Boar semen can tolerate rapid cooling rates prior to freezing

Two experiments were performed in the present study that demonstrated that boar spermatozoa are capable of surviving rapid cooling rates within a range of 15-5 °C before freezing. Boar ejaculates diluted in Beltsville thawing solution (BTS) (1:1, v/v) were held at 17-20 °C and shipped over a 24-h time period from two AI centres to a cryobiology laboratory, where they were pooled (Experiment 1) or cryopreserved individually (Experiment 2) using a standard 0.5-mL straw freezing protocol. The effects of cooling before freezing were assessed after thawing through the objective evaluation of sperm motility and flow cytometric analysis of membrane integrity, acrosomal status, changes in membrane lipid architecture monitored by merocyanine and annexin V binding and intracellular production of reactive oxygen species. In Experiment 1 (six replicates), two semen pools (five ejaculates per pool) were cooled from 15 to 5 °C at rates of 0.08, 0.13, 0.40 and 1.50 °C min(-1). These cooling rates did not result in any significant differences (P>0.05) in any of the post-thaw sperm assessments, even in thawed samples incubated under capacitation conditions. In Experiment 2, three individual ejaculates from 16 boars were slowly (0.08 °C min(-1)) or rapidly (1.5 °C min(-1)) cooled before freezing. A consistent interboar variability (P<0.01) was detected, which was independent of the cooling rate used. Cooling rate only significantly influenced (P<0.05) sperm assessments in four of 16 boars, which exhibited slightly higher percentages of motile cells and intact plasma and acrosomal membranes in the samples that had been cooled slowly. These findings demonstrate that boar spermatozoa undergoing cryopreservation can withstand rapid cooling rates before freezing.     

Characterization of viability,mitochondrial activity,acrosomal integrity and capacitation status in boar sperm during in vitro storage at different ambient temperatures

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen-thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4 degrees C, 15 degrees C, 20 degrees C and 39 degrees C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 +/- 1% of the sperm did not take up the Hoechst 33258, whereas 95 +/- 2% were stained by SYBR-14 and 96 +/- 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15 degrees C and 20 degrees C. There were 62 +/- 2% (15 degrees C) and 89 +/- 2% (20 degrees C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.     

Testing an egg yolk supplemented diet on boars to aid in sperm adaptation at 5°C

Abstract

In many species, extended semen can be stored at low temperatures to slow bacterial growth. However, boar semen performs poorly at temperatures below 15°C and this poses unique challenges, as it is not easy to maintain a constant 15–19°C during shipment. Some extenders have been formulated with egg yolk for storage at 5°C but the addition of egg yolk is not applicable in the majority of commercial operations. The purpose of this study was to evaluate if boar dietary supplementation with powdered egg yolk imparts any protective effects on sperm quality when stored at 15°C and 5°C for up to 11 days in a conventional extender. Ten boars were fed a commercial diet with the addition of 0.11Kg of powdered egg yolk for 10 weeks. Ejaculates collected on weeks 4, 6, 8, and 10 were processed for storage at both 15°C and 5°C and compared with ejaculates from boars fed a standard diet. Throughout an 11-day storage period, sperm quality was assessed including several motility and morphologic parameters and select plasma membrane properties (fluidity, integrity, and triacylglycerol content). Linear regression models were used to describe effects of treatment, storage day, week and temperature on all sperm parameters. Overall, there were minimal beneficial effects of egg yolk treatment on sperm quality parameters. Sperm from egg yolk supplemented boars did have a slower decline in viability and plasma membrane fluidity than that observed in the control sperm when stored at 5°C (p?<?0.001). Additionally, there was an increase in total morphologic abnormalities in sperm from egg yolk fed boars compared to controls at week 10 (p?<?0.001). In conclusion, the results of this study do not support a significant benefit to sperm quality or resistance to cold storage when feeding a 10-week dietary supplementation of 0.11Kg powdered egg yolk to crossbred boars.     

Changes in the Quality of Rabbit Semen in 14 Consecutive Ejaculates Obtained Every 15 Minutes

Modifications of semen quality related to ejaculation frequency is one of the most important and neglected factors from the standpoint of artificial insemination or sperm competition. New Zealand white rabbits ( Oryctolagus cuniculus ) offer an advantageous experimental model because they have characteristic sexual behavior, they present rapid ejaculation after a single intromission, they have a very short interval between successive ejaculations, and semen can be easily collected. The authors studied the modifications on sperm quality (semen volume, sperm concentration, sperm motility) produced by 14 consecutive ejaculations recovered every 15 min using stimulus females and an artificial vagina. Bucks were exposed every 15 min to a sexually receptive female. After each ejaculation the female was removed and reintroduced 15 min later. Sperm concentration showed a clear biphasic conduct. The amount of spermatozoa per milliliter decreased rapidly until ejaculate number 6, showed a highly significant increase in ejaculates 7-9, and decreased to nil in the last 2 ejaculates. Total number of ejaculated spermatozoa was 557 &#50 10 6, 76% of which were recovered from the first 4 ejaculates. Ejaculate volume also showed a biphasic conduct. In the first ejaculates the volume decreased linearly until ejaculate number 6, showed a significant increase in ejaculates 7-10, and then decreased. The total semen volume recovered during the experiment was 2.44mL, 40% of which (0.98mL) was recovered from the first 2 ejaculates. Individual motility in the first 6 ejaculates was preferentially progressive (60% of the sperms) and turned to random or in situ from the seventh ejaculate up. The proportion of spermatozoa with cytoplasmic droplets increased from ejaculates 6 and 7 up. The results seem to reflect an acceleration of semen transport through the epididymis when the demands for spermatozoa increase.     

Semen characteristics and sperm morphology in the Arabian leopard (Panthera pardus nimr) and how these vary with age and season

The Arabian leopard is a critically endangered species. Since there are only an estimated 200 animals remaining in the wild, careful management of the captive population is necessary to minimise inbreeding. The objective of this study was to characterise sperm morphology and ejaculate quality in captive males. Semen was collected by electroejaculation from 8 adult captive male leopards (aged 2-16 years) during the summer and winter months, and semen parameters, including sperm morphology, were assessed. Two-year-old leopards showed lower total sperm counts per ejaculate than older animals and these counts declined at > 8 years. Ejaculates collected during the hot summer showed significantly lower sperm concentrations, total sperm counts, sperm motility and viability and percentage of spermatozoa showing normal morphology than ejaculates collected in the cooler winter. The results showed that the male leopard attains sexual maturity between 2 and 3 years of age and exhibits good semen quality until 8 years. Collection of semen for artificial breeding or banking would best be carried out in the cooler winter months.     

The usefulness of Real Time Morphology software in semen assessment of teratozoospermic boars

The aim of the present study was to compare two different techniques of sperm cell morphology evaluation in teratozoospermic boars: computer assisted semen morphology analysis and conventional assessment of stained semen smears. The semen samples were collected manually from 30 boars with reduced semen quality. In all samples the percentage of morphologically normal spermatozoa was below 70%. Computer assisted semen morphology assessment was performed using the Real Time Morphology (RTM) software (IVOS ver. 12.2, Hamilton Thorne Bioscience). The assessment was made by phase contrast optics, with the magnification of 20 x 3.8 and without staining. Conventional morphology assessment was performed by bright field microscopy with 1,000 x magnification after staining with Giemsa. At least 200 spermatozoa were evaluated per slide in both methods. The Bland-Altman plot indicated a general agreement between both methods of sperm morphology evaluation. The plots revealed the widest limits of agreement (mean?±?1.96 SD) for the percentage of midpiece anomalies (from ?16 to 13.2), and the narrowest for the percentage of looped tail (from ?1.49 to 1.09). The Bland Altman plot indicates general agreement between RTM and Giemsa staining in the percentage of major and minor defects. However, it was not possible to evaluate acrosomes using RTM. Otherwise, RTM proved to be a valuable tool in sperm morphology assessment, with accuracy equal to typical conventional methods.     

男性不育患者精浆锌含量与精液主要参数相关性研究

目的:探讨不育男性精浆的锌含量与精液质量的关系。方法:回顾性分析2011年8月～2012年1月在广西壮族自治区妇幼保健院生殖中心就诊的343例男性不育患者的相关资料,依据精浆锌含量分为正常A组(n=274例)和异常B组(n=69例),比较两组间精液参数的差异;同时根据精液黏稠度分为黏稠C组(n=54例)与非黏稠D组(n=289例),比较两组间精浆锌含量及其他精液参数的差异。结果:A组与B组患者年龄比较差异无统计学意义(P>0.05)。A组的精液量、每次射精精子总数、前向运动精子总数显著高于B组,而黏稠精液的比例明显低于B组,差异均有统计学意义(P<0.05)。其他精液各指标比较差异无统计学意义(P>0.05);C组与D组精液圆细胞浓度、精液量、精子浓度、精子总数比较差异无统计学意义(P>0.05);D组的前向运动精子百分率、前向运动精子总数、活动率、精浆锌明显高于C组,差异有统计学意义(P<0.05)。精浆锌含量与精液量、每次射精精子总数、前向运动精子总数显著正相关,与其他参数无显著相关性。结论:精浆锌含量直接影响精液量、精子总数、前向运动精子总数和精液黏稠度,精浆锌含量是男性生殖力的重要评估指标。     

Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 +/- 4% purity) of X- and Y-bearing spermatozoa was 3400 +/- 850 spermatozoa sex(-1) s(-1). In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen-thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.     

Pre-pubertal Di(2-ethylhexyl) Phthalate (DEHP) Exposure of Young Boars Did Not Affect Sperm In vitro Penetration Capacity of Homologous Oocytes Post-puberty

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinylchloride (PVC) products (e.g., plastic bags and medical equipment), has been reported to have toxic effects on animal reproduction and is considered an environmental hazard based, mostly, on rodent studies. However, the doses used in these studies are often considerably higher than that presumed in human exposure. In the present study we used young boars as model animals to assess the effects of pre-pubertal DEHP exposure on the ability of spermatozoa to penetrate homologous oocytes in vitro. Eight pairs of cross-bred male boar siblings were used. One brother in each pair became, at random, the test animal exposed to DEHP per os, three times a week, from 3 to 7 weeks of age while the other acted as the control, i.e., placebo-exposed. Semen was collected and frozen between 8 and 9 months of age and stored until spermatozoa were evaluated for their ability to in vitro penetrate in vitro-matured homologous oocytes post-thaw. Both the penetration rate and the number of spermatozoa per oocyte were considered within expected ranges for frozen boar semen of good quality. Penetration rate did not significantly differ (p > 0.05) between the groups with DEHP-exposed: 50%; control: 59%, which could be owing to a large variation between boars, and between replicates. The number of spermatozoa in the ooplasm was low and similar (p > 0.05) between the groups with DEHP-exposed: 1.5 and the control: 1.7. Under the conditions of the present experiment, pre-pubertal exposure to DEHP does not seem to cause a deleterious effect on the in vitro fertilizing ability of frozen spermatozoa post-puberty.     

分段射精法收集少精子症患者精液质量比较

目的:探讨分段射精法在精子优选过程中的有效性。方法:采用分段射精法收集少精子症患者前后两部分精液,分别检测前后两部分精液的精子参数。结果:前部分精液的精子密度、精子活动率、精子形态和精子顶体完整性均显著高于后部分,而精子存活率、精子膜的完整性前后两部分无显著差异(P>0.05)。结论:对于少精子症患者,采用分段射精法优选精子,能有效改进精子的质量参数,提高精液中精子密度、精子活动率、正常形态精子和正常顶体精子比例。     

Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls

Flow cytometry was utilised for the first time to independently measure five sperm parameters of individual spermatozoa of bull ejaculates to differentiate between outcome successes after artificial insemination (AI). These parameters included plasma membrane and acrosome integrity, mitochondrial functionality and DNA damage measured by sperm chromatin structure assay (SCSA) and terminal deoxynucleotide transferase-mediated dUTP nick end labelling (TUNEL) assays. For each parameter, results of 142 ejaculates (30 bulls) were ranked into three groups according to their flow cytometric measures: (1) ejaculates with the 25% lowest measures; (2) the 50% middle measures; and (3) the 25% highest measures. In total, 20 272 first-service inseminations (18 ;10(6) spermatozoa per AI dose) were performed, where fertility was defined as non-return within 60 days after first insemination. While plasma membrane and acrosome integrity, and mitochondrial functionality were not significantly related to fertility, data from SCSA and TUNEL assays were significantly associated with fertility. Ejaculates in SCSA group 1 had higher odds of AI success (1.07, 95% CI = 1.02-1.12), whereas those in group 3 had lower odds of AI success (0.94, 95% CI = 0.89-0.99), compared with the average odds of all three groups. Ejaculates in group 2 did not have significantly higher odds of AI success compared with the average odds. For TUNEL-positive spermatozoa, the odds of AI success was higher in group 1 compared with the average odds (1.10, 95% CI = 1.02-1.13), whereas odds of AI success in groups 2 and 3 were not significant compared with the average odds. In conclusion, despite the high number of spermatozoa per AI dose from high-quality bulls, both SCSA and TUNEL assays were valuable measures in this study for evaluating sperm quality in relation to fertility after AI.     

广西地区正常生育力男性精液质量的调查分析

目的了解广西地区正常生育力男性的精液质量状况。方法选择广西地区有生育力的男性进行精液检测,对精液的各主要参数进行分析,比较壮族和汉族不同居住地间的差别。结果 687例正常生育力男性的精子密度、存活率、精液量和pH值与世界卫生组织(World Health Organization,WHO)——第4版的正常参考值符合率较高,液化时间、正常精子形态率和活动力的符合率偏低。其中有321例(46.72%)达到世界卫生组织的精液分析正常值标准。汉族正常精子形态率则低于壮族,差异具有统计学意义(P<0.05)。居住在农村的男性和城市的相比较,除pH值无明显差异(P>0.05)外,其他参数均明显优于城市,差异有统计学意义(P<0.05)。结论广西地区正常生育力男性的精液参数与国内其他调查分析结果相似;广西汉族、壮族间精液质量差别不大,农村男性的精液质量优于城市男性。     

Use of multivariate statistics to identify unreliable data obtained using CASA

In order to identify unreliable data in a dataset of motility parameters obtained from a pilot study acquired by a veterinarian with experience in boar semen handling, but without experience in the operation of a computer assisted sperm analysis (CASA) system, a multivariate graphical and statistical analysis was performed. Sixteen boar semen samples were aliquoted then incubated with varying concentrations of progesterone from 0 to 3.33 µg/ml and analyzed in a CASA system. After standardization of the data, Chernoff faces were pictured for each measurement, and a principal component analysis (PCA) was used to reduce the dimensionality and pre-process the data before hierarchical clustering. The first twelve individual measurements showed abnormal features when Chernoff faces were drawn. PCA revealed that principal components 1 and 2 explained 63.08% of the variance in the dataset. Values of principal components for each individual measurement of semen samples were mapped to identify differences among treatment or among boars. Twelve individual measurements presented low values of principal component 1. Confidence ellipses on the map of principal components showed no statistically significant effects for treatment or boar. Hierarchical clustering realized on two first principal components produced three clusters. Cluster 1 contained evaluations of the two first samples in each treatment, each one of a different boar. With the exception of one individual measurement, all other measurements in cluster 1 were the same as observed in abnormal Chernoff faces. Unreliable data in cluster 1 are probably related to the operator inexperience with a CASA system. These findings could be used to objectively evaluate the skill level of an operator of a CASA system. This may be particularly useful in the quality control of semen analysis using CASA systems.     

Transforming growth factor-β (TGFβ) in porcine seminal plasma

Bioactive factors in seminal plasma induce cellular and molecular changes in the female reproductive tract after coitus. An active constituent of seminal plasma in mice and humans is the potent immune-modulating cytokine transforming growth factor-β (TGFβ). To investigate whether TGFβ is present in boar seminal plasma, TGFβ(1) and TGFβ(2) were measured by immunoassay. High levels of TGFβ(1) and TGFβ(2) were detected in 100% of seminal fluid samples from 73 boars. Both were predominantly in the active, not latent form. Interferon-γ (IFNγ) and lipopolysaccharide (LPS), agents that interact with TGFβ signalling, were detectable in 5% and 100% of samples, respectively. TGFβ(1) and TGFβ(2) concentrations varied widely between boars, but correlated with each other and with sperm density, and remained relatively constant within individual boars over a 6-month period. Frequent semen collection substantially diminished the concentration of both TGFβ isoforms. Using retrospective breeding data for 44 boars, no correlation between TGFβ content and boar reproductive performance by artificial insemination (AI) with diluted semen was found. It is concluded that TGFβ is abundant in boar seminal plasma, leading to the speculation that, in pigs, TGFβ may be a male-female signalling agent involved in immune changes in the female reproductive tract elicited by seminal fluid.     

Studies on effect of different seasons on expression of HSP70 and HSP90 gene in sperm of Tharparkar bull semen

ObjectiveTo assess the effect of different seasons on expression of HSP70 and HSP90 genes of spermatozoa in Indian breed, Tharparkar bull.MethodsTotal numbers of 60 ejaculates from 3 bulls were collected through artificial vagina method twice a week during summer and winter season (30 ejaculates from each season). The semen samples were pooled and diluted with the standard TEYC extender and these semen samples were allowed to study the expression of HSP 70 and HSP 90 genes of spermatozoa with commercially available kit.ResultsNo significant difference was observed in spermatozoal mRNA expression of HSP 70 and HSP 90 during winter and summer season in this bull semen. But the mRNA expression of both HSP 70 and HSP 90 during summer season was found non-significantly higher in comparison to winter season.ConclusionIt was concluded from the present study that there was no significant difference in the mRNA expression of HSP 70 and HSP 90 between the winter and summer season, presence of similar type of stress resistant spermatozoa in Tharparkar bull semen and the semen can be cryopreserved throughout the year in this prestigious Indian breed.     

Relationship between phospholipase C-zeta,semen parameters,and chromatin status

The need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca²⁺ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.     

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²⁺ concentration in boar spermatozoa during cryopreservation

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.     

The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender

Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 μg mL?1) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 10? and 800 × 10? spermatozoa mL?1. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.     

Semen quality in captive Houbara bustard,Chlamydotis undulata undulata

Semen quality in captive-bred Houbara bustards, Chlamydotis undulata undulata, was assessed during three consecutive breeding seasons. In any one season, sperm quality, in terms of the proportion of eosin-permeable spermatozoa and of spermatozoa with abnormally large nuclei, varied among individual males, but not among their ejaculates. Neither the proportion of spermatozoa with large nuclei, nor those permeable to eosin were related to the total sperm output of males. The fertilizing ability of males was related to their mean seasonal proportion of eosin-permeable spermatozoa, but not the proportion of spermatozoa with large nuclei. The ranking of males on the basis of the proportion of spermatozoa with large nuclei in their ejaculates was significantly positively correlated between seasons, although ranking on the basis of sperm eosin-permeability was not. The cause or consequence of producing spermatozoa with large nuclei (and excess DNA) remains to be elucidated, but appears to be a trait that is characteristic of houbara bustard males that is maintained between breeding seasons.