Epididymal glycoprotein involved in rat sperm association

Recently, a new head-to-head sperm association was described in the rat during epididymal transit. This association was called a rosette and a filamentous and PAS-positive material was also described joining the sperm heads. The begining of rosette formation in the epididymis and the linking material between heads have remained unclear. Epididymides of adult rats were fixed by vascular perfussion and thin sections of the principal regions were studied by transmission electron microscopy (TEM). The first evidence of rosette formation was observed in the distal corpus. Rosettes were isolated from the distal corpus and processed for immunogold and immunofluorescence microscopy to detect an epididymal glycoprotein called DE. This glycoprotein is secreted by the corpus epididymis and appears to be involved in sperm maturation. Colloidal gold marks and fluorescence were observed in the linking material between the sperm heads. The results presented here show that rosettes begin to appear following the sites of DE secretion and permit us to postulate that DE is involved in rosette formation and constitutes another example of gamete-epididymal interaction. © 1994 Wiley-Liss, Inc.     

Major morphological sperm abnormalities in the bull are related to sperm DNA damage

The influence of sperm morphology and chromatin integrity on bull fertility suggests a strong but undefined biological relationship between these two parameters. In this study we explore this relationship, making use of the Sperm Chromatin Dispersion test, which allows simultaneous observation of sperm abnormalities and DNA fragmentation. Based on spermatozoa from 17 Holstein-Friesian bulls, we determined a relationship between DNA fragmentation and the presence of the “so called” major-type sperm defects. Values for DNA fragmentation index (mean ± SEM) calculated from cells with major-type abnormalities were significantly (P < 0.05) higher (85.05 ± 5.00%) than those from abnormal forms classified as minor-type (17.89 ± 5.55%). Some of the sperm abnormalities, such as double forms, narrow base heads, small heads, shortened tails and proximal cytoplasmic droplets, were only associated with sperm showing fragmented DNA. The simultaneous assessment of sperm morphology and DNA fragmentation has the potential to improve the efficacy of sperm quality assessment in this species.     

Automatic evaluation of ram sperm morphometry

This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.     

Automated sperm head morphology analyzer for open-source software

Sperm head morphology has been identified as a characteristic that can be used to predict a male's semen quality. In the present study, we have developed an automated sperm head morphology analysis (ASMA) plug-in for open-source ImageJ software (http://rsbweb.nih.gov/ij/). We describe the plug-in's functionality, and confirm its validity for sperm head morphology analysis using fish sperm. Sperm head morphological measurements (length and width) made with the ASMA plug-in did not differ from manual measurements. Using the plug-in to measure sperm head-shaped objects of known size, the associated plug-in error rate was < 0.5%. Brightness and contrast ratios influenced sperm head measurements, suggesting the need for standardized protocols. This plug-in was effective at measuring elliptical (i.e., Atlantic cod) as well as slightly irregular (i.e., Chinook salmon) shaped sperm heads. In conclusion, our ASMA plug-in represents a versatile alternative to costly sperm morphology software.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.     

Effect of age on spermiogram of Holstein Friesian × Sahiwal crossbred bulls

This study was conducted on 94 Frieswal (5/8 Holstein Friesian 3/8 Sahiwal) crossbred bulls of three different grades, categorized based on their semen freezability visualising Group 1 (consistently freezable semen producer bulls, N = 11), Group 2 (inconsistent freezable, N = 16) and Group 3 (Non freezable, N = 67). Each group was further divided into two classes that is young (up to 30 months) and adult (31 to 70 months) bulls depending upon their age. Sperm morphology was studied by using the eosin-nigrosin staining technique. Bulls age significantly (P < 0.01) affected semen quality and sperm morphology. In adult bulls, semen volume, mass activity and sperm concentration were 36%, 17.56% and 19.6%, respectively, higher than young. Initial progressive motility (%) and livability showed significant (P < 0.01) improvement with the advancement of age (43.37 ± 1.21 and 67.71 ± 1.11, respectively, in young; 53.02 ± 1.11 and 74.17 ± 1.03, respectively, in adult). In young bulls, sperm head, mid piece, tail abnormality and total abnormal sperm percent (12.38 ± 0.92, 4.87 ± 0.24, 11.01 ± 0.60 and 28.26 ± 1.34, respectively) were 1.85, 1.27, 1.20 and 1.44 folds higher than that of their mature stage (6.69 ± 0.64, 3.82 ± 0.32, 9.14 ± 0.64 and 19.66 ± 1.31, respectively). Significant reduction (P < 0.01) in micro cephalic sperm, free heads, bent mid piece, looped mid piece and proximal protoplasmic droplets were observed at mature age as compared with their younger stage. In bulls of consistent freezing category, abnormal sperm heads significantly decreased from 4.40 ± 0.31% to 3.28 ± 0.02% on maturity. Similarly, in inconsistent freezing grade bulls sperm head abnormality (9.28 ± 0.75% to 5.13 ± 1.20%) and total abnormal sperm percent (24.89 ± 1.43 to 18.73 ± 3.40) was decreased over the age. On the contrary, in non-freezing category bulls' sperm morphology did not show significant (P > 0.05) improvement with age advancement, rather some abnormalities like long slender head, under developed/deformed head, abaxial implantation of mid piece, double mid piece, stump tail and distal protoplasmic droplets tend to increased significantly (P < 0.05) with age of bulls. Results indicated that in potential Frieswal bulls semen quality and sperm morphology were improved from young to mature stage, where as, in poor quality (non-freezing) semen producer bulls neither the morphology nor the semen quality showed any improvement with maturity. It was recommended that crossbred bulls producing more than 25% morphologically abnormal sperms in young age (below 30 months) along with poor progressive motility (<50%) and low sperm concentration (<1000 million/ml) need immediate culling with out any expectation of further improvement in semen quality with age advancement.     

Effect of method and clinician on stallion sperm morphology evaluation

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.     

Maturation of hamster epididymal sperm motility and influence of the thiol status of hamster and rat spermatozoa on their motility patterns

A method for objective quantification of hamster sperm movement parameters as an indicator of maturation along the epididymis was established using a computerised system. Analysis of spermatozoa released into medium from five epididymal regions showed that the most drastic increases in percentage motility and curvilinear velocity (VCL) occurred from the distal corpus to the beginning of the proximal cauda and in straight-line velocity (VSL) from the beginning to a more distal site within the proximal cauda region. Both high osmolarity (400 mOsm/kg) and the thiol-oxidising agent diamide (10 μM) increased flagellar straightness of distal corpus spermatozoa, but VSL was increased only with the latter. The thiol-reducing agent dithiothreitol (DTT, 1mM) stimulated and maintained percentage motility and velocities of spermatozoa from the caput, stimulated only percentage motility of distal corpus sperm, but decreased velocities of those from the proximal cauda in prolonged incubation. In rats, diamide increased path straightness but not velocities of caput spermatozoa and yet caused immotility within 15 min, whereas DTT prolonged the maintenance of in vitro motility. The slight increases in kinematic parameters in the presence of DTT were enhanced by a 2-min preincubation with diamide. The finding that the effects of DTT and diamide were not compensatory suggests that the influence of the SH/S-S status on sperm movement is multifaceted, with decreasing sensitivity to stimulation upon sperm maturation. © 1994 Wiley-Liss, Inc.     

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water

Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP™), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP™ (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP™ and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.     

Sperm head phenotype and male fertility in ram semen

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter²/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.     

Study of nuclear and acrosomal sperm morphometry in ram using a computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method

The aim of this study was to develop a new method that allows morphometric assessment of the sperm nucleus and acrosome in the ram using fluorescence microscopy and free software. The study was divided into three experiments. In the first experiment, semen smears from 20 ejaculates were fixed and labeled with a propidium iodide–pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed using the ImageJ program. The computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method used allowed the differentiation, capture, and morphometric analysis of most sperm nuclei, acrosomes, and whole heads with high precision and the assessment of the acrosomal status. In the second experiment, sperm nuclear morphometry by CASMA-F was compared by staining with the PI/PSA combination and staining with Hoechst 33342 as in previous studies. Similar results were obtained using both methods. In the third experiment, CASMA-F with PI/PSA was compared with a more conventional CASMA method (semen smears stained with Hemacolor (HEM) and processed with the ISAS commercial software, HEM). Spermatozoa displayed a bigger size when processed with CASMA-F than with HEM method in all primary sperm head morphometric parameters, but results using both methods were correlated. It was concluded that the CASMA-F method allows the simultaneous assessment of sperm nucleus, acrosome, and head in the ram.     

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26 306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.     

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter²/[4 × π × area]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.     

Mutagenic profiles of carbazole in the male germ cells of Swiss albino mice

Mutagenic effect of carbazole was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice were treated for five consecutive days either with 30 or 60 mg/kg body weight (b.w.) of carbazole by single intraperitoneal (i.p.) injection. For the sperm head abnormality assay mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w as a single i.p. injection. Treatment of adult male mice with carbazole resulted in induction of dominant lethal mutation and abnormal sperm heads. The results show that carbazole is mutagenic in male germ cells of mice.     

雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究

对雌核发育银鲫和两性融合彩鲫精子蛋白组份进行了比较分析。通过分级抽提得到精子的不同组份精浆、精头的膜、鞭毛和脱膜精头等,然后经不同的凝胶电泳系统,比较分析了银鲫精子和其两性亲缘种彩鲫精子相应组份可溶性蛋白成份的差异。研究表明,经分级抽提的银鲫精子和彩鲫精子的各个组份都含有其特定的蛋白谱带。精浆蛋白在两种鱼之间和两种鱼的不同个体之间都存在一定差异。精头膜、鞭毛和脱膜精头的可溶性蛋白在同种鱼不同个体间高度一致,但在两种鱼之间表现出差异。两种鱼精头膜的可溶性蛋白在SDS－PAGE电泳图谱上基本一致,而在非变性聚丙烯酰胺凝胶电泳图谱上则具有各自的特征性谱带。鞭毛可溶性蛋白的SDS－PAGE分析在雌核发育银鲫中揭示出一条特异的蛋白带。脱膜精头的可溶性蛋白在SDS－PAGE电泳图谱上差异明显,存在几条特征性蛋白带,并经Acid-Urea PAGE系统分析,证实这些特征性蛋白为碱性蛋白。这些发现为进一步鉴定雌核发育银鲫雄鱼精子的特异性蛋白和揭示其分子机制打下了基础。     

Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm

The effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.     

The integrity of sperm chromatin in young tropical composite bulls

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r = 0.34, P = 0.0076), Mot (r = 0.36, P = 0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r = 0.31, P = 0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r = −0.28, P = 0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r = −0.53, P < 0.0001), the percentage of sperm with head abnormalities (r = 0.68, P < 0.0001) and the percentage of intact sperm (Int) with SBH (r = −0.26, P = 0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.     

Morphometric characterization and classification of alpaca sperm heads using the sperm-class analyzer computer-assisted system

Sperm morphology has been identified as one characteristic which can be useful in the prediction of sperm fertility, therefore, we hope that this study aimed at establishing standardized morphological criteria might serve in future studies dealing with the search for sperm parameters which facilitate an estimation of sperm quality. For this purpose, ejaculates from fertile alpacas were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer (SCA) computer-aided image analysis system. We defined three morphological categories according to sperm head size (normal 50%, small 26%, large 24%) and five categories according to sperm head shape (normal 47%, pyriform 3%, short 20%, round 1%, long 29%). Sperm classification according to shape was performed by first morphometrically characterizing sperm heads clearly falling into each of the shape categories. Thereafter, discriminant analysis was performed on the data from these typical sperm heads and the resulting classification functions were used to categorize 2,200 spermatozoa from 11 alpacas. Classification of sperm heads by this method agreed in 88% of the cases with most of the misclassifications being due to pyriform heads classified as long heads. Morphometric values obtained from samples of 50, 100, 150, 175 and 200 sperm heads were compared. At least 150 sperm heads should be evaluated to overcome sample size influence on sperm measurements. Significant differences in sperm morphometry were found between individuals (CV for morphometric parameters ranging from 1.3 to 13.0) and there were marked differences in the sperm morphological composition of the ejaculates. Within-animal CV ranged from 4.7 to 17.8 thus showing the high degree of sperm polymorphism present in the alpaca ejaculate.     

Relationship between sperm motility, morphology and the fertility of stallions

Sperm quality has an important role in determining fertility. Although there have been numerous studies to document the relationship between sperm quality and fertility, the methods of determining this association and conclusions vary. In the present study, computer-assisted sperm analysis (CASA) was used for evaluation of sperm motility, and differential interference contrast (DIC) microscopy was used for evaluating sperm morphologic features of breeding stallions. Fertility was measured using three endpoints: seasonal pregnancy rate (PR), percent pregnant/cycle (PC), and percent pregnant/first cycle (FCP). Increased total sperm motility (P = 0.08) and progressive path velocity (P = 0.06) tended to be associated with higher PR, whereas percent coiled tails (P = 0.02) was associated with a lower PR. Sperm motility variables associated with an increase in PC and FCP included total, progressive, and rapid sperm motility, and increased path and progressive velocity. Percent pregnant/first cycle was the only fertility measure able to discriminate among high, average, and low fertility groups, based on total and progressive sperm motility. Percent normal sperm was the only morphology variable associated with an increased PC and FCP, whereas increased levels of most sperm morphologic abnormalities (including abnormal and detached heads, proximal and distal droplets, general midpiece abnormality, and coiled tails) were associated with a decline in PC and FCP. Sperm quality variables most highly correlated with fertility included percent total sperm motility (PR, r = 0.37, P < 0.05; PC, r = 0.59, P < 0.05; and FCP, r = 0.64, P < 0.05), and percent morphologically normal sperm (PC, r = 0.42, P < 0.05; and FCP, r = 0.39, P < 0.05).