obtained from a field trial conducted during 10 week of breeding period on a commercial farm using multiparous sows (n = 948) through single‐sire mating with 2 × 10⁹ motile sperm cells per artificial insemination (AI) dose. Sperm motion parameters, evaluated with computer‐assisted semen analysis system in raw and stored semen at 17°C for 240 h, in addition to morphological sperm defects, measured on the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate cluster, canonical and discriminant analyses. Four clusters of boars were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The other boars were separated in two distinct clusters (four and seven boars), including boars with intermediate TB (12.64 and 13.22) but divergent values for FR (95.9% vs 91.8%). Semen traits with higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement and cytoplasmatic droplets. Through multivariate discriminant analysis, more than 80% of the 140 ejaculates were correctly classified into their own group, showing that this analysis may be an efficient statistical tool to improve the discrimination of potential fertility of boars. Nevertheless, the validation of the relationship between fertility and semen traits using this statistical approach needs to be performed on a larger number of farms and with a greater number of boars.     

Die Genauigkeit von Ejakulatsuntersuchungen zur Bestimmung der Befruchtungsfähigkeit von Bullensperma

Contents Investigations on 3432 ejaculates from 57 A. I. sires showed that there were practically significant relations between semen traits and conception rate only between bulls but not within bulls. Therefore the first five ejaculates already provided a satisfactory estimate of the fertility of normal A. I. bulls. The conception rate could be estimated by five examinations with an accuracy of R = 0.65 and by 60 examinations per bull with an accuracy of R = 0.70.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Multivariate Cluster Analysis Regression Procedures as Tools to Identify Motile Sperm Subpopulations in Rabbit Semen and to Predict Semen Fertility and Litter Size

Computerized motility analysis (CASA) shows that four separate subpopulations of spermatozoa with different motility characteristics co-exist in rabbit ejaculates. There were significant (p < 0.01) differences in the distribution of these subpopulations among separate genetic lines, total sperm abnormalities and the percentage of altered acrosomes. Furthermore, logistic and linear multivariate regressions among several parameters of rabbit semen quality analysis were tested for use as predictive tools for the fertilizing ability of a specific artificial insemination semen sample. Logistic regression analysis rendered two mathematical, significant (p < 0.01) models: one between sperm viability and conception rate and the other between total sperm abnormalities and conception rate. Multiple linear regression analyses also yielded some significant relationships between both fertility (p < 0.001) and litter size (p < 0.05), with respect to some semen characteristics. Our results support the hypothesis that the predictive in vivo fertility use of the standard rabbit semen quality analysis coupled with a CASA determination could be reasonably achieved by applying linear and logistic regression analyses among several parameters of rabbit semen quality analysis.     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage

Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.     

The thermoresistance test of spermatozoa and fertility in bulls

The spermiograms of 17 bulls were studied and 160 ejaculates were subjected to the thermoresistance test (38 degrees C) to evaluate sperm survival after thawing. After the first insemination of 10 682 cows, statistically significant differences were found in the fertilizing capacity of the ejaculates with various values of the thermo-resistance test. The best sperm fertilizing capacity was obtained in the ejaculates which retained progressive movement in 40% of the spermatozoa after two hours of exposure to the thermoresistance test. Out of the 1496 cows inseminated, 971 (i.e. 64.9%) got in calf, whereas after the insemination of 4216 cows with semen where only 30% of spermatozoa moved progressively at the end of the test, the number of pregnant dams was 2403, i.e. 56.98%; this difference is statistically significant (p0.05). At a lower sperm activity in the test the fertility after the first insemination was even lower. Although there was some difference in the individual fertility of bulls (54 to 67%), a positive relationship between the results of the thermoresistance test and fertility was recorded in all bulls.     

Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled‐stored semen as well as motility and kidding rate of frozen‐thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk‐based and egg yolk‐free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above‐mentioned bucks, split and diluted with egg yolk‐free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3‐ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk‐free extenders containing 0.6 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120–140 × 10⁶ progressively motile spermatozoa was used for a single cervical insemination of cloprostenol‐synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk‐deficient (2.5%) extenders significantly improved viability of chilled‐stored semen together with motility (48.5%) and fertility (62.5%) of frozen‐thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled‐stored semen but also post‐thaw motility (47.5%) and fertility (53.75%) of frozen‐thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.     

Fertility of a stallion with low sperm motility and a high incidence of an unusual sperm tail defect

At the beginning of the breeding season an eight-year-old standardbred stallion had semen with virtually zero sperm motility and an approximately 90 per cent incidence of midpiece and tail defects. The motility of the sperm improved to 7 per cent when semen was collected daily but its morphology did not improve. Electron microscopy revealed that the defects consisted mainly of a loss of microtubules in the axoneme and of disorganised midpieces. A pregnancy rate of 24 per cent per cycle and 44 per cent for the season was achieved in 32 mares after the insemination of whole ejaculates collected from the stallion frequently. The fertility was much higher than would have been expected from the characteristics of the semen. It is concluded that this sperm defect, reminiscent of the 'Dag defect' in bulls and the defect in T-locus mice, does not render the animal infertile.     

Semen quality and fertility after heat stress in boars

Sperm morphology and the fertilizing capacity of ejaculated spermatozoa were examined in 6 Swedish Landrace boars before and after heat stress. The boars were exposed to 35° C during 100 h in a climatic room. Fertility was measured by insemination of gilts before and at various times after heat stress. Each gilt (n = 44) was inseminated with a total of 5×10⁹ spermatozoa diluted to 10O ml with EDTA-glucose diluent and fertilization was assessed by examining recovered ova 2 days after insemination.Changes in semen quality varied among the boars from a very weak response in 2 boars to pronounced semen alterations occurring 2–6 weeks after heat stress in the other boars. A close relationship was found between seminal changes and fertilization rates, all ejaculates which had high fertilization rates being of the same quality as the pre-exposure ejaculates. The ejaculates that had poor fertility were characterized by lowered sperm motility and increased numbers of spermatozoa with abnormal heads, proximal cytoplasmic droplets and nuclear pouch formations.     

Prediction of first season stallion fertility of 3-year-old Dutch Warmbloods with prebreeding assessment of percentage of morphologically normal live sperm.

In the selection procedure to acquire a breeding licence, 3-year-old Dutch Warmblood stallions have to undergo a breeding soundness test It is questioned whether this evaluation is predictive of the stallion's fertility results in the first breeding season. Therefore, semen parameters at the beginning of their first breeding season were evaluated and correlated to nonreturn at first cycle and foaling rate of mares bred by stallions (n = 13). The total number of mares inseminated with chilled semen from those stallions was 1055. Semen parameters were recorded on 2 ejaculates, collected 1 h apart. Percentage progressive sperm motility, % morphologically normal from unstained spermatozoa (MNA), % sperm cells with abnormal acrosomes and the total number of spermatozoa were correlated with first cycle nonreturn rate and foaling rate. Mean motility at evaluation was 72 +/- 6%. Mean MNA was 62 +/- 13%. Mean first cycle nonreturn rate and foaling rate were 58 +/- 15% and 69 +/- 12%, respectively. A significantly positive correlation (P<0.05) was found between the MNA and first cycle nonreturn rates. Foaling rates were not significantly correlated with semen characteristics and first cycle nonreturn rates. In conclusion, the breeding soundness test is of predictive value for the breeding results in the breeding season following the test. First cycle nonreturn rates reflect fertilising capacity better than foaling rates.     

Application of computer-assisted semen analysis to explain variations in pig fertility

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P < 0.05) effects of progressive motility, velocity curvilinear, and beat cross frequency on farrowing rate (FR). Total motility, velocity average path, velocity straight line, and amplitude of lateral head displacement affected (P < 0.05) total number of piglets born (TNB). Boar- and semen-related parameters explained 5.3% of the variation in FR and 5.9% of the variation in TNB. Motility parameters, measured by CASA, explained 9% of the boar- and semen-related variation in FR and 10% of the boar- and semen-related variation in TNB. Individual boar and genetic line of the boar affected (P < 0.0001) the variation in FR and TNB. No differences (P > 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.     

Semen collection and characterization in rockhopper penguins (Eudyptes chrysocome chrysocome).

Low egg fertility and hatchability is a common problem in captive populations of rockhopper penguins (Eudyptes chrysocome chrysocome). These conditions make sustaining a captive population challenging. A method for collecting and evaluating semen from rockhopper penguins was developed to assist in the evaluation of low egg fertility found in one captive population. Six adult male rockhopper penguins were conditioned to allow semen collection once a week from the start of breeding season until ejaculates no longer contained sperm. A total of 59 ejaculates was collected between 17 September and 31 December 2004. Forty-five of these samples were evaluated for volume, pH, sperm concentration, and sperm quality (motility, viability, and morphology). There was a large variation between individuals and between collections for each individual. The mean motility was 34.5% (+/- 22%). Mean volume of ejaculate was 0.23 ml (+/- .31 ml). Mean concentration was 16.9 x 10(6) sperm/ml (+/- 48.7 x 10(6) sperm/ml). Mean number of sperm per collection was 1.7 x 10(6) (+/- 4.2 x 10(6)). Mean percentage of living sperm was 82.9% (+/- 18.1%). Mean percentage of sperm with normal morphology was 82.1% (+/- 18.8%). Mean pH was 6.47 (+/- 0.49). During this season, only one of these males paired with a female. The pair produced one fertile egg, but the embryo died early in incubation. Male rockhopper penguins had low sperm concentration and low motility indicating that low male fertility may be contributing to the poor egg fertility rate. This work represents the first step in an ongoing study to improve captive breeding of rockhopper penguins.     

Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender

The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.     

Assessment of the morphology of frozen-thawed bull sperm in relation to its cryopreservation for artificial insemination

The morphology of sperm in raw semen was compared with that of the live sperm in semen which had been frozen and thawed. The thawed semen was stained with 6-carboxyfluorescein diacetate and propidium iodide and examined by fluorescence microscopy; smears of the raw semen were stained with eosin and nigrosin. Thirty-four ejaculates from 24 bulls of various breeds were examined. There were fewer abnormal heads, detached heads, coiled tails and proximal cytoplasmic droplets/pseudodroplets in the thawed semen than in the raw semen, there was no change in the number of bent tails, but the number of distal cytoplasmic droplets/pseudodroplets increased. There were no significant differences in morphology between ejaculates which passed or failed the osmotic resistance test after thawing, but failed batches tended to have more distal cytoplasmic droplets/pseudodroplets.     

Seminal Characteristics in Spanish Purebred Stallions: A Retrospective Study

In this work, we studied retrospectively the influence of several factors on semen quality of the Spanish Purebred (SPB) stallion. Among these factors, we considered stallion age, season, and interval between two consecutive collections. The semen was collected from 11 SPB stallions (634 ejaculates). Semen quality was evaluated based on volume, concentration, and motility of the ejaculates. Results showed that the semen collected in autumn and winter was more concentrated in comparison with that collected in spring and summer, when the volume of ejaculate was the highest. Those ejaculates collected from younger stallions were characterized by having the smallest volume and the highest sperm concentration. When incorporating an SPB stallion into a program of artificial insemination, it is important to take into account all of these factors to achieve the maximum reproductive potential of the animal.     

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield,motility and survival

Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.     

Zusammenhang zwischen Fertilität und Kopfschwanzkoppelung bei Eberspermien

Abstract: Relationship between the implantation of the tails and the fertility in boars In this study ejaculates of 47 different breed and age boars were examined. The aim of this study was to determine the relationship between the implantation of the tails and the fertility in boars. From the evaluation of 115 ejaculates it was observed that the rate of abaxial implantation of the tails in ejaculates of boars was very fluctuating. The rate of axial and abaxial implantation of the tails was stable in the ejaculates of the same boars. Although some studies indicate that abaxial implantation can be the reason for infertility the present authors observed better fertilisation results with ejaculates having higher percentages of abaxial spermatozoa.