Short communication: A comparison between purebred Holstein and Brown Swiss × Holstein cows for milk production, somatic cell score, milking speed, and udder measurements in the first 3 lactations.

Brown Swiss × Holstein (BS × HO) crossbred cows (n = 55) and purebred Holstein (HO) cows (n = 50) were compared for milk yield, fat and protein production, somatic cell score, milking speed, and udder measurements for the first 3 lactations. Cows from a designed experiment were housed in a freestall barn at the experimental station of the federal state of Saxony-Anhalt, Germany, and calved from July 2005 to August 2008. Best prediction was used to determine actual production for 305-d lactations from test-day observations. For the first 3 lactations, BS × HO cows and HO cows were not significantly different for milk yield, fat and protein production, or SCS. Average milking time was significantly longer for BS × HO cows than for HO cows for first, second, and third lactations by 35, 51, and 30s, respectively. Average milking speed expressed as average yield per minute was significantly lower for BS × HO cows than for HO cows for the first 3 lactations by 0.19, 0.35, and 0.19 kg/min, respectively. Front and rear teats were significantly longer for BS × HO cows than for HO cows. Furthermore, front and rear udder clearance was significantly lower for BS × HO cows compared with HO cows in first and second lactations.

Brown Swiss × Holstein crossbreds compared with pure Holsteins for calving traits, body weight, backfat thickness, fertility, and body measurements.

Brown Swiss × Holstein crossbred cows and pure Holstein cows were compared in a designed experiment. All cows were housed in a freestall barn at the experimental station of the federal state of Saxony-Anhalt, Germany, and calved from July 2005 to August 2008. Brown Swiss × Holstein crossbred cows were mated to Holstein AI bulls for first calving and mated to Fleckvieh artificial insemination (AI) bulls for second and third calvings. Pure Holstein cows were consistently mated to Holstein AI bulls. At first calving, Holstein-sired calves from Brown Swiss × Holstein crossbred dams (282 d) had longer gestation length than Holstein-sired calves from Holstein dams (280 d). For second and third calvings, gestation length was significantly longer for Fleckvieh-sired calves from Brown Swiss × Holstein crossbred dams (284 d) than for Holstein-sired calves from Holstein dams (278 d). Holstein-sired calves from Brown Swiss × Holstein crossbred dams (43 kg) and Holstein-sired calves from pure Holstein dams (42 kg) were not significantly different for calf weight at birth for first calving. For second and third calvings, Fleckvieh-sired calves from Brown Swiss × Holstein crossbred dams (50 kg) had significantly heavier calf weight at birth than Holstein-sired calves from pure Holstein dams (44 kg). For calving difficulty and stillbirth, Brown Swiss × Holstein crossbred cows were not different from pure Holstein cows at first calving or at second and third calving. Brown Swiss × Holstein crossbred cows (71 d) were not significantly different from pure Holstein cows (75 d) for days to first breeding during first lactation; however, Brown Swiss × Holstein crossbred cows (81 d) had significantly fewer days to first breeding than pure Holstein cows (89 d) during second lactation, and the crossbred cows (85 d) tended to have fewer days to first breeding than pure Holstein cows (92 d) during third lactation. For days open, Brown Swiss × Holstein crossbred cows were not significantly different than pure Holstein cows during any of the first 3 lactations. For body weight, Brown Swiss × Holstein crossbred cows were significantly heavier than pure Holstein cows during first lactation (621 kg versus 594 kg) and second lactation (678 kg versus 656 kg). Also, Brown Swiss × Holstein crossbred cows (18.20mm) had significantly more backfat thickness than pure Holstein cows (15.81 mm) during first lactation. Brown Swiss × Holstein crossbred cows (48 cm) had significantly greater chest width than pure Holstein cows (46 cm). Furthermore, Brown Swiss × Holstein crossbred cows had significantly longer front heel walls (5.2 cm versus 5.0 cm), significantly longer rear heel walls (4.2 cm versus 4.0 cm), and significantly more depth of the front heel (4.4 cm vs. 4.1cm) than pure Holstein cows. This study has shown that F(1) of Brown Swiss × Holstein cows are competitive with pure Holstein cows for all traits analyzed here. For fertility, crossbred Brown Swiss × Holstein cows exhibited fewer days to first breeding during second lactation than pure Holstein cows.

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males.

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 100x10(6) 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.

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni).

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.

Germany/Australia index of sperm sex sortability in elephants and rhinoceros.

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.

Moderate seasonality in testis function of domestic cat.

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.

Seasonal variation in expression and localization of testicular transforming growth factors TGF-{beta}1 and TGF-{beta}3 corresponds with spermatogenic activity in roe deer.

Adult roe deer males show hormonally controlled seasonal cycles of testicular growth and involution. Mediation of endocrine signals likely requires variable production of testicular growth factors for regulation of testis function. Here we studied the expression pattern of transforming growth factors (TGFs) beta1 and beta3. Total RNA from testis parenchyma was extracted monthly and analysed using quantitative reverse transcriptase PCR. The localization of mRNAs was determined by in situ hybridization, and corresponding proteins were visualized immunohistochemically. Both factors showed different expression levels and different seasonal expression patterns. The TGF-beta1 mRNA content was up to 45 times higher than that of TGF-beta3. Compared with its lowest level in May, TGF-beta1 expression was slightly enhanced during pre-rut (June/July). TGF-beta3 expression increased 5-fold from April to June/July and decreased thereafter to its low in December. This corresponded with changing numbers of spermatocytes and round spermatids, in which both TGF-beta3 mRNA and the protein were mainly localized. The TGF-beta1 mRNA was found in interstitial cells, mainly during the non-breeding season, but also in spermatocytes and spermatids during activated spermatogenesis. The translation product was localized in few spermatogenic cells only. The results suggest that TGF-beta1 and -beta3 are important in regulating seasonal spermatogenesis of roe deer with diverse functions affecting interstitial and spermatogenic cells.

Testicular FGF-1 protein is involved in Sertoli cell-spermatid interaction in roe deer.

Roe deer (Capreolus capreolus) is a seasonal breeder showing extreme changes in spermatogenic activity. It is an excellent model to study the regulation of testicular activation and regression by endocrine signals and paracrine effectors such as growth factors. Previous studies on FGF-1 mRNA showed a special seasonal expression pattern in roe deer testis. This was difficult to explain by their exclusive localization in interstitial and Sertoli cells and without detection of the translation product. Therefore, the cellular localization of the FGF-1 protein was studied during a complete annual cycle. Parenchyma samples were collected bimonthly and prepared for histological and immunohistochemical investigations. A polyclonal rabbit anti-bovine FGF-1 antibody was used for indirect immunohistochemistry. Seasonal changes in the cellular composition of roe deer testis parenchyma were quantified by morphometry and means of computer aided image analysis. In the tubular compartment FGF-1 protein was exclusively (and stage-specific) present in elongating spermatids. This cell type occurs shortly before (June) and during the rutting season (August) only. In interstitial cells FGF-1 is detectable throughout the whole year. Results suggest FGF-1 being involved in Sertoli cell-spermatid communication and could also serve as a survival factor for somatic cell populations within the testis. The occurrence of the protein indicates an increased expression of this factor during activated spermatogenesis.

[Prevalence of anti-Yersinia antibodies in European brown hares in North-Rhine Westphalia, Germany]. / Prävalenz von Anti-Yersinia-Antikörpern bei Feldhasen in Nordrhein-Westfalen.

Yersinia (Y.) pseudotuberculosis infections may lead to significant lethality in European brown hare (Lepus europaeus, Pallas) populations especially during the cold and wet seasons. In recent decades, also Y. enterocolitica was isolated from hares found dead. Consequently, a Western-blot technique proved to be valuable for the detection of antibodies against all pathogenic Yersinia isolates was applied to monitor the prevalence of antibodies in hare populations in North-Rhine Westphalia, Germany. A total of 89.6% of the 230 animals tested was seropositive. Further investigations should be performed to elucidate the role of subclinical yersiniosis in the decline of European brown hare populations in Germany.

Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment.

Seasonally regulated breeding in roe deer, Capreolus capreolus, is associated with significant changes in testis mass, structure and function. This study has quantified seasonal changes of morphometric parameters and cellular composition in roe deer testis parenchyma. Tissue samples were collected bimonthly during a complete annual cycle. Morphometric parameters of seminiferous tubules were measured and the number of different cell types was counted using a computer-aided image-analyzing system. A scheme of eight tubular epithelium stages for active spermatogenesis was devised according to the spermatid development. Stage I is characterized by the occurrence of new round spermatids, stage IV by spermiation and stage VIII by the meiotic division of spermatocytes. The average diameter of seminiferous tubules varied between 88.4+/-3.6 micro m (February) and 216.8+/-9.2 micro m (June). Also numbers of spermatogonia, spermatocytes and spermatids per tubule cross-section showed considerable seasonal changes. In December and February the germinative epithelium mainly consists of Sertoli cells and spermatogonia. In February, the first differentiated spermatogonia enter meiosis, and in April even spermatids occasionally occur, which reach their highest numbers during the rut in August. Both the expansion and the proportion of tubular and interstitial compartment change seasonally and result in differing cell densities. Assuming numerically constant populations of Sertoli cells and interstitial cells during the entire year, the hypothetical cell numbers per mm(2) of the tubular and interstitial areas were calculated for the seasonally variable total areas of tissue cross-sections. The concordance of these theoretical values with measured cell densities provided evidence that the total numbers of Sertoli cells, as well as interstitial cells, remain really constant throughout the seasonal cycle. The exact quantification of variable and constant components provides basic data for characterization of cell type and stage-specific processes of spermatogenesis.

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation.

The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 +/- 12.7% vs. 66.8 +/- 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 +/- 5.8% in May and 15.6 +/- 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 +/- 43.9 vs. 164.4 +/- 66.4 microm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) > stallion (P < 0.001) > season (P < 0.05).

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters, morphology and function of testis and accessory glands.

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.

Effect of sperm cryopreservation and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes.

Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent trials that compared acrosome reaction, hyperactivation and chromatin integrity of fresh or cryopreserved stallion spermatozoa after treatment with heparin or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturation time and cumulus morphology on the penetration rate and fertilization rate. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound guided follicle aspiration or by scraping of follicles from ovaries obtained at a slaughterhouse. Upon recovery, we evaluated the cumulus morphology, and the COCs were matured in vitro for 18 to 24 or 26 to 40 h. Fresh semen and cryopreserved semen were treated either with heparin (200 microg/mL) or calcium ionophore (7.14 microM). Overall, 28.4% (99/349) of the oocytes were penetrated, and 12.9% (45/349) were fertilized. Fresh spermatozoa treated with calcium ionophore showed a higher penetration rate than cryopreserved spermatozoa (36.0 vs. 0%). Fresh and heparin-treated spermatozoa showed a penetration rate of 29.1%, and the same treatment for cryopreserved spermatozoa showed a penetration rate of 33.7%; none of these differences was significant (P>0.05). Fertilization rates after the calcium and heparin treatment followed the same trend and also showed no significant differences. Prolonged maturation period resulted in higher penetration (P<0.05) and fertilization rates in compact (26 to 40 h: 37.7 and 13.1% vs. 18 to 24 h: 13.1 and 2.8%) and in tendency in expanded COCs (26 to 40 h: 40.0 and 30.3% vs. 18 to 24 h: 29.4 and 13.5%). In oocytes with only a few cumulus cells, the rates tended to be higher after the shorter incubation (18 to 24 h: 33.5 and 18.8% vs. 26 to 40 h: 17.2 and 6.5%). We observed hyperactivation more frequently in fresh than in cryopreserved semen after different treatments (43.2, 39.1 and 35.4% for heparin, calcium ionophore and control vs. 15.7, 10.8 and 5.7%, respectively). We observed significant changes in the acrosome reaction of fresh spermatozoa after heparin treatment (62.6 vs. 48.2%, P<0.05), as well as in cryopreserved spermatozoa after calcium ionophore treatment (31.7 vs. 17.6%, P<0.05). The chromatin integrity was significantly reduced after heparin treatment of fresh spermatozoa, in comparison to control and calcium ionophore (81.0 vs. 87.3 and 86.6, P<0.02). We also observed a similar reduction of chromatin quality after heparin treatment in cryopreserved spermatozoa, but the difference was significant only between heparin and calcium ionophore treatment [77.4 vs. 86.4 (P<0.02) and 84.9]. The results in the this retrospective study show that capacitating fresh spermatozoa with calcium ionophore, or using heparin in cryopreserved spermatozoa, results in higher penetration and fertilization rates of in vitro matured horse oocytes. A prolonged maturation time of 26 to 40 h is necessary for compact cumulus oocyte complexes to achieve the fertilization capacity. Further investigation is needed to show the developmental capacity of these fertilized oocytes.

Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season.

The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test. In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%, respectively) were higher than during the breeding season in May (59 and 65.9%; P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%), suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors.

Establishment of assisted reproduction technologies in female and male African wild dogs (Lycaon pictus).

Transrectal ultrasonography, electroejaculation and cryopreservation of spermatozoa were applied to the African wild dog (Lycaon pictus) to establish non-invasive protocols for assessing the reproductive health of one of the most endangered African canids. Transrectal ultrasonography was performed on immobilized male (n = 2) and female (n = 5) captive wild dogs. The testes and epididymides of the male dogs were imaged transcutaneously, followed by electrostimulation and cryopreservation of spermatozoa. The sonomorphology of the female and male urogenital tracts was characterized. In females, the vagina, cervix, non-pregnant uterus and ovary were imaged and the reproductive health of each female was evaluated. The sonographic assessment helped to identify one pyometra and extensive abdominal fat deposits in two other individuals in which pyometra had been suspected. Images of the adrenal glands showed differences in size among individuals of the same breeding group. Whether these differences were related to the dominance hierarchy remains to be determined. In males, visualization of the prostate gland, testis and epididymis indicated sexual maturity. Three ejaculatory fractions (1.0, 1.5 and 0.5 ml, with 50, 95 and 95% motility, respectively; 1.125 x 10(8) spermatozoa per ejaculate) were collected from one male. The motility of each of these fractions after thawing was 0, 30 and 40%, respectively. Electrostimulation of the second male, in which a cystic structure in a testis had been identified by sonography, resulted in an aspermic ejaculate (0.5 and 1.0 ml). These technologies provided basic data on reproduction in female and male African wild dogs and were an efficient way to evaluate reproductive health.

Detection of growth factors in the testis of roe deer (Capreolus capreolus).

Roe deer is a seasonal breeder characterised by a short rutting season in summer. Mature males show synchronised cycles of testicular involution and recrudescence. Therefore, this species is a valuable model to study seasonal regulation of spermatogenesis in ruminants. It is hypothesised that a time-dependent production of testicular growth factors is required to regulate seasonal changes in testis growth and spermatogenesis. To identify potential candidates, total RNA from roe deer testis tissue was extracted at three different seasonal periods (April, August, December), and using RT-PCR the presence of several growth factors (aFGF, bFGF, IGF-I, IGF-II, TGF-alpha, TGF-beta1, TGF-beta3 and two isoforms of VEGF) was detected. Sequencing of the growth factor PCR fragments revealed a high sequence homology between cattle and roe deer. To further explore the expression patterns of the identified growth factors in roe deer their expression levels were standardised using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. The study demonstrates the expression of several growth factors in roe deer testis and supports the assumption of their seasonally diverse regulation. These results provide the basis to investigate the role of growth factors in the regulation of circannual changes of testicular activity.

Testicular mitosis, meiosis and apoptosis in mink (Mustela vison) during breeding and non-breeding seasons.

Testes of mink were compared between the breeding (March) and non-breeding seasons with the start (November) and cessation (May) of spermatogenic activity. Testicular mass and spermatozoa per gram testis were assessed. Percentages of haploid (1C), diploid (2C) and tetraploid (4C) cells were monitored using DNA flow cytometry and the proportions of somatic and spermatogenetic cells were determined after selective labelling of somatic cells with a vimentin antibody. Apoptosis was examined by cell death detection ELISA, and testosterone concentrations were measured with an enzyme-immunoassay. The significantly higher testis mass during the breeding period coincided with higher numbers of testicular spermatozoa per gram testis and peak of testicular testosterone concentration in comparison with non-breeding periods. The proportions of 1C, 2C and 4C cells showed corresponding strong differences between these periods with the maximum of 1C cells during breeding. The proportions of testicular cells in G2-M phase of mitosis were very low during the period of peak spermatogenesis; they were markedly increased in the time of autumnal resumption in November but were even higher during testis involution in May. However. the meiotic transformation (1C:4C ratio) is maximal in March. The total as well as the relative proportions of spermatogenic and somatic cells differed significantly not only between breeding and non-breeding periods but also between the periods at the start and at the end of active spermatogenesis. The intensity of apoptosis was also seasonally dependent. The highest level in March indicates a stimulated apoptosis even during the breeding period. In conclusion, the production of spermatozoa in mink is intensified by enlargement of gonads as well as enhanced efficiency of spermatogenesis during breeding. In this time, the testosterone concentration and the meiotic transformation show high levels, but the mitotic activity of spermatogenic cells is already significantly diminished and an intensified apoptosis seems to precede the forthcoming testis involution after breeding. The results suggest that the regulation of seasonal testicular activity is characterised by co-ordinated shifts in the relationships between mitosis, meiosis, apoptosis and testosterone production.

Influence of chlorocholinechloride-treated wheat on selected in vitro fertility parameters in male mice.

The aim of this study was to evaluate the influence of feeding with food and water containing chlorocholinechloride (CCC) on the fertility of male mice in a two-generation study. For this purpose the number of testicular spermatozoa and the relative proportion of primary and secondary spermatocytes involved in spermatogenesis were measured. Furthermore, the fertility of epididymal spermatozoa from tested male mice was investigated in a special in-vitro fertilization system. The experimental food was composed of CCC-treated wheat in the first experiment and CCC-free wheat and water mixed with pure CCC in the second experiment. The CCC residue content in the treated food and water was 0.21 mg/kg and 0.2 mg/L, respectively. Under the influence of feeding with CCC-treated wheat (Experiment 1) the fertilization and cleavage rates of oocytes incubated with spermatozoa from CCC-fed mice were reduced: the fertilization rate 65.1% vs. 21.1% and the cleavage rate 51.9% vs. 20.3%, p < 0.01 (control feeding vs. CCC feeding, respectively). Feeding of sperm donors with pure CCC mixed with untreated wheat pellets or water (Experiment 2) led to a reduction in the fertilization and cleavage rate (control: 60.8%, 32.4%; CCC-food: 29.8%, 12.1%; CCC-water: 30.1%, 10.2%; CCC-food/water: 36.6%, 12.5%; p < 0.01, respectively). The normal course of spermatogenesis was unchanged after the exposure to CCC. Testicular weight, the number of spermatozoa, and the proportion of haploid, diploid, and tetraploid testicular cells were not influenced. However, the functional competence of epididymal spermatozoa from CCC-fed donors was reduced, resulting in a significantly diminished fertilization and cleavage rate in vitro. The results suggest that CCC could interfere with epididymal protein secretion and the process of sperm maturation during passage through the epididymis.

Influence of environmental cadmium on testicular proliferation in roe deer.

The influence of cadmium on spermatogenesis in roe deer was studied, comparing animals from areas with high (n = 37) and low (n = 23) cadmium exposure. Testes were evaluated during pre-rut (May) and rut (July/August). During these periods the cadmium accumulation (mg/kg kidney) in polluted regions averaged 3.41 and 3.52 in comparison with 1.61 and 1.81 in controls. Cadmium accumulation was positively correlated with testis weight during both seasons. In May the proliferation (units of tissue polypeptid specific antigen/g parenchyma) was lower in animals with higher cadmium (24.02 compared with 78.20; P < 0.01). During the rut, testicular proliferation and spermatozoa/g testis as well as apoptosis showed no significant changes with increased cadmium contamination. The results suggest delayed proliferation during the pre-rutting period in animals with high cadmium exposure, but other indications of effects on the testis were not significant.

Calculation of spermatogenic transformations based on dual-flow cytometric analysis of testicular tissue in seasonal breeders.

The percentages of somatic and spermatogenic cells were quantified at different time intervals in testes of three seasonal breeders: roe deer, brown hare and mink. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry. Somatic and spermatogenic cells were distinguished by dual-parameter flow cytometry after DNA staining and selective labelling of the somatic cells with an antivimentin antibody. The portions of spermatogenic diploid and tetraploid cells were used to calculate total germ cell transformation (haploid:diploid cell ratio) as well as meiotic transformation (haploid:tetraploid cell ratio). During the breeding season the means of both ratios were 10.27 and 38.42 in roe deer, 7.55 and 15.81 in brown hare, and 11.25 and 53.47 in mink. The equivalent quotients calculated on the base of total diploid and tetraploid cells were considerably lower: 4.41 and 7.79, 5.13 and 7.86, 5.68 and 13.75, respectively. Portions as well as proportions of germinative and somatic cells changed during the annual cycle. The results demonstrate that the distinction of somatic and germinative cells in flow cytometric studies of spermatogenesis is a prerequisite for exact calculations of mitotic and meiotic processes and their alterations under different conditions.