Diethylstilbestrol-treated adult rats with altered epididymal sperm numbers and sperm motility parameters, but without alterations in sperm production and sperm morphology

In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 microg, 40 microg, 8 microg, 1.6 microg, or 320 ng x rat(-1) x day(-1) for 12 days. The control group received corn oil only. DES effects were similar in the 8-microg group and higher dose groups and included significant (P < or = 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-microg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the > or = 8-microg groups and to a very low level in the 1.6-microg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 microg x rat(-1) x day(-1) for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-microg and 8-microg groups and 1 in the 1.6-microg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13-15 pups/litter in the controls. DES at a rate of 8 microg x rat(-1) x day(-1) for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-microg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.     

Influence of anaesthetic drugs on the epididymal sperm quality in domestic cats

The present study investigated the effect of different anaesthetic agents commonly used in cats on the fresh and frozen-thawed epididymal sperm. Seventeen male domestic cats were castrated using pentobarbital, ketamine HCl or isoflurane. Sperm samples were recovered from epididymides and evaluated before and after freezing, determining the vigor, motility, morphology, acrosome status, sperm viability and functional membrane integrity. Fresh epididymal sperm was influenced by the drugs used, noting that motility features, i.e. vigor (p≤0.05) and progressive motility (p≤0.05), were higher for the inhalation anaesthetic while the others did not showed statistical differences. In frozen-thawed sperm samples, cats treated with barbiturics showed lower values for acrosome status (p≤0.05) and integrity and functionality of membrane (p≤0.05 and p≤0.01, respectively) than in the others groups. Results suggested that drugs used for castration in cats could affect the sperm quality and this should be considered when implementing sperm cryopreservation in the feline.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.     

Morphometric classification of Spanish thoroughbred stallion sperm heads

This work used semen samples collected from 12 stallions and assessed for sperm morphometry by the Sperm Class Analyzer (SCA) computer-assisted system. A discriminant analysis was performed on the morphometric data from that sperm to obtain a classification matrix for sperm head shape. Thereafter, we defined six types of sperm head shape. Classification of sperm head by this method obtained a globally correct assignment of 90.1%. Moreover, significant differences (p<0.05) were found between animals for all the sperm head morphometric parameters assessed.     

Identification of sperm morphometric subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm chromatin integrity?

A statistical approach using sequentially principal component analysis (PCA) clustering and discriminant analysis was developed to disclose morphometric sperm subpopulations. In addition, we used a similar approach to disclose subpopulations of spermatozoa with different degrees of DNA fragmentation. It is widely accepted that sperm morphology is a strong indicator of semen quality and since the sperm head mainly comprises the sperm DNA, it has been proposed that subtle changes in sperm head morphology may be related to abnormal DNA content. Semen from four mongrel dogs (five replicates per dog) were used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (ASMA). Each sperm head was measured for nine primary parameters: head area (A), head perimeter (P), head length (L), head width (W), acrosome area (%), midpiece width (w), midpiece area (a), distance (d) between the major axes of the head and midpiece, angle (theta) of divergence of the midpiece from the head axis; and four parameters of head shape: FUN1 (L/W), FUN2 (4pi A/P2), FUN3 ((L - W)/(L + W)) and FUN 4 (pi LW/4A). The data matrix consisted of 2361 observations, (morphometric analysis on individual spermatozoa) and 63,815 observations for the DNA integrity. The PCA analysis revealed five variables with Eigen values over 1, representing more than 79% of the cumulative variance. The morphometric data revealed five sperm subpopulations, while the DNA data gave six subpopulations of spermatozoa with different DNA integrity. Significant differences were found in the percentage of spermatozoa falling in each cluster among dogs (p < 0.05). Linear regression models including sperm head shape factors 2, 3 and 4 predicted the amount of denatured DNA within each individual spermatozoon (p < 0.001). We conclude that the ASMA analysis can be considered a powerful tool to improve the spermiogram.     

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.     

Quantitation of specific parameters of motility in large numbers of human sperm by digital image processing

The CellSoft computer-assisted digital image analysis system was validated for quantitating specific motility parameters in large numbers of human sperm. Motility patterns ranging from linear head trajectories (Type 1) to nonlinear, asymmetric patterns with overlapping trajectory (Type 5) were subjectively identified in semen and washed samples prepared for in vitro fertilization. A representative of each type was used for optimizing the digital imaging set-up parameters, tracking rate, and frequency. Each cell type was also characterized according to the following motility parameters: curvilinear velocity (Vcl), straight line velocity (Vsl), linearity of forward progression (Lin), maximum and mean lateral head amplitude (maxLHA; mean LHA), and beat cross frequency (BCF). Comparison of all parameters that could be determined both digitally and manually (Vcl, Vsl, Lin, and BCF) indicated no differences (p greater than 0.05) in Vcl, Lin, or BCF and only slight differences (5-6%) in Vsl measurements. After validation of the digital imaging technique, populations of seminal and washed cells were studied. Replicate analysis of the same sample demonstrated no significant intraassay variability. A comparison of semen and washed cells from 10 different donors indicated that all of the motility parameters, with the exception of Lin, were significantly higher (p less than 0.05) in washed cells. It was concluded that the digital imaging system can adequately and rapidly quantitate a large number of cells with heterogeneous motility patterns. This technique may prove to be useful in defining motility characteristics associated with capacitation, the acrosome reaction, and fertility of human sperm.     

Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity

Although sperm head shape and relative dimensions are considered reliable indicators of sperm quality, their quantification is most often operator-driven, e.g., subjective. Artificial insemination semen doses from 35 mature stud boars of known fertility and belonging to three breeds and two hybrid breeds (Duroc, Large White, Landrace, respectively, Yorker and Risco) were used in this study. Sperm samples were extended to 100x10(6) cells per mL and 10microL of the sperm suspension used to made smears which, stained, were examined using phase contrast microscopy interfaced with an automated sperm morphology analyzer (ASMA, ISAS). Each sperm head was measured for four primary parameters [area (A) microm(2), perimeter (P) mum, length (L) microm, width (W) microm], and four derived parameters of head shape [(L/W), (4piA/P(2)), ((L-W)/(L+W)), (piLW/4A)]. Definition of head size was statistically performed. The threshold for each class was established on the basis of the area values, considering the 25th percentile as small and the 75th percentile as large spermatozoa. In a second step, sperm head shape was determined as normal, elliptic, abnormal (rugose) contour, long or irregular and percentiles set as above to define spermatozoa with normal values for each shape parameter. Significant differences were found among breeds in the size of morphologically normal spermatozoa, which were significantly larger and more elliptic (P<0.001) in the Duroc breed. Sperm chromatin integrity was studied using the SCSA-assay, with significant differences observed in the degree of fragmentation intensity (DFI) although this value was consistently low in all animals studied. The hereby-validated ASMA was able to determine significant differences in sperm shape and dimensions among breeds, which were not accompanied by deviations in chromatin structure neither within nor between fertile AI-boars.     

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.     

Influence of the uterine environment on rat sperm motility and swimming speed

The present study compared several rat sperm parameters in semen samples recovered from a natural uterine environment (i.e., intact estrous female) to those recovered from an artificially induced uterine environment (i.e., ovariectomized hormonally primed female). The sperm parameters measured were percent motile, percent exhibiting forward progressive motility, actual swimming speed, and linear swimming speed. The comparisons were conducted at four postcopulatory time points (0.25, 1.5, 3, and 6 hours) in order to detect differences as a function of residence time within the uterus. No significant differences (P less than 0.05) in the parameters were seen between the two types of uterine environments. Residence time within the reproductive tract had no significant effect on the parameters with the exception of percent motile, which was significantly increased (P less than 0.01) at the 1.5-hour postcopulatory time point.     

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP-106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.     

Cryopreservation and fertility of ejaculated and epididymal stallion sperm

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides was flushed to obtain EP-0h sperm. The contralateral epididymis was stored at 5°C for 24h before being flushed to obtain EP-24h sperm. The sperm samples were analyzed at three different times: immediately after sperm recovery, after dilution in the freezing extender, and post-thawing. A fertility trial was performed using 39 estrous cycles. After ovulation induction with 1mg of deslorelin acetate (i.m.), mares were inseminated with 800×10(6) sperm. The total number of sperm recovered was 7.8±4.7×10(9) for EJ-0h sperm, 12.9±9.2×10(9) for EP-0h sperm and 12.0±8.0×10(9) for EP-24h sperm. The sperm motility, evaluated by total motility, progressive motility and the percentage of rapid cells, was similar among the samples before and after freezing (P>0.05). However, the plasma membrane integrity was different between EJ-0h and EP-0h pre-freezing and between EJ-0h and EP-24h post-thawing (P<0.05). The conception rates were similar between groups inseminated with sperm recovered from the epididymal cauda immediately after orchiectomy (EP-0h), after 24h of storage at 5°C of the epididymal cauda (EP-24h) and with ejaculated sperm (EJ-0h) (P>0.05). In conclusion, the viability and fertility of cauda epididymal sperm are similar to those of ejaculated sperm.     

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): A model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.     

Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility

Sperm plasma membrane lipids have an important role to play in determining membrane fluidity and sperm motility. The objective of the present study was to determine whether there are differences in the lipid and fatty acid (FA) composition of boar sperm and seminal plasma in the ejaculates of boars having different sperm motilities. Semen was collected from two groups of boars having normal (> 60%; n = 53) or low (< 60%; n = 53) motility sperm and the semen was evaluated for motility, morphology and vitality. The semen was then centrifuged to separate the sperm from the seminal plasma and both were kept at −20 °C until analyzed for lipid content and FA profile by gas chromatography. Total antioxidant status (TAS) of seminal plasma was determined using a commercial kit. There were differences (P ≤ 0.05) in sperm total lipids, cholesterol, saturated fatty acids (SFA), phospholipids, n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and the ratio of n-6:n-3 PUFA between boars with normal and low motility sperm. Total lipids, cholesterol, phospholipids, PUFA, DHA and n-3 PUFA were positively correlated with sperm motility, viability, normal morphology and normal plasma membrane. In contrast, SFA and the ratio of n-6: n-3 PUFA were negatively correlated (P ≤ 0.05) with sperm motility, viability, normal morphology and normal plasma membranes. The TAS of seminal plasma from boars having normal motility sperm was higher (P ≤ 0.05) than that of boars having low motility sperm and TAS was positively correlated (P = 0.0001) with sperm motility, viability, normal morphology and normal plasma membranes. In summary, differences in sperm motility were related to n-3 PUFA content in the sperm plasma membrane and extracellular antioxidants in seminal plasma which protect sperm plasma membranes from lipid peroxidation during periods of oxidative stress.     

Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.     

Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

Comparison of the semen characteristics of Fengjing, Meishan and Yorkshire boars

Semen characteristics of Chinese Fengjing, CHinese Meishan and American Yorkshire boars were examined. Samples were collected from 24 boars: 6 Fengjing, 12 Meishan and 6 Yorkshire. Three semen characteristics and 6 biochemical evaluations of semen or seminal plasma were analyzed. The whole semen parameters measured were gelatinous (gel) and gel-free volume and progressive motility. Fengjing boars were higher in gel volume than Meishan (P < 0.05) but not Yorkshire boars (P > 0.25), while Yorkshire boars were higher than Meishan boars in gel volume (P < 0.10). The gel-free volume was higher in Yorkshire and Fengjing boars than Meishan boars (P < 0.10), but Fengjing gel-free volumes did not differ from Yorkshire gel-free volumes (P > 0.80). However, the only difference detected for progressive motility was between Fengjing and Meishan boars (78.5 vs 74.5%; P < 0.10). Sperm concentration was higher in Meishan than Yorkshire boars (P < 0.01) although these breeds did not differ from Fengjing boars (P > 0.18). The biochemical messurements made were fructose, galactose, inositol, total carbohydrate and total protein in seminal plasma and pH in gel-free semen. Fengjing boars had higher concentrations of galactose in seminal plasma than Meishan or Yorkshire boars (P < 0.05), while Meishan seminal plasma had higher galactose concentrations than Yorkshire seminal plasma (P < 0.10). Fructose, inositol and total carbohydrate concentrations were all higher in Fengjing and Meishan seminal plasma than Yorkshire seminal plasma (P < 0.05). Fengjing seminal plasma had higher levels of inositol than Meishan seminal plasma (769 vs 566 mg/100 ml; P < 0.10). Furthermore, Fengjing and Meishan semen had similar fructose and total carbohydrate concentrations (P > 0.80). The pH level and total protein concentration did not differ among the 3 breeds (P > 0.30). This study indicated that breed differences were detected for several seminal parameters, although no single breed had consistantly higher values for all the parameters measured.     

Unilateral intrauterine insemination with prostatic fluid-sensitized frozen caudal epididymal sperm in beagle dogs

The effects of the absence or presence of prostatic fluid (PF) during cauda epididymal sperm retrieval were assessed as regards semen quality after freezing semen in egg yolk Tris-fructose citrate solution (EYT-FC). Epididymal spermatozoa from the left testis of each of 10 dogs was retrieved into PF, whereas that from the right testis into EYT-FC only. At sperm recovery, the only difference between the two groups was that the incidence of spermatozoa with cytoplasmic droplets (immature sperm) was lower in the PF group (P < 0.01). In contrast, after freezing-thawing, (mean +/- S.E.) sperm motility (32.0 +/- 1.4 versus 12.5 +/- 2.0%, P < 0.01) and viability (58.2 +/- 3.5 versus 41.8 +/- 5.6%, P < 0.05) were higher in the PF group than in the EYT-FC group, respectively. Furthermore, 25.6 +/- 2.7% spermatozoa in the PF group were still motile after being maintained at 20 degrees C for 6 h. The incidence of immature spermatozoa post-thaw was lower compared to that after recovery in the EYT-FC group (P < 0.01), but was still higher than that in the PF group (P < 0.05). Frozen-thawed spermatozoa (2 x 10(8)) were used for unilateral intrauterine AI. The conception rate of PF-unsensitized sperm was 20% (2/10), but that of PF-sensitized sperm was 80% (8/10; P < 0.01). Therefore, sperm recovered in PF and frozen-thawed were of good quality. Sensitization of epididymal sperm with PF before freezing clearly improved the conception rate to AI of spermatozoa derived from the cauda epididymus.