Assessment Of Sperm Concentration Research Articles

ASSESSMENT OF SOME MICROSCOPIC PARAMETERS OF RAM SEMEN CORRELATED WITH THE AGE OF THE ANIMALS

"The study was carried out to evaluate some microscopic parameters (mobility, concentration, viability), to assess the metabolic intensity of spermatozoa (Redox test), and sperm resistance test related with ram fertility and the quality of ram semen in different age groups. The study was conducted in a farm located in Cluj County, on 34 rams of Turcana Alba breed, grouped according to age into 4 batches. Weekly an ejaculate and the mentioned variables were measured. Semen samples were collected from each animal using the artificial vagina (AV). For sperm mobility, the best values were observed for 3 years old rams (X ± S = 88.4 ± 3.02). Variations in sperm viability showed some changes, but for all age groups were obtained values above those indicated in the literature. Assessment of sperm concentration revealed that rams in B6 (X ± S = 2.75 ±0.31) and B5 (X ± S = 2.7 ± 0.38) had the best values. Higher metabolic intensity rate in B6, B5, B3 groups was correlated with higher values of concentration and mobility in these age groups. Thus, the best values regarding sperm resistance were recorded for the rams aged 6 and 5 years, in which the average values were equal to 7022.22. With increase in age, ram showed increase percentages of motility and viability of sperm in all studied batches."

Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.

Aldose Reductase B1 in Pig Seminal Plasma: Identification, Localization in Reproductive Tissues, and Relationship With Quality and Sperm Preservation.

Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C.

A prospective study of variations in conventional semen parameters among local andrology laboratories

Introduction: Wide variations in semen parameters exist among different andrology laboratories. This study sought to determine the inter- and intra-technician variations in conventional semen parameters among local andrology laboratories. Methods: Pooled semen samples were prepared and sent in two batches to participating andrology laboratories. One technician who routinely performed semen analysis in the participating laboratories was asked to analyze the study samples. The inter-technician and intra-technician coefficients of variation (CVs) were calculated. Information on the qualification and training of the participating technicians, the workload of the centers, their techniques and participation in external quality assurance programs were collected and correlated with the CVs. Results: Eleven andrology laboratories participated in the study. The inter-technician CVs ranged from 14.3% to 44.1% for concentration, 13.8% to 26.2% for progressive motility, and 38.8% to 95.3% for morphology. Andrology laboratories which participated in external quality assurance programs had lower inter-technician CVs for concentration (P = 0.004) and progressive motility (P = 0.002), but not for morphology (P = 0.232). Technicians with more experience or higher workload did not demonstrate lower intra-technician CVs. Conclusion: There were considerable inter- and intra- technician variations in the assessment of sperm concentration, progressive motility and morphology among local andrology laboratories, independent of the workload and experience of the technicians. Participation in external quality assurance programs reduced inter-technician variations in sperm concentration and progressive motility but not morphology.

Short term cold storage and sperm concentration assessment of lumpfish (Cyclopterus lumpus. L) Milt

There is increased commercial interest in the production of lumpfish (Cyclopterus lumpus) as a biological control against sea lice infections in Atlantic salmon farming. To ensure a sustainable supply of lumpfish, reliable captive reproduction is required however, optimal husbandry and conditions for captive broodstock performance remain unknown. Artificial fertilisation of gametes remains the preferred management strategy for lumpfish, but this requires an effective milt management protocol. The present study tested several milt extender solutions for short term cold storage and validated sperm concentration assessment of lumpfish milt. Results demonstrated lumpfish sperm has a long motility survival time (motile for up to 3 h) when compared to other marine teleosts. Importantly, all extenders used in the study were non-activating on dilution of the milt. Lumpfish milt was successfully stored at 4 °C in Modified Turbot Extender (MTE), Herring Ringers Solution and Aquaboost SpermCoat (Cryogenetics, Norway) for up to 14 days post stripping. MTE performed more effectively with regards to maintenance of sperm activation time in comparison to the other tested extenders. There was a significant positive correlation between sperm concentration identified through cell counts using a haemocytometer and both packed cell volume (spermatocrit) and measured optical density at 640 nm (r2 = 97.42 ± 2.14). This suggests both packed cell volume and spectrophotometric measurements can be effective methods for rapidly assessing sperm concentration in lumpfish. This study validated several options for quantifying sperm concentration and short-term cold storage of lumpfish milt that can be used for hatchery management in lumpfish aquaculture.

Reproductive Function in a Population of Young Faroese Men with Elevated Exposure to Polychlorinated Biphenyls (PCBs) and Perfluorinated Alkylate Substances (PFAS)

Semen quality may be adversely affected by exposure to environmental chemicals such as polychlorinated biphenyls (PCBs) and perfluorinated alkylate substances (PFASs) that are persistent and may act as endocrine disrupting compounds. The aim of this study was to explore whether PCBs or PFASs exposure were associated with abnormalities in semen quality or reproductive hormones in Faroese men. This population based cross-sectional study includes 263 Faroese men (24–26 years) who delivered a semen sample for assessment of sperm concentration, total sperm count, semen volume, morphology and motility. A blood sample was drawn and analyzed for reproductive hormones, PCBs and PFASs. Exposure to ∑PCBs and perfluorooctane sulfonate (PFOS) was positively associated with sex hormone-binding globulin (SHBG) and luteinizing hormone (LH). In addition, total testosterone (T) was positively associated with ∑PCB. Both PCBs and PFOS appear to lead to increased SHBG, perhaps mediated via the liver. The higher total T associated with PCB may represent a compensatory adaption to elevated SHBG levels to maintain an unchanged free testosterone concentration. The positive association to LH for both PCBs and PFOS may indicate a direct adverse effect on the testosterone producing Leydig cells.

Role of Genetic Testing in Male Infertility

Male-factor infertility is responsible for 30-55% of all infertility cases. The causes of male infertility include varicocele, endocrine disorders, genital tract infections, genetic disorders and idiopathic. It is estimated that genetic abnormalities contribute to 50% of male infertility. In daily practice, the diagnosis of male infertility has been based on history taking, relevant physical examination, hormone tests and basic semen analysis with a strong emphasis on the assessment of sperm concentration, motility, and morphology. Although recent development in assisted-reproductive technologies such as in vitro fertilization and intrauterine insemination increases the chance of clinical pregnancy and live birth, genetic counseling and testing should always be performed whenever genetic risks are related to the cause of infertility for the identification of possible genetic abnormalities and to assess the risk of transmitting the genetic defects to future generations. Genetic defects affect male infertility by disrupting hormonal homeostasis, spermatogenesis, and sperm quality. These genetic defects include chromosomal abnormalities (e.g. Klinefelter Syndrome), Y chromosome deletions, and cystic fibrosis transmembrane conductance regulator gene mutations. The utilization of genetic counseling and testing is also important to predict the success of sperm retrieval in men with certain genetic abnormalities. To name a few, genetic analysis at the chromosomal level (karyotyping), androgen receptor gene mutations test, cystic fibrosis test, and Y chromosome microdeletions analysis should be considered in the diagnosis of male factor infertility where genetic risks are present.

Novel centrifugal technology for measuring sperm concentration in the home

To evaluate the analytical performance and usability of the Trak Male Fertility Testing System, a semiquantitative (categorical) device recently US Food and Drug Administration (FDA)-cleared for measuring sperm concentration in the home by untrained users. A three-site clinical trial comparing self-reported lay user results versus reference results obtained by computer-aided semen analysis (CASA). Simulated home use environments at fertility centers and urologist offices. A total of 239 untrained users. None. Sperm concentration results reported from self-testing lay users and laboratory reference method by CASA were evaluated semiquantitatively against the device's clinical cutoffs of 15M/mL (current World Health Organization cutoff) and 55M/mL (associated with faster time to pregnancy). Additional reported metrics include assay linearity, precision, limit of detection, and ease-of-use ratings from lay users. Lay users achieved an accuracy (versus the reference) of 93.3% (95% confidence interval [CI] 84.1%-97.4%) for results categorized as ≤15M/mL, 82.4% (95% CI 73.3%-88.9%) for results categorized as 15-55M/mL, and 95.5% (95% CI 88.9%-98.2%) for results categorized as >55M/mL. When measured quantitatively, Trak results had a strong linear correlation with CASA measurements (r = 0.99). The precision and limit of detection studies show that the device has adequate reproducibility and detection range for home use. Subjects generally rated the device as easy to use. The Trak System is an accurate tool for semiquantitatively measuring sperm concentration in the home. The system may enable screening and longitudinal assessment of sperm concentration at home. ClinicalTrials.gov identifier: NCT02475395.

Uncertainty of measurement and clinical value of semen analysis: has standardisation through professional guidelines helped or hindered progress?

This article suggests that diagnostic semen analysis has no more clinical value today than it had 25-30years ago, and both the confusion surrounding its evidence base (in terms of relationship with conception) and the low level of confidence in the clinical setting is attributable to an associated high level of 'uncertainty'. Consideration of the concept of measurement uncertainty is mandatory for medical laboratories applying for the ISO15189 standard. It is evident that the entire semen analysis process is prone to error every step from specimen collection to the reporting of results and serves to compound uncertainty associated with diagnosis or prognosis. Perceived adherence to published guidelines for the assessment of sperm concentration, motility and morphology does not guarantee a reliable and reproducible test result. Moreover, the high level of uncertainty associated with manual sperm motility and morphology can be attributed to subjectivity and lack a traceable standard. This article describes where and why uncertainty exists and suggests that semen analysis will continue to be of limited value until it is more adequately considered and addressed. Although professional guidelines for good practice have provided the foundations for testing procedures for many years, the risk in following rather prescriptive guidance to the letter is that unless they are based on an overwhelmingly firm evidence base, the quality of semen analysis will remain poor and the progress towards the development of more innovative methods for investigating male infertility will be slow.

Sperm membrane integrity in fresh and frozen–thawed canine semen samples: a comparison of vital stains with the NucleoCounter SP-100

The objective of this study was to assess sperm membrane integrity in canine semen samples using three different vital stains and the NucleoCounter SP-100 (NC). In addition, the occurrence of half-stained sperm heads, the influence of investigator, and storage-related artifacts using stained smears were examined. Forty fresh (30 dogs) and 40 frozen–thawed (28 dogs) canine semen samples were analyzed. The vital stains eosin (E), eosin-nigrosin (EN), and bromphenolblue-nigrosin (BN) were compared. Two smears per stain were prepared and a total of 200 sperm per slide were classified using bright field microscopy. Each slide was examined twice by three investigators. Spermatozoa with completely red (E, EN) or blue (BN) stained sperm heads were classified as “dead”. Half-stained sperm heads were counted separately. Sperm concentration and viability were determined using the NC. The NC works with a built-in fluorescence microscope using propidium iodide as a fluorescence dye. Statistical analysis for comparison of results was made using mean values with standard deviation, Bland-Altman plot and coefficient of variation (CV). Staining with E led to a significant higher percentage of dead sperm compared with EN and BN (P < 0.05), which gave comparable results. Vital stains revealed higher CVs (range 8.8%–32.1%) than the NC (<6.5%). Interobserver viability ranged from 17.5% to 45.4% and was within the same range between stains. If only completely stained sperm heads were considered, best agreement was found between the E and the NC. In case of EN and BN, inclusion of half-stained sperm heads reduced the difference compared with NC. In general, the agreement between methods was better in samples with a low percentage of dead spermatozoa. In smears of fresh semen stored up to 3 months, no increase in the percentage of dead spermatozoa could be observed. In some smears of frozen–thawed samples stained with E (n = 12) or BN (n = 2), all previously unstained spermatozoa were additionally stained during storage. In conclusion, all vital stains examined in this study technically can be used for differentiation between live and dead spermatozoa in canine semen samples, but the relatively high CVs have to be kept in mind. It is recommended to examine smears of frozen–thawed semen soon after preparation. Half-stained sperm heads should be counted as live sperm in E-stained smears. The NC allows assessment of sperm concentration and viability with a reasonable repeatability.

Implementing a QC vial/external proficiency testing (PT) program to help understand and reduce user variation in the assessment of post-thaw sperm motility and concentration

CCB has strived to reduce inter-technician/-facility variation in assessments of sperm concentration and motility, which can be problematic in sperm banking. The aim of this QC study was to objectively contrast inter-facility variation assessing a Makler chamber (n=8 labs) or microCell slides (n=5 labs), while validating a customized mini Dry Heat Bath (DHB; Southwest Science) for standardized thawing.

Image cytometer method for automated assessment of human spermatozoa concentration

In the basic clinical work-up of infertile couples, a semen analysis is mandatory and the sperm concentration is one of the most essential variables to be determined. Sperm concentration is usually assessed by manual counting using a haemocytometer and is hence labour intensive and may be subjected to investigator bias. Here we show that image cytometry can be used to accurately measure the sperm concentration of human semen samples with great ease and reproducibility. The impact of several factors (pipetting, mixing, round cell content, sperm concentration), which can influence the read-out as well as inter-operator and -cytometer variation on two different image cytometers (NC-3000 and SP-100) were evaluated. Furthermore, 725 semen samples were assessed both by manual assessment (WHO recommended method) and by image cytometry and tight correlations between the measured concentrations were shown. Moreover, by evaluation of repeated measurements it appeared that image cytometry produced more consistent and accurate measurements than manual counting of human spermatozoa concentration. In conclusion, image cytometry provides an appealing substitute of manual counting by providing reliable, robust and easy measurement of human sperm concentration.

SELECTED ORAL COMMUNICATION SESSION, SESSION 70: ANDROLOGY AND SEMINAL FACTORS Wednesday 6 July 2011 14:00 - 15:45

Introduction: Couples who have not achieved a pregnancy within one year of unprotected intercourse qualify for a fertility work-up. A semen analysis is part of this work-up and includes the assessment of sperm concentration, motility and morphology. The predictive value of sperm morphology has been

Is photometry an accurate and reliable method to assess boar semen concentration?

Sperm concentration assessment is a key point to insure appropriate sperm number per dose in species subjected to artificial insemination (AI). The aim of the present study was to evaluate the accuracy and reliability of two commercially available photometers, AccuCell™ and AccuRead™ pre-calibrated for boar semen in comparison to UltiMate™ boar version 12.3D, NucleoCounter SP100 and Thoma hemacytometer. For each type of instrument, concentration was measured on 34 boar semen samples in quadruplicate and agreement between measurements and instruments were evaluated. Accuracy for both photometers was illustrated by mean of percentage differences to the general mean. It was −0.6% and 0.5% for Accucell™ and Accuread™ respectively, no significant differences were found between instrument and mean of measurement among all equipment. Repeatability for both photometers was 1.8% and 3.2% for AccuCell™ and AccuRead™ respectively. Low differences were observed between instruments (confidence interval 3%) except when hemacytometer was used as a reference. Even though hemacytometer is considered worldwide as the gold standard, it is the more variable instrument (confidence interval 7.1%). The conclusion is that routine photometry measures of raw semen concentration are reliable, accurate and precise using AccuRead™ or AccuCell™. There are multiple steps in semen processing that can induce sperm loss and therefore increase differences between theoretical and real sperm numbers in doses. Potential biases that depend on the workflow but not on the initial photometric measure of semen concentration are discussed.

Evaluation of fish sperm quality

The development of cheaper technical tools and the availability of well established assay kits provide today the spermatologists with several devices for objective and quantifying assessment of sperm quality. The present paper reviews modern methods on sperm concentration assessment, morphometry, motility processes through flagellum studies, motility objective assessment by computer assisted sperm analysis (CASA), sperm integrity and energetic state measurements.

Assessment of sperm morphology without quality control may be meaningless for clinicians

Dear Editor, Recently we read several articles related to sperm morphology 1, 2, 3 published on the Special Issue of Asian Journal of Andrology on Semen Analysis in 21st Century Medicine and were quite enlightened; herein we would like to present some of our views and suggestions. Assessment of sperm morphology, motility and concentration provides a basis for decision on patient care. A number of studies have shown a statistical association between morphologically normal spermatozoa and fertility 4, 5. Data from assisted reproductive technology programs also suggest that, if the percentage of sperm with normal morphology falls below 15% assessed with methods and definitions described in the Manual, the fertilization rate in vitro decreases 6. Assessment of sperm morphology is much more difficult compared with assessment of sperm motility and concentration. Although quality control (QC) for sperm morphology is suggested to be essential according to the revised QC chapter in the WHO manual 5th edition 7, it is not well used for clinic in China and few people paid attention to its potential in evaluating male fertility. It is even worse that results from different laboratories vary significantly due to the poor implementation of QC for sperm morphology. The lower reference limit of 4% for sperm morphology proposed in the new edition of the WHO manual 7 makes QC even more difficult 2. We are convinced that the assessment of sperm morphology without QC may be meaningless for clinicians. We would like to share some of our views on how to improve morphology assessment based on experience of our lab. To begin with, four issues could be key to sperm morphology assessment. (1) Preparation of smears: the quality of smears will impact the result of staining. The smears must not be taken too thick. Otherwise, the debris and a large amount of particulate material will make it difficult to analyze. (2) Staining: how to make spermatozoa and other cells well-stained with a definite border depends on the choice of staining methods and frequency in changing the staining solutions. A prepared undyed smear for QC should be added in each dyeing batch to evaluate the dyeing effect. (3) Morphological evaluation: it is essential to establish a uniform and consistent definition of morphology. To minimize the variability of technicians, quality-controlled slides for sperm morphology should be used. Data from QC should be reviewed regularly by laboratory supervisors. If results were found to fall outside control limits, the problems should be addressed in time. (4) QC training: QC training for standardization of sperm morphology criteria could reduce individual interpreting difference. In our laboratory, the mean percentage difference reported among three technicians from our own laboratory was 4.57% ± 3.69% at the beginning, but after training, it decreased to 1.96% ± 1.19%. Secondly, sperm morphology is usually assessed only in one sample in many fertility centers in China. We suggest that two samples should be collected to assess sperm morphology just like the assessment of sperm concentration and motility. Finally, it is more meaningful to assess the morphology of forward progressive sperms than other ones since the forward progressive sperms are able to fertilize ova. The percentage of morphologically normal spermatozoa was obviously improved after sperm selection using Percoll gradient centrifugation and swimming up technique 8, 9. We assume that the total number of forward progressive and morphologically normal sperm per ejaculate is a more valuable indicator for male fertility potential, but it needs more data.

Assessment of Sperm Concentration and Determination of Optimum Sperm to Egg Ratio in Heteropneustes fossilis (Bloch)

Assessment of Sperm Concentration and Determination of Optimum Sperm to Egg Ratio in Heteropneustes fossilis (Bloch)

Comparison of Different Methods for Assessment of Sperm Concentration and Membrane Integrity With Bull Semen

Assessing semen quality is crucially important for the exploitation of genetically superior sires in an artificial insemination (AI) program. In this study, we compare modern and conventional techniques to estimate bovine sperm concentration and membrane integrity. First, the NucleoCounter SP-100 was validated for sperm concentration and provided statistically reliable and repeatable estimates among aliquots and replicates of 25 fresh ejaculates. Sperm concentrations in 78 ejaculates were then determined with hemacytometer, flow cytometer, and NucleoCounter SP-100 and were significantly correlated (P < .001), with regression coefficients among these 3 techniques close to 1 (P < .01). However, the sperm concentration determined by hemacytometer was lower (P < .01) than by flow cytometer and NucleoCounter SP-100. Forty frozen-thawed semen samples were then assessed for sperm concentration and membrane integrity with hemacytometer, flow cytometer and NucleoCounter SP-100. Significant relationships were found for sperm concentration determined by hemacytometer and NucleoCounter SP-100 and for sperm membrane integrity determined by flow cytometer and NucleoCounter SP-100 (P < .01). Finally, the standard curves of sperm concentrations in 6 spectrophotometers, comparing optical density against counts drawn by hemacytometer and NucleoCounter SP-100 (n = 94 fresh ejaculates) showed different (P < .01) intercepts and regression coefficients (linear, quadratic, cubic). It was calculated that a breeding station can improve its production potential by 13% with the use of NucleoCounter SP-100 instead of hemacytometer for calibration of spectrophotometers. Flow cytometer and NucleoCounter SP-100 can be used with equal confidence to estimate sperm concentration and membrane integrity in domestic animals and human semen.

Feasibility study for the centralized measurement of sperm concentration

Despite several efforts to standardize methods of semen analysis, sperm count is known to be subject to large interlaboratory differences. This is especially a problem in multicentre clinical trial settings and protocols for the preparation of semen for centralized assessment of sperm concentration are suggested here. The stability of semen has been tested after fixation with formalin at different dilutions and at different temperatures for different sperm concentrations. Sperm concentrations in formalin-fixed semen (at dilutions 1 + 0.5 to 1 + 19) were stable (<20% difference from original) at all concentrations (0.1 x 10(6)/mL to 100 x 10(6)/mL) for at least 5 days at room temperature and 4 degrees C. Prolonged stability up to 5 weeks at 4 degrees C was demonstrated for the lower dilutions (1 + 0.5 and 1 + 1). Freezing of fixed semen samples was unacceptable. These results illustrate that centralized assessment of sperm concentrations is feasible.

High Ejaculation Frequency Enhances Semen Production in Taiwan Country Chickens

The objective of this study were to investigate the effect of ejaculation frequency on semen characteristics and to establish a method for quick assessment of sperm concentration in TCC using packed cell volume (PCV) as the parameter (Trial 1). Eighty senior roosters, averaging 61 wk-old, were used and the sperm concentrations were determined using a hemacytometer. The PCV value was measured in a capillary (0.75 mm in inner diameter) by centrifugation. A simple linear regression analysis suggested that the sperm concentrations were significantly correlated with PCV values (r=0.62, p<0.001). Trial 2 was conducted to determine the optimal ejaculation frequency of TCC roosters in a weekly semen collection program. The male birds were subjected to 1, 2, 3 or 6 ejaculations per week for four consecutive weeks and semen characteristics including ejaculation volume (EV, mL), sperm motility (%), PCV (%), sperm concentration (ESC, ×10 9 /mL), weekly sperm production (WSP, 10 9 /wk) and average motile sperm numbers (AMSN, ×10 9 /ejac) were determined. Average EV was greater in the group with 3 ejac/wk than with only 1 ejac/wk in weeks 1 and 3 of the collection period. WSP increased with ejaculation frequency during the first 3 weeks of collection (p<0.05). Sperm motility was better in the birds with 6 ejac/wk than in single ejaculation group for the first 2 wk and no significant differences were found for the last 2 wk of study. In contrast, the PCV value showed a trend of reduction for the first 2 wks in the 6 ejac/wk group. Surprisingly, no significant differences were detected in the AMSN among treatment groups. The weekly motile sperm production (WMSP) increased with ejaculation frequency. Based on our observation, PCV values could be used for a quick estimation of sperm concentration and an intensive semen collection program enhanced weekly sperm production in TCC roosters.