Semen Fractions Research Articles

315 REACTIVE OXYGEN SPECIES AND OXIDATIVE STRESS IN CANINE SEMEN FRACTIONS

Reactive oxygen species (ROS) are physiologically produced by spermatozoa and leukocytes present in the seminal plasma. In low concentrations, ROS play an important role in sperm function because they are required for sperm fertilizing capacity, mainly during sperm capacitation and hyperactivation (Saleh RA and Agarwal A 2002 J. Androl. 23, 737-752). However, an imbalance between the formation of free radicals and the capacity for defense of the antioxidant mechanisms may lead to cell damage (Rover Jr L et al. 2001 Química Nova 24, 112-119). Spermatozoa are sensitive to lipid peroxidation due to its high content of polyunsaturated fatty acids and low concentrations of protective enzymes (Sharma RK and Agarwa LA 1996 Urology 48, 835-850). The aims of the present study were to compare ROS content among the 3 fractions of canine semen and to correlate these values with sperm variables. Semen samples were collected from 15 healthy dogs of distinct breeds aged 2 to 6 years. Sperm analysis was performed through motility, forward progressive velocity, morphology, and the percentage of viable sperm with the use of the eosin/nigrosin stain. The determination of thiobarbituric acid reactive substances (TBARS) was used to estimate the degree of lipid peroxidation in each sperm fraction. Values were compared using ANOVA and Tukey’s test for multiple comparisons at a significance level of 5%. Pearson correlation was used to calculate the relationship between sperm variables in the second fraction. Sperm motility, velocity, and percentage of viable sperm were within the normal range for canine semen: 84±2%, 3.4±0.1%, and 83 ± 2%, respectively. The sperm-rich fraction presented statistically higher concentrations of TBARS (1474.19 ± 245.78 ng mL-1) compared to the first and third fractions (579.41 ± 171.23 and 399.62 ± 58.08 ng mL-1, respectively; P < 0.05), indicating that spermatozoa and epididymal secretions are the main source of free radicals. No statistical correlation among TBARS and sperm motility and velocity were verified. However, a positive correlation was observed between the percentage of sperm proximal droplets and TBARS (r = 0.44, P = 0.7). This result suggests that a high incidence of sperm proximal droplets can enhance ROS formation in seminal plasma. Hence, canine sperm presenting delayed maturation in the epididymes produce higher concentrations of free radicals. In fact, sperm production of ROS occurs mainly by abnormal cells, especially the ones containing cytoplasm residues (Gomez E et al. 1998 Int. J. Androl. 21, 81-94). However, no protective effect of the sperm distal droplets was verified in canine semen as observed elsewhere for the bovine spermatozoa (Nichi M et al. 2007 Theriogenology 67, 334-340). In conclusion, spermatozoa and epididymal fluids are the primordial source of free radicals in canine seminal plasma, mainly when sperm proximal droplets are present.

Comparison of Post-Thaw DNA Integrity of Boar Spermatozoa Assessed with the Neutral Comet Assay and Sperm-Sus Halomax Test Kit

In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm-Sus-Halomax (SSH) test kit could provide similar measurements of post-thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm-rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose-LPFo-G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post-thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post-thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post-thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo-induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen-thawed boar semen quality.

Characteristics of spermatozoa of whole ejaculate and sperm-rich fraction of dog semen following exposure to media varying in osmolality

This study aimed to analyze the effects of different osmolalities on the characteristics of spermatozoa originating from whole ejaculates (WE; including the prostatic fluid) and the sperm-rich fractions (SRF). Ejaculates, collected from four mixed-breed dogs, were exposed for 10 min at room temperature to Tris-fructose-citrate (TFC) solution with osmolality ranging from 150 to 1100 mOsm. After treatment spermatozoa were evaluated by microscopic analysis of motility and fluorescent assessments of plasma membrane integrity (carboxyfluorescein diacetate and propidium iodide, CFDA/PI) and mitochondrial function (rhodamine 123, R123). Irrespective of the sperm source, there was a complete loss of motility when spermatozoa were exposed to TFC solution with 1100 mOsm. There were no marked differences in the sperm characteristics between media with 300 and 350 mOsm, regardless of the ejaculate collection procedure. However, a marked reduction in motility of spermatozoa retrieved either from the WE or SRF was observed after exposure to different anisosmotic conditions (150, 550 and 800 mOsm). In all dogs, spermatozoa from the WE exhibited greater osmotolerance in terms of plasma membrane integrity and mitochondrial function when exposed to anisosmotic conditions (150, 550, 800 and 1100 mOsm). There were inter-dog variations in response to various osmotic conditions. The findings of this study indicated that spermatozoa from the WE tolerated exposure to a wider range of osmolality than those from the SRF. It seemed that the presence of prostatic fluid of dog semen rendered the sperm membrane structures less susceptible to osmotic stress.

Peptidase activities in the semen from the ductus deferens and uterus of the neotropical rattlesnake Crotalus durissus terrificus

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.

Sites of Persistence of Lumpy Skin Disease Virus in the Genital Tract of Experimentally Infected Bulls

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell-rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell-rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.

Hepatitis C virus detection in the semen of infected patients

Though HCV infection is a serious public health problem, some aspects of its biology are still not well understood, such as its transmission through seminal fluid and sexual transmission. We looked for HCV in the semen of infected patients. Thirteen patients were included. Semen fractions (seminal plasma, leukocytes and spermatozoa) were separated with 45% and 90% Percoll gradients. The HCV-RNA in blood and semen fractions was extracted using the same protocol (AMPLICOR Roche) and was detected using the qualitative Roche Amplicor test and by agarose gel electrophoresis, with ethidium bromide staining. The mean age of the patients was 40.7 years. Risk factors for the acquisition of HCV included injectable and inhaled drug use in six (42.8%), blood transfusion in four (28.6%), and no risk factors in four (28.6%) patients. Genotype 1 was detected in 62% of the patients, followed by genotype 3 in 23% and genotype 2 in 15%. All blood samples were positive, regardless of the technique used for detection. All semen samples identified by Roche Amplicor and analyzed by agarose gel electrophoresis were negative. Among the 52 semen samples (total and fractions) identified by the Roche Amplicor method, 45 (87%) were inhibited. A negative result was recorded for one (1.9%) total semen sample, one (1.9%) leukocyte and four (7.7%) seminal plasma fractions. Only one (1.9%) sample of the spermatozoon fraction was positive. The results obtained suggested false-negative reactions for the semen samples.

Distribution of alpha-, gamma (+beta)- and delta-tocopherol in the seminal plasma, spermatozoa and seminal vesicles of rabbit

Dietary vitamin E supplementation plays a key role in animal reproduction by protecting germ cells from oxidative damage. Recently, alpha-tocopherol homologues (namely, beta-, gamma- and delta-tocopherol) have been the object of increasing research because of their peculiar nonantioxidant properties. We found that these tocol-derived compounds are not homogeneously distributed among semen components. Alpha-T was the major vitamin E homologue found in all semen fractions. Half of the total gamma (+beta)-T was found in germ cells, while more than 50% of total delta-T was preferentially accumulated in seminal plasma. The concentration of various tocol-derived compounds depended on their relative amounts in diet and the competition for saturable enzymes implicated in their metabolism. A higher concentration of delta-T in seminal plasma may be related to its more polar nature. However, the biological function of this compound in semen remains to be cleared. To our knowledge, this is the first study aimed at identifying alpha-tocopherol homologues in rabbit semen fractions.

LPS Introduced at Mating Regulates KC Production in the Murine Uterus During Early Pregnancy.

An inflammatory cascade is elicited in the female reproductive tract following mating in mice. The resulting expression of cytokines and recruited leukocytes have roles in facilitating implantation through activating immunological tolerance and endometrial tissue remodelling. We have previously shown that seminal plasma acts to induce synthesis of GMCSF and IL6 in the female reproductive tract in response to TGFβ present in seminal fluid. Recently we have shown that chemokines, specifically the neutrophil chemoattractant KC (mouse homologue of human IL8), is dramatically upregulated after mating. The purpose of this study was to identify the active constituent of semen responsible for KC induction. Female mice were mated with either intact, seminal vesicle deficient or vasectomized males and uterine lavages were collected approximately 8 hours later when KC content was measured by KC specific ELISA. KC production was increased 13-fold, 6-fold and 10-fold respectively, indicating that neither the seminal plasma nor the sperm fraction of semen is independently necessary for induction. To investigate more precisely the identity of the KC inducing factor, an in vitro primary uterine epithelial cell culture system was employed. Uterine epithelial cells were harvested from estrous female mice and exposed to a range of doses of seminal vesicle fluid, recombinant TGFβ1 or lipopolysaccharide (LPS). Following addition of seminal vesicle fluid or TGFβ1, KC production was decreased by 100% and 58% respectively, however synthesis was increased ≈2-fold in a dose-responsive manner to LPS. In addition, instillation of LPS into the uterine luminal cavity of estrous mice acted to induce a 2-fold increase in KC production and to elicit neutrophil recruitment into the endometrial tissue. To further evaluate the contribution of LPS signalling via its receptor, toll-like receptor 4 (TLR4), to the post insemination induction of uterine KC, uterine fluid lavages were collected 8 hours after mating from female LPS hyporesponsive (C3H/HeJ) mice. Uterine KC production was increased 6-fold, similar to that observed in wild type mice, indicating that the increase in uterine KC content in response to mating is independent of LPS signalling via TLR4. Neutrophil recruitment into the uterine tissue following mating in C3H/HeJ mice was similar to that observed in wild type mice. Together these data indicate that while LPS derived from the male or lower female reproductive tract accessing the uterus after insemination can contribute to KC induction, factors other than LPS may contribute to this response, and/or LPS signalling may be mediated by a signalling pathway that is independent of the conventional LPS receptor, TLR4. These findings highlight the potential importance of microflora in the male and female reproductive tracts in influencing the immune environment during early pregnancy. This work was supported by Program Grant 453556 from the NHMRC of Australia.

Development and validation of a duplex real-time PCR assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples

A duplex real-time quantitative PCR (qPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and an exogenous internal positive control (IPC) in porcine semen samples was developed. The IPC was included to monitor DNA extraction and PCR inhibition and consisted of a mutated PCV2 plasmid clone which differed from the target PCV2 in the probe binding region and thus was detected by the use of a second probe with different end-labeling. The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested PCR (nPCR) methods for the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable tool for accurate and quantitative detection of PCV2 DNA in boar semen.

Caspase-3 activation and phosphatidylserine membrane translocation in human spermatozoa: is there a relationship?

The presence of biochemical signs of apoptosis in ejaculated spermatozoa suggests that apoptosis may be one of the pathways for sperm death. The relationship between the phosphatidylserine (PS) externalization and the presence of the active form of caspase-3 (CP-3) was studied in human spermatozoa after exposure to hydrogen peroxide (H 2O 2). Semen from 27 normozoospermic men was examined, as the neat semen, after swim-up isolation and after H 2O 2 incubation, for the translocation of PS, activation of caspase-3 and mitochondrial membrane potential. The percentage of vital spermatozoa expressing PS translocation was lower than the percentage of vital spermatozoa with the active form of the caspase-3, either in neat (4.9 ± 2.3% versus 19.7 ± 6.2%, P < 0.001) or in swim-up semen (2.2 ± 2.3% versus 4.8 ± 2.9%, P < 0.01). After swim-up isolation, the percentage of vital spermatozoa with active caspase-3 decreased ( P < 0.01). After H 2O 2 stimulation of the swim-up semen fraction, a decrease in the mitochondrial membrane potential was observed ( P < 0.001). Only the midpiece revealed PS translocation after H 2O 2 stimulation, and it was also the only part to reveal the presence of the active form of caspase-3. All spermatozoa expressing the PS translocation revealed the presence of the active form of caspase-3.

Pregnancy percentage following deposition of sex-sorted sperm at different sites within the uterus in estrus-synchronized heifers

Our objective was to assess the effect on heifer pregnancy rate of deposition at three sites within the uterus of frozen-thawed sex-sorted sperm at a fixed time after estrus synchronization. Estrus was synchronized in 209 heifers by administration of PGF2a 14 days apart. At 80–82 h after the second PGF2a injection, X-chromosomes bearing fractions of semen with 2.2 × 10 6 sperm in insemination dose were used for single insemination into the uterine body (UB-AI, n = 91) or for intracornual deposition in the middle of the uterine horn (MH-AI, n = 57) or close to the utero-tubal junction (UTJ-AI, n = 61). The overall pregnancy rate was 43.1%. Pregnancy rates did not differ ( P > 0.05) among sites of sperm sperm deposition, between the two farms at which the heifers were kept or between the two bulls producing the semen. Within UB-AI, MH-AI and UTJ-AI treatments, pregnancy rates were 41.8%, 49.1% and 39.3%, respectively ( P > 0.05). Pooled across classes for deposition site, pregnancy rate was 25.1% higher ( P < 0.01) for heifers showing strong signs of estrus than for heifers showing weak signs of estrus (45.9 versus 20.8%, respectively). Embryonic and fetal loss from diagnosis of pregnancy to term and at calving equalled 5.6%. Of 88 calves of identified sex, 93.2% were female. In conclusion, pregnancy rates of heifers did not differ significantly following deposition of 2.2 × 10 6 sex-sorted sperm 80–82 h after the second PGF2a injection near the utero-tubal junction, in the middle of the horn or into the uterine body.

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing

Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

Validation of an automated real-time PCR protocol for detection and quantitation of HIV and HCV genomes in semen

The ability to detect and quantify HIV and HCV genomes is important for checking spermatozoid preparation protocols also known as “sperm washing”. But no commercial assay is available. A method was developed for detecting HIV and HCV in semen fractions using the COBAS Ampliprep™ and COBAS Taqman™ instruments. It will detect fewer than 200 copies of HIV RNA per ml of semen plasma and fewer than 200 copies/3 × 10 6 semen cells. The sensitivity for HCV is similar at more than 200 IU/ml and below 200 IU/3 × 10 6 semen cells. No inhibitor of PCR amplification was detected. This automated protocol permits a convenient, standardized testing for HIV and HCV in semen. The performance is the same as that of the previous generation of automated assays but the cost and operating time are both reduced.

Inadvertent Germline Transmission of AAV2 Vector: Findings in a Rabbit Model Correlate with Those in a Human Clinical Trial

The risk of germline transmission of vector sequences in humans is a major safety concern, because the enrollment of subjects of reproductive age in early-phase clinical trials of gene transfer continues to increase. In a study of adult men with hemophilia B, adeno-associated virus serotype 2 (AAV2) delivered to the liver via the hepatic artery resulted in unexpected transient vector dissemination to the semen. Here we report that intravenous AAV2 injection in rabbits proved a useful model to assess biologic parameters of vector dissemination to the semen. Detectable vector sequences in semen disappeared in a dose-dependent and time-dependent fashion. AAV infectious particles were present only as long as day 4 after injection and were undetectable thereafter. The kinetics of vector clearance was faster in the semen fractions enriched for motile sperm than in the total semen. In addition, increased frequency of semen sampling accelerated the clearance of vector sequences from semen. Long-term follow-up, spanning hundreds of spermatogenesis cycles, showed that there was no recurrence of detectable vector sequences in semen, thus reducing the probability of inadvertent transduction of early spermatogonia not committed to differentiation at the time of vector injection. We conclude that AAV2 presents minimal germline transmission risk for humans.

Medically assisted procreation and transmission of hepatitis C virus: absence of HCV RNA in purified sperm fraction in HIV co-infected patients

The risk of hepatitis C virus (HCV) transmission in medically assisted procreation (MAP) is debated and some researchers have proposed to exclude MAP for HCV-positive infertile patients. The objectives of this study were to assess the presence of viral RNA in the final preparation of density gradient semen fractions collected from men with chronic HCV and HIV co-infection participating in a MAP program, and to assess whether HIV co-infection influences the rate of the presence of HCV RNA in the semen. The study was based on a cohort of 170 HCV-infected male patients (93 HIV co-infected) participating in a MAP program in a French center. Semen samples were subjected to standard MAP sperm preparation, using density-gradient centrifugation with 40 and 90% layers. All aliquots were tested with a commercially available HCV RNA assay (Roche Monitor), adapted for use with semen after a nucleic HCV RNA extraction modification (Organon Technika). Seminal plasma samples from 19 (11%) patients were HCV RNA positive. The positive HCV viral load in semen was less than 600 IU/ml. None of the 90% fractions from HCV-infected patients were HCV RNA positive. Among the 93 co-infected patients, 10 were positive for HCV RNA in semen and three were HIV/HCV RNA positive in semen. Although HCV RNA was found in the semen of 11% of patients, no purified sperm fraction, or spermatozoa used in MAP were HCV RNA positive. The 90% purified sperm fraction discards the virus and must be used with care in MAP.

Multicenter quality control of the detection of HIV-1 genome in semen before medically assisted procreation

Couples in whom the man is HIV-1-positive may use medically assisted procreation in order to conceive a child without contaminating the female partner. But, before medically assisted procreation, the semen has to be processed to exclude HIV and tested for HIV nucleic acid before and after processing. The performance was evaluated of the technical protocols used to detect and quantify HIV-1 in 11 centers providing medically assisted procreation for couples with HIV-1 infected men by testing panels of seminal plasma and cells containing HIV-1 RNA and/or DNA. The performance of these tests varied due to the different assays used. False positive results were obtained in 14-19% of cases. The sensitivity for RNA detection in seminal plasma was 500-1,000 RNA copies/ml, over 500 RNA copies/10(6) cells in semen cells, and for DNA detection in semen cells 50-500 DNA copies/10(6) cells. The use of silica-based extraction seemed to increase the assay performance, whereas the use of internal controls to detect PCR inhibitor did not. This first quality control highlights the need for technical improvements of the assays to detect and quantify HIV in semen fractions and for regular evaluation of their performance.

SEMEN COLLECTION IN RHINOCEROSES (RHINOCEROS UNICORNIS, DICEROS BICORNIS, CERATOTHERIUM SIMUM) BY ELECTROEJACULATION WITH A UNIQUELY DESIGNED PROBE

Electroejaculation in rhinoceroses has historically yielded inconsistent results, with the collection of high-quality, sperm-rich samples rare. The goal of this study was to develop a reliable method of electroejaculation in the rhinoceros by designing a rectal probe that appropriately fits the anatomy of this taxon and refining the procedure. A curved probe handle ending in an oblate, ellipsoid head was built using readily available supplies. A combination of rectal massage, penile massage, and electrical stimulation with a specially designed probe was employed in attempts to collect semen on 14 occasions from greater one-horned rhinoceroses (Rhinoceros unicornis; n = 4), black rhinoceroses (Diceros bicornis; n = 2) and a southern white rhinoceros (Ceratotherium simum; n = 1). During 13 of the 14 attempts, ejaculates were collected in multiple fractions. All but one of the ejaculates contained spermatozoa, and seven ejaculates contained good-quality fractions of semen (-60% sperm motility; > or =20 x 106 spermatozoa/ml) suitable for sperm banking and assisted reproduction procedures. Mean (+/-SEM) values for volume, pH, osmolality, and total sperm number for ejaculates containing good-quality fractions (98.2 +/-21.8 ml, 8.5+/-0.1, 290.4+/-6.7 mOsm, and 37.1+/-12.0 x 10(9), respectively) did not differ (P > 0.05) from those containing only poor-quality samples. Urine and/or erythrocyte contamination was not uncommon in fractions of both ejaculate types. Males producing good-quality samples ranged in age from 7 to 34 yr. None of the samples contained > or =75% morphologically normal spermatozoa. Electroejaculation with a uniquely designed probe consistently produced ejaculates in the rhinoceros. However, the production of high-quality samples continued to be challenging, occurring in only 50% of collection attempts. Regardless, the technology has progressed to a stage at which good-quality semen samples can be produced for sperm banking and assisted reproduction, and thereby can be integrated into intensive rhinoceros management strategies for the ultimate survival of this taxon.

Lipid composition of the main fractions of rabbit semen

Rabbit semen contains mature spermatozoa and several other fractions (seminal plasma, droplets and vesicles) which are separated by various procedures. These fractions have a variable lipid profile: spermatozoa contain the largest amount of phospholipids (PL), whereas seminal plasma, droplets and vesicles accounted for 49.8% of the total PLs. The cholesterol content in raw semen was 811 μg/10 9 but was only 21–23% in spermatozoa. The main PL classes of rabbit spermatozoa were PC, LPC, PE, PS, SM and PI, which varied according to the separation procedures used. Percoll-separated spermatozoa (Sp p) showed an increase of LPC, a higher LPC/PC ratio but a lower lipid content compared to the theoretical amount. This membrane modification did not affect the number of live cells but greatly influenced the functional properties of the rabbit spermatozoa, i.e. the HOS-test and induced acrosome reaction. PC, followed by PE and LPC were the most abundant PL classes of seminal plasma, droplets and vesicles. These fractions have higher PE and SM levels and lower PC/PE + PC ratios than in the germinal cells. Some physiological implications are discussed.

Quantitative study of caspase-3 activity in semen and after swim-up preparation in relation to sperm quality

There are no studies relating the apoptotic marker caspase-3 in human sperm to different degrees of abnormal sperm concentration, morphology and rapid progressive motility. Semen from 67 males with abnormal semen analyses (n = 61) and normozoospermia (n = 6) were used. In each case, sperm from the neat semen (semen fraction) and after gradient centrifugation and swim-up (swim-up fraction) were incubated with a caspase-3 profluorogenic substrate. Caspase-3 activity was quantified in 119 850 sperm, 67 488 from semen and 52 362 from swim-up fractions. Logistic regression was used to estimate odds ratio (with 99% confidence intervals) for the presence of caspase-3-positive sperm. In semen fractions, no relationship was found between abnormal semen analysis subgroups and sperm caspase-3 activity. On the contrary, a significantly increased number of sperm with caspase-3 activity was found in the swim-up fractions from samples with poor sperm morphology. When analysis was restricted to single semen analysis defects, a significant increase of caspase-3-positive sperm was found in the semen fractions of cases with asthenozoospermia, and in the swim-up fractions of cases with teratozoospermia. Sperm caspase-3 activity seems to be associated with teratozoospermia and asthenozoospermia, thus suggesting that nuclear, mitochondrial and cytoskeletal abnormalities induce caspase-3 activation during spermiogenesis or sperm maturation.

63 IMPROVEMENT OF SEMEN-DERIVED EPITHELIAL CELL PROLIFERATION BY FIBROBLAST CO-CULTURE

We previously isolated epithelial-like cells (ELC) from sheep (Ovis aries) and eland (Taurotragus oryx) semen (Nel-Themaat et al. 2004 Reprod. Fertil. Dev. 16, 152) and subsequently developed a system to separate ELC before plating (Nel-Themaat et al. 2005 Reprod. Fertil. Dev. 17, 314). Cells attached and proliferated in only 50% and 31% of the attempts, respectively. Therefore, the purposes of the present study were to improve ELC proliferation by co-culture with inactivated 3T3 mouse fibroblasts and to characterize the obtained cells. Semen fractions from two mature Gulf Coast Native rams (n = 20 ejaculates) and one common eland bull (n = 2 ejaculates) were plated on feeder layers (A), on collagen with feeder cell inserts (B), or on collagen alone (C). For B and C, cell attachment and division were assessed; proliferation and passage 1 (P1) confluence were evaluated for A, B, and C. No difference in attachment rates between B (80%) and C (70%) were found for ovine cells, but (P &lt; 0.05) cells divided more times in B (80%) than in C (35%). All colonies in A (60%) and B (70%) reached P1 confluence and no difference was detected between A and B, but less proliferation (10%) and P1 confluence (5%) were observed in C. Therefore, contact between epithelial cells and feeders was not necessary for growth stimulation by the feeder cells. No difference among A, B, and C was detected for eland ELC proliferation (100%), but no P1 confluence was observed in C. Ram cells were subsequently characterized by immunohistochemical detection of keratin and vimentin, as well as morphology. The 3T3 cells cross-reacted with keratin, and characterization was thus performed mainly on morphology and vimentin expression. Distribution of vimentin microfilaments differed between different epithelial morphologies and fibroblasts. Expression in epithelial cells was faint and patchy in confluent colonies and located around cytoplasmic extremities in semi-confluent colonies. In 3T3 cells, expression was very prominent throughout the cytoplasm and around the nucleus. For treatments A and B, 63 and 57%, respectively, were characterized as only epithelial cells; 25 and 36%, respectively, appeared to contain a mixture of epithelial and fibroblast cells; and 13 and 7%, respectively, contained only fibroblast cells. Only one sample was evaluated from treatment C and only keratin was detected in the epithelial-like colony. We conclude that culture of semen-derived ELC is markedly improved by 3T3 fibroblast co-culture. Further research on conditioned media may simplify the system and reduce chances of 3T3 cell contamination.