Ultrasound and electroejaculation have become accepted methods for the assessment of the reproductive status of male rhinoceroses. The activity status of the accessory sex glands, the amount of testicular fibrosis as sign of ageing and the semen quality in the ejaculate are characteristics, which were regarded as sufficient to allow an accurate evaluation of the current breeding status of a bull (HERMES et al., 2005) . However, a recent report on testicular neoplasia gave a first indication of the occurrence of reproductive pathology in male rhinoceroses and possible necessity of advanced diagnostic procedures to evaluate reproductive status and health (PORTAS et al., 2005). In this study routine reproductive assessments of 6 male rhinoceroses of four different species (black n=1, white n=3, Indian n=1, Sumatran n=1) produced findings, aspermatic ejaculates (n =3), non-nomgenicity of the testicluar parenchyma (n=3) and epididymal cysts (n=1), which required further diagnostic procedures. Ultrasound-guided biopsy of the testes was performed in 4 males as part of the routine reproductive assessment using a 18/17 G needle-trocar biopsy system (Somatex® Medical Technologies GmbH, Berlin, Germany) to further assess the activity of the spermatogenic tissue or to determine the histopathological character of a neoplastic lesion. The testis biopsy samples (1-3 per testis) was immersion-fixed in cold Bouin's solution and embedded in paraffin and stained with the standard Haematoxylin & Eosin ( HE ) Staining Protocols . In the aspermatic white rhinoceros the seminiferous tubules showed no pattern of active spermatogenesis. The spermatogenic activity appeared arrested at the level of elongating spermatids. The aspermatic Sumatran rhinoceros with epididymal cysts showed a similar arrested spermatogenesis. While this primary infertility in the white rhino seemed related to very poor body condition the aetiology in the Sumatran rhino remains speculative, specifically, with the high libido this male had expressed. In the aspermatic Indian rhinoceros all stadiums of spermatogenesis were present in the tubuli semeniferi. Despite this active spermatogenesis the stage of spermiation, the presence of adluminal sperm as last step in spermatogenesis was absent. This seemingly reduced spermatogenesis in combination with low testosterone concentrations suggested a temporary infertility. Histopathology combined with the irregular ultrasound appearance of a neoplasia in the black rhinoceros characterised this lesion as malignant, resulting in the decision to hemi castrate this male. However, due to the small sample size and a consequent lack of tumour tissue connection to the basal membrane a specification of the tumour type from a fine-needle biopsy sample proved difficult. It is concluded that ultrasound of testis and epididymis and testicular biopsy in rhinoceros have the potential to specifically diagnose transient or permanent reductions in

Vascular endothelial growth factor A (VEGFA) influences spermatogenesis, but its impact on seasonally regulated sperm production is still not fully understood. Thus, we investigated both expression levels and localisation of VEGFA and its receptors VEGFR1 and 2 in roe buck testis via real-time reverse transcription polymerase chain reaction and immunohistochemistry in relation to seasonal changes in the cellular composition of the testis. VEGFA was expressed by interstitial cells while its receptors were found on endothelial and perivascular cells. Inside the tubules, VEGFA was located in spermatogonia and spermatocytes, VEGFR1 was present on elongating spermatids and VEGFR2 on Sertoli cells. VEGFR1 mRNA was expressed tenfold lower than VEGFR2 and VEGF mRNAs. Relative VEGF and VEGFR2 expression (divided by the number of VEGFA and VEGFR2 expressing cells) showed an increase towards the rut (July/August) and a decrease thereafter. The results suggest involvement of VEGFA in the adjustment of vascular permeability as well as in spermiogenesis and the proliferation of spermatogonia.

In seasonal breeders such as the European roe deer (Capreolus capreolus) the well timed regulation of testicular functions is crucial to repro-ductive fitness and successful breeding. Such functions are in particular the production of spermatozoa and testosterone. Having only a very short rutting season from mid-July to mid-August, roe deer testis undergo drastic cyclic changes during the transitions between breeding and non-breeding seasons. These changes require mechanisms to stimulate both cell proliferation and spermatogenesis during testis growth (re-crudescence) and to initiate repression of spermatogenesis during testis involution. The expression of genes responsible for these local con-trol mechanisms needs to be seasonally regulated. Due to the lack of a fully sequenced roe deer genome, we used an 8K expression microar-ray from cattle as its evolutionary closest and sequenced relative to investigate the gene expression changes in roe deer testis during the tran-sition between the two extreme phases of the circannual testicular development: the arrest of spermatogenesis (December) on one hand and the fully activated spermatogenesis (June) on the other. During the transition from December to June, 622 genes were significantly differen-tially expressed (p < 0.05). Out of the 407 genes found to be up-regulated, 197 exhibited an increase in expression of twofold or more, and 132 out of the 215 down-regulated genes showed a decrease of twofold or more. Based on database queries, 51 of the differentially expressed genes could be assigned to 50 broad biological processes, 32 of which covered molecular functions and 28 were assigned to different cell components.

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.

ABSTRACT: Teratospermia (>60% morphologically abnormal sperm/ejaculate) is associated with increased sperm output in the domestic cat. The objective of this study was to determine whether increased sperm production in teratospermic donors was associated with disturbances in germ cell apoptosis, the usual mechanism for sperm cell elimination. Apoptosis was measured by evaluating DNA fragmentation, expression of Caspase‐3, and anti‐apoptosis repressor with caspase recruitment domain (ARC) in the testes of normospermic compared with teratospermic cats. Testes (n = 6 males/group) were obtained by bilateral castration and immediately fixed in Bouin solution. Results revealed that greater than 97% of cells labeled as DNA fragmented were tubular regardless of male type. Fewer ( P < .05) apoptotic spermatogenic cells per tubule (0.52 ± 0.11 cells/tubule, x̄ ± SEM) and per 100 Sertoli cells (3.79 cells/100 Sertoli cells) were observed in teratospermic compared with normospermic (1.25 ± 0.36 cells/tubule and 6.44 cells/100 Sertoli cells) cats. Among the spermatogenic cells, fewer ( P < .03) spermatocytes were positively labeled in teratospermic (0.3 ± 0.07/tubule) compared with normospermic (0.83 ± 0.28/tubule) counterparts. Neither donor type differed in Caspase‐3 or ARC expression activity. However, each factor was both cell‐ and stage‐specific in expression. Specifically, Caspase‐3 was located in Sertoli cells, A‐spermatogonia, and round spermatids at stage V. The ARC was found in primary spermatocytes at each stage of the spermatogenic cycle. These results demonstrate that the high incidence of morphologically abnormal sperm in teratospermic male cats is accompanied by a reduced elimination of defective spermatogenic cells via apoptosis.

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n = 16) or frozen-thawed semen (Group B, n = 9) at a concentration of 100 × 106 spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.

Movement tracks of spermatozoa of human, lion, tiger, cow, pig and sheep are recorded by dark field photography (fluid layer thickness 16.7 microns, exposure time 1 s). Comparison before and after capacitation by an incubation of 2 h in modified tissue culture medium TCM 199 with 10% fetal calf serum resulted in two quite different patterns of hyperactivated spermatozoa: 1) Tracks are broadened due to enlarged lateral head displacement or radius of rotating head movements respectively (tiger, lion) and show beside that a markedly increase in erratic motility (human). 2) In the studied species of artiodactyla, cow, pig and sheep, a qualitative new, panicle-like pattern arised as a result of superposition of spermatozoa head pendular movements around the axis of forward motility and the other one around the head axis. This new type of tracks allows a simple quantitative analysis of hyperactivation of bull spermatozoa, first described in this report. The method is applied to investigations on efficacy of capacitation media and provides evidence for high individual differences of semen donors in capacitation success.

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p < 0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p < 0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20–39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p < 0.01, p < 0.03, p < 0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p < 0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.

Adult roe deer males show seasonal cycles of testicular growth and involution. The exact timing of these cycles requires endocrine regulation and local testicular control by autocrine/paracrine factors. Recent findings suggest that the vascular endothelial growth factor (VEGF) might have effects on both vascular and germinative cells in testis. Thus, we studied the expression pattern of vascular endothelial growth factor (VEGF) in roe deer testis using quantitative RT-PCR. The strength of VEGF mRNA expression depended on season. It reached its highest level at the peak of spermatogenesis during the pre-rutting period and had its nadir at the end of the rut when involution already began. The results suggested that VEGF may directly affect the regulation of spermatogenesis but may not be involved predominantly in testicular microvasculature as initially expected.

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

Roe deer (a seasonal breeder, rut: July to August) is a well characterized model for studying the seasonal regulation of testicular activity. However, not much is known about the impact of estrogens on seasonally determined sperm production. We therefore explored the time and cell type specific expression of estrogen receptors and of enzymes involved in steroid biosynthesis in roe deer testicular parenchyma and in the epididymis. Every second month during the entire seasonal cycle five roe bucks were castrated (n = 30). Estrogen receptor (ER) α, ERβ and the enzymes P450Aromatase and P450C17 were localized immunohistochemically. The expression levels of ERα, ERβ and P450Aromatase were evaluated by semi-quantitative Western blot. Contrary to the enzyme required for androgen production (P450C17), which is expectedly located only in the Leydig cells and shows an expression increase towards rutting season, a seasonal expression difference of the enzyme required for the conversion into oestradiol (P450Aromatase) is visible only in the epididymis. In the testis, ERα expression shows a striking dependency on tubular cell composition, and the single cell expression activity increases towards rut. This implicates that estrogens are directly involved in the regulation of spermatogenesis in the roe buck. In the epididymis, expression of ERα is seasonally determined particularly in the ductuli efferentes. ERβ was detected throughout the year with no distinct dependency on season or the stages of germinative epithelium cycle. We conclude that estrogens in the roe buck influence the seasonally determined sperm production predominantly by the regulated expression of ERα.

The roe deer shows a distinct seasonal breeding pattern accompanied with significant changes in testicular structure and function during the annual cycle. It serves as a uniquely well-characterized ruminant model system to investigate the regulation of testicular activity. However, data regarding the seasonal variations taking place in the epididymis of the roe buck are not available. Therefore, this study provides a detailed morphological description of the roe buck's epididymis (cell types and segments) and a qualitative as well as quantitative characterization of the seasonal changes in the different parts of the duct. For every second month of the complete seasonal cycle, five roe bucks were castrated (n=30). Seasonal changes in the cellular composition of the epididymis were studied by computer aided image analysis of histological preparations. With regard to morphological criteria we defined 6 segments (S) within the epididymis (ductuli efferentes and S1–5) during the active period. S1–3 are located in the caput, 4 represents the corpus and 5 the cauda epididymidis. The epithelium consists of principal cells, basal cells, macrophages, lymphocytes and apical cells, except for the ductuli efferentes (cuboidal epithelium composed of ciliated and unciliated cells) and S5 (no apical cells). The quantification of the three functional compartments within the organ (lumina, epithelium and interstitial tissue) revealed distinct and region-specific seasonal changes in the cellular composition of caput, corpus and cauda epididymidis. As expected, the duct with its surrounding tissue expands towards rutting season. In the caput this enlargement of the duct is primarily caused by the growth of the epithelial compartment, whereas in the cauda it is predominantly attributed to the dilatation of the lumen, which is filled with testicular and epididymal fluid and spermatozoa towards the rut. This leads to distinct changes in the tissue composition of samples taken from the three main regions of the epididymis at different times of the year. This morphometric study provides the prerequisite for investigations of regulation mechanisms in epididymis function.

Adult male domestic cats are known to produce sperm throughout the year, although sexual activity is influenced by geographical location. In the northern hemisphere, feral domestic cats reproduce usually between January and July. Thus, seasonality in testicular activity might be suggested. The aim of the present study was to investigate gametogene and endocrine activity of cat testis throughout the entire year. Testes and epididymides (n = 10-12 per month) were collected after castration. Spermatogenesis was quantified by assessment of testicular sperm per testis and by flow cytometric analysis of the cells with different DNA content. Sperm from cauda epididymis were evaluated according to motility and morphological integrity. Testicular testosterone concentration was determined by enzyme immunoassay. Testis mass and sperm production varied moderately throughout the year. Significant seasonal variations were observed in the proportion of cells in the G2/M phase of cell cycle (p = 0.004) and the meiotic transformation (ratio of haploid : tetraploid cells; p = 0.021). Changes in testicular testosterone concentration were more pronounced and showed periods with high (spring) and significantly reduced testosterone levels (autumn). A marked seasonal alteration (p < 0.001) with a peak in March was assessed in the percentage of progressively motile sperm. The proportion of morphological intact sperm was also significantly higher in spring compared with winter time (p < 0.001). In conclusion, the study suggests moderate seasonal changes in quantity of sperm, more pronounced annual variation in hormone production and a distinct seasonal influence on functional sperm parameters in domestic cat.

Apoptosis is involved in the regulation of spermatogenesis. The involution of testes in seasonal breeders might be expected to involve enhanced apoptotic cell elimination. We have compared seasonally changing testicular apoptosis in roe deer with that in non-seasonally breeding cattle. Apoptotic cells were detected as TUNEL-positive cells by both flow-cytometric analysis and in situ localisation of fragmented DNA in tissue sections. Apoptosis-induced DNA fragments were also assessed by enzyme-linked immunosorbent assay (ELISA) in homogenised testicular parenchyma. As expected, the testis mass and the percentage of haploid cells in roe deer showed a seasonal pattern with a significant maximum during the rut (August), whereas no annual variation of these parameters was found in bulls. All three methods for determining apoptosis showed similar findings. Roe deer exhibited significant seasonal fluctuation of total apoptotic activity (ELISA, apoptotic cells per tubule cross section) with a maximum during the breeding season. However, the seasonal differences in the number of apoptotic cells corresponded to the variable total numbers of spermatogonia and spermatocytes per tubule cross section. Thus, the percentages of TUNEL-positive cells related to the combined number of both germ cell types showed no seasonal variance, as confirmed by percentages of apoptotic cells analysed flow-cytometrically. The maximum level of apoptosis during the rut in roe deer was similar to the values obtained during the invariably high spermatogenic activity in cattle. These results suggest that, in roe deer, apoptosis is not the cause of the seasonal involution of testes.

Plasmalogens (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphocholines and -phosphoethanolamines) are important constituents of spermatozoa membranes and possess significant antioxidative properties. This particularly holds as plasmalogens from spermatozoa also possess a very high content of highly unsaturated fatty acyl residues (especially 22:6). The organic spermatozoa extracts of two different ruminants (cattle and roe deer) were analyzed for their contents of characteristic choline plasmalogen oxidation products by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry. It will be shown that 1-hydroxy-2-docosahexaenoyl-sn-glycero-3-phosphocholine (LPC 22:6) and formyl-LPC 22:6 are reliable measures of lipid oxidation of spermatozoa and allow, accordingly, conclusions about the storage conditions. All data on spermatozoa were also confirmed by the investigation of the oxidation behavior of selected reference compounds. It will be shown that, equally if plasmalogens or diacyl PC species are used, oxidation takes place primarily at the double bond next to the glycerol backbone. These data were additionally confirmed by recording the corresponding post source decay (PSD) fragment ion spectra.

Seasonal changes in spermatogenesis were studied with respect to testicular production of both testosterone and epidermal growth factor (EGF) in mink. The testes were collected in November (n = 15; testis recrudescence), February (n = 15; before breeding season), March (n = 14; breeding season), and May (n = 11; testis involution) and the following parameters of testicular activity were quantified: testicular mass, number of testicular spermatozoa, percentages of haploid, diploid, and tetraploid (G2/M-phase) cells and content of testosterone and EGF. The growth factor was immunohistochemically localized in the parenchyma. Testis mass, spermatogenic activity, and the production of both testosterone and EGF were maximal in March, but were not significantly different from the levels in February. The correlation between testis weight and sperm per testis was r = 0.825 (P < 0.001). Testosterone and EGF levels were correlated to each other (r = 0.78; P < 0.001) and had significant positive correlations to testis mass, number of sperm and proportion of haploid cells; and negative correlations to percentages of mitotic cells. EGF was localized in interstitial cells and in the luminal region of seminiferous tubules, where it occurred during the last steps of spermiogenesis. We inferred that intensified seasonal spermatogenesis was stimulated by testosterone and by autocrine/paracrine effects of EGF.

In mature male seasonal breeders, the circannual cycles of testicular growth and involution involve significant changes in structure and function of both the tubular and interstitial testicular compartment. Roe deer (Capreolus capreolus) are seasonal breeders with a short defined rutting season from mid-July to mid-August and represent a unique non-rodent model for studying testicular functions during the course of a complete reproductive cycle with naturally changing photoperiod. Germ cells and Sertoli cells of the seminiferous tubules and the interstitial Leydig cells all display significant morphological and physiological alterations during the seasonal changes. In contrast to the germ cell population, Sertoli and Leydig cells persist as a numerically constant cell population in the roe deer testis. This report emphasizes the intricate relationship between seasonal cellular differentiation, intratesticular growth factor networks and their impact on the functional dynamics during the seasonal changes in roe deer testis.