R. Thun

Comparison of Biociphos-Plus® and TRIS-egg yolk extender for cryopreservation of bull semen

For optimizing routine freezing of bull semen, we examined three different cryopreservation methods using either TRIS-egg yolk-citrate extender or Biociphos-Plus. Biociphos-Plus (IMV, France) has been marketed as an extender, in which egg yolk is replaced by a sterile soybean extract to reduce the contamination risk derived from animal borne substances. We used 78 bulls of various breeds (Brown Swiss, Holstein, Simmental) between 12 and 23 months of age, and we produced a total of 800-1000 straws (0.25 ml, 20 x 10(6) spermatozoa) from each bull using three different methods. In method A, we used TRIS-egg yolk as extender and packaged at 4 degrees C. In method B, we also used TRIS-egg yolk but packaged at room temperature (RT) between 18 and 22 degrees C. In method C, Biociphos-Plus served as extender and we packaged at RT. We compared methods A, B and C by using post-thaw motility, viability, morphology and osmotic resistance as semen quality parameters. In addition, we recorded 75-day nonreturn rates (NR75) to detect the effect of extenders on fertility. With the exception of primary defects, all laboratory parameters investigated were significantly (P < 0.05) better in methods A (TRIS-egg yolk, 4 degrees C) and B (TRIS-egg yolk, RT), compared to method C (Biociphos-Plus, RT). We recorded no significant difference between methods A and B. We could not verify the differing laboratory results by fertility data (NR75). However, when we analyzed NR75 for a single breed, significant (P < 0.05) differences existed between methods A and B compared to method C in Simmental and Holstein but not in Brown Swiss. We obtained best results in Simmental using method A (69%, n = 3384), while method C (61.4%, n = 763) was superior to methods A (57.6%, n = 698) and B (57.3%, n = 737) in Holstein. After considering various factors like preparation of extender, cost of materials and ambient working temperature, we concluded from our data that bull semen processing using TRIS-egg yolk extender and RT for packaging (method B) produced the best semen quality and field fertility.

Seasonal changes in semen quality and freezability in the Warmblood stallion

The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawed semen samples motility as well as viability (SYBR-14/PI) was tested, and the hypoosmotic swelling test (HOS) was performed. To analyze seasonal differences 4 periods of 3 months each were defined: autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1 year experiment all semen quality parameters showed a clear seasonal pattern. The volume, total sperm count and motility in fresh semen were significantly higher (P<0.05) in summer than in winter, while sperm concentration was significantly lower in summer compared to the other seasons. Regarding morphology, normal sperm was significantly lower (P<0.05) in summer than at any other time of the year and higher values (P<0.05) were found for major defects in summer than in spring and autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in autumn when compared to spring and summer. Viability was lowest in summer and differed significantly (P<0.05) from other seasons. The HOS test revealed significantly more (P<0.05) membrane damaged spermatozoa in winter than in spring, summer and autumn. Our results demonstrate that in our climatic conditions clear seasonal differences occur in semen quality of fresh and frozen-thawed semen and that cryopreservation of stallion semen should preferably be performed in autumn.

Suppression of testicular function and sexual behavior by vaccination against GnRH (Equity™) in the adult stallion

The aim of this study was to evaluate the effects of an anti-GnRH vaccine on testosterone concentration, antibody titer, scrotal width, semen quality and sexual behavior in the stallion. Adverse reactions to the vaccine were also determined. Eight clinically healthy sexually experienced stallions aged between 6 and 15 years from the National Stud in Avenches (Switzerland) were used. Five stallions were immunized 3 times at an interval of 4 and 8 weeks, respectively, with 200 microg of a GnRH-protein conjugate (Equity, CSL Limited, Australia) intramuscularly in the neck and 3 control animals received an equivalent volume of saline solution. Plasma testosterone concentrations and GnRH antibody titers as well as semen quality and libido were determined weekly during 1 year (52 weeks). In addition, scrotal width was measured in all stallions before and 4, 8 as well as 12 months after first vaccination. Our results demonstrate that in 4 stallions plasma testosterone started to decrease after the second vaccination and remained suppressed for at least 6 months whereas one stallion showed no effect. Until the end of the experiment 2 stallions reached prevaccination testosterone values. Antibody titers varied individually in all 5 stallions and reached peak concentrations within 2 weeks after the third vaccination. Scrotal width was significantly (P<0.05) lower in vaccinated than in control stallions 8 months after first vaccination. Semen quality started to decreased after the second vaccination and improved towards the end of the experiment. In 4 stallions libido was clearly reduced after the second immunization but normalized in 2 animals before the end of the study while 2 stallions continued to show poor libido. From our results we conclude that three immunizations with Equity are well tolerated in the stallion and may effectively suppress testosterone secretion and reduce semen quality as well as sexual behavior. The inhibiting activity of Equity on these parameters is individually different and may last for a minimum of 6 months.

Seasonal changes of semen quality and freezability in Franches–Montagnes stallions

The objective of this study was to investigate seasonal changes of semen quality parameters in Franches-Montagnes stallions and to compare the freezability of ejaculates collected in autumn and winter. Experiments were performed using 15 stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and evaluated every month during 1 year as well as cryopreserved in autumn and winter (September to February). In fresh semen the gel-free volume, concentration, motility and morphology (normal sperm, major defects, vacuoles and acrosome defects) were evaluated and in frozen-thawed semen the motility as well as the viability (SYBR-14/PI) were performed. To analyse seasonal differences four periods of 3 months each were defined as autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1-year experiment all fresh semen quality parameters demonstrated a clear seasonal and individual pattern. The gel-free volume was significantly (P<0.05) higher in spring and summer compared to autumn and winter while sperm concentration was significantly (P<0.05) lower in spring than at any other time of the year. Total sperm number was significantly (P<0.05) higher and sperm motility significantly (P<0.05) lower in summer than in other seasons. Regarding sperm morphology, normal sperm was significantly (P<0.05) higher in autumn than in winter and summer and major defects were lowest (P<0.05) in autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in the ejaculates collected in autumn compared to winter, while viability showed no obvious differences. Our results clearly demonstrate that individual and seasonal differences occurred in semen quality of Franches-Montagnes stallions. Ejaculates collected in autumn (September, October, November) demonstrated good quality, especially regarding sperm morphology, and were more suitable for cryopreservation because of better motility in frozen-thawed semen collected during autumn than in winter.

Analysis of the Pathogenetic Basis for Shedding and Transmission of Ovine Gamma Herpesvirus 2

ABSTRACT Ovine herpesvirus 2 (OvHV-2), a member of the viral subfamily Gammaherpesvirinae, shares numerous similarities with human herpesvirus 8 (HHV-8). Both viruses are apathogenic in their healthy original host, may cause lymphoprolipherative diseases, cannot routinely be propagated in cell culture, and may be sexually transmitted. However, the pathways of sexual transmission of these viruses, as well as the underlying pathogenetic dynamics, are not well understood. Organs from naturally OvHV-2-infected, as well as OvHV-2-free, sheep were quantitatively analyzed for OvHV-2 by the DNA amplification techniques. The dynamics of OvHV-2 multiplication and excretion were monitored after experimental infections and, most importantly, subsequent to vasectomy. The OvHV-2 DNA load in various tissues and internal organs was not merely reflecting the viral DNA load in the bloodstream, which suggested compartmentalization of OvHV-2. Moreover, OvHV-2 DNA was detected at several portals for virus shedding, i.e., the respiratory, alimentary, and urogenital tracts. Transient OvHV-2 excretion was detected in ejaculates of experimentally infected rams. Upon vasectomy, OvHV-2 DNA reappeared in the ejaculatory plasma, but the titers did not decline after reaching a peak. Spiking and fractionation experiments revealed an inhibitory activity, associated with the spermatozoa, which was able to suppress detection of viral DNA but which was no longer present in samples from vasectomized animals. Therefore, epidemiological studies on viruses that may be transmitted by the ejaculatory pathway and for whose tracing nucleic acid amplification methods are used, i.e., OvHV-2, HHV-8, and the human immunodeficiency virus, should include vasectomized males.

Histomorphological and immunohistochemical findings in testes, bulbourethral glands and brain of immunologically castrated male piglets.

The aim of this study was the histological and immunohistochemical evaluation and comparison of testicular, bulbourethral and brain tissue in immunized and intact control boars. Fourteen male piglets, aged between 10 and 16 weeks, were vaccinated twice subcutaneously 4 to 5 weeks apart with Improvac, an anti-GnRH vaccine. The pigs were sacrificed 1 to 16 weeks following the second injection. Testicular weight was recorded and various tissue samples were collected and fixed in formalin and Bouins fixative for histological examination. In addition, 2 boars were immunized five times and slaughtered 60 weeks after the last injection. Histological and immunohistological studies performed on testes and epididymes showed clear signs of atrophy in the immunized animals and a significant reduction in paired testes weight was seen in treated boars. Microscopically, the mean diameter of the seminiferous tubules was markedly reduced. Spermatogonia as well as few spermatocytes were visible between the Sertoli cells and Leydig cells were atrophic. None or only few spermatozoa were detected in the epididymis. The bulbourethral glands of immunocastrated pigs were smaller than in control pigs and showed histological evidence of atrophy. Immunohistological detection of LH and FSH in the pituitary gland of treated and control boars showed no quantifiable difference in the amount of these two gonadotropins and no lesions were visible in the hypothalamus and the pituitary gland. From our findings it can be concluded that the anti-GnRH vaccine Improvac induces severe atrophy of testes and bulbourethral glands in immunized pigs. This effect appears to be reversible, depending on the immune response of each animal and the time elapsed after the last booster injection.

Vaccination against gonadotropin-releasing factor (GnRF) with Bopriva significantly decreases testicular development, serum testosterone levels and physical activity in pubertal bulls.

The aim of this study was to evaluate the effects of vaccination against gonadotropin-releasing factor (GnRF) on testicular development, testosterone secretion, and physical activity in pubertal bulls. The experiment was performed using 44 bulls aged between 6 and 7 mo. Twenty-three animals were vaccinated twice 4 wk apart with 1 mL of Bopriva (Pfizer, Animal Health, Parkville, Australia) and 21 bulls served as matched controls. Serum GnRF antibody titer and testosterone concentration as well as body weight and scrotal circumference were determined in all bulls for 24 wk from the first vaccination. In addition, physical activity was analyzed in 11 vaccinated and in 10 control animals using the ALPRO DeLaval activity meter system (DeLaval AG, Sursee, Switzerland). The results show that vaccination significantly (P < 0.05) influenced all parameters evaluated except body weight. Antibody titers to GnRF began to rise 2 wk after the first vaccination and reached peak values 2 wk after the second injection. Significant group differences in anti-GnRF titer were present for 22 wk following the first vaccination. Testosterone concentrations were significantly lower between weeks 6 to 24 after first vaccination in bulls with Bopriva compared with control animals. In vaccinated bulls testicular development was impaired after the second injection and scrotal circumference was significantly smaller between weeks 8 to 24 after first vaccination. Physical activity of vaccinated bulls was reduced after the booster injection with significant group differences for a continuous period of 106 days. In conclusion, vaccination against GnRF with Bopriva in pubertal bulls decreased testosterone levels in peripheral blood, testicular development, and physical activity but did not affect weight gain.

Die Kastration männlicher Lämmer mittels Immunisierung gegen GnRH

Das Ziel dieser Arbeit war es, die Wirksamkeit einer GnRH-Vakzine am mannlichen Lamm abzuklaren. Fur die Untersuchungen standen 20 Widderlammer zur Verfugung, die zufallig einer Versuchs- (GnRH-Impfung) und Kontrollgruppe (physiologische NaCl-Losung) zugeordnet wurden. Beim Erreichen eines Korpergewichtes von 20 Kg (Alter 2-3 Monate) erfolgte bei Tieren der Versuchsgruppe eine zweimalige Impfung mit 2 ml Improvac ® (CSL Limited, Parkville, Victoria, Australien) im Abstand von 3 Wochen. Das Korpergewicht und der Testosterongehalt im Blut wurden bei allen Lammern wochentlich wahrend 16 Wochen bis zum Erreichen des Schlachtgewichtes bestimmt. Anschliessend wurde die Testosteronkonzentration im Blut bei den geimpften Lammern in monatlichen Abstanden gemessen. Nach der 2. Impfung wurde die Hodenentwicklung gehemmt und der Testosterongehalt im Blut blieb wahrend mindestens 12 Wochen unter 0.1 ng/ml. Bei Kontrolltieren schwankten die durchschnittlichen Testosteronkonzentrationen zwischen 0.1 und 0.9 ng/ml Plasma...

Effect of vaccination against gonadotropin-releasing factor (GnRF) with Bopriva® in the prepubertal bull calf.

The aim of this study was to evaluate the effect of immunization against gonadotropin-releasing factor (GnRF) with Bopriva(®) (Pfizer Animal Health, Parkville, Australia) in prepubertal bull calves. For the study, 6 calves were vaccinated at the age of 3 and 6 weeks with 1 mL Bopriva(®), and 6 animals served as matched controls. Concentrations of GnRF antibodies, testosterone and LH were determined in serum samples out to 30 weeks after the first immunization. Body weight and scrotal circumference were measured for 59 weeks. At slaughter, 65 weeks after the first immunization, the quality of epididymal sperm was evaluated. The results showed that vaccination against GnRF influenced (P<0.05) anti-GnRF titer, LH and testosterone concentrations as well as scrotal circumference. Antibody titers significantly (P<0.05) increased after the booster vaccination and reached peak values 2 weeks later. Compared to control animals, inhibition (P<0.05) of the prepubertal LH secretion was observed in vaccinated calves at weeks 10 and 12-14 after the first vaccination. In vaccinated calves testosterone concentrations decreased after the booster injection to values below 0.5 ng/mL serum and remained for at least 22 weeks at this low level. Animals vaccinated with Bopriva(®) showed a delay in testes growth and smaller scrotal circumference. Puberty occurred at the age between 46 and 55 weeks in vaccinated and between 38 and 52 weeks in control animals and body weight gain was similar in both groups. All vaccinated bulls attained spermatogenic capacity at slaughter when they were 68 weeks old.

Effect of a gonadotropin-releasing factor vaccine on follicle-stimulating hormone and luteinizing hormone concentrations and on the development of testicles and the expression of boar taint in male pigs.

The objective of this study was to determine the effect of using a gonadotropin-releasing factor (GnRF) vaccine on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in plasma, the size of testicles, and the expression of boar taint in male pigs. Vaccinated pigs were compared with surgically castrated pigs and entire males. Pigs were randomly assigned to three treatment groups: surgically castrated during the first week of life (T01, n=274), immunized twice during the fattening period with a GnRF vaccine, the first when 13 to 14 wk of age and the second when 20 to 21 wk of age (T02, n=280), and entire males (T03, n=56). From a subgroup of both T01 and T02 and from all pigs of group T03, blood samples were collected immediately before second vaccination (T02) and again before slaughter at either 24 to 25 or 26 to 27 wk of life to determine the plasma concentrations of LH and FSH. Testicles were removed after slaughter and their size was determined. Meat and fat samples from all pigs of T02 and T03 as well as 25% of the pigs of T01 were examined with the cold cooking and fat melting test. Immediately before the second vaccination (T02 only), LH and FSH concentrations were not significantly different between T02 and T03. However, LH and FSH concentrations were significantly higher in T01 compared with T02 and T03. Before the first slaughter date, LH and FSH concentrations were significantly lower in T02 than in T03. Testicle size was significantly lower in T02 compared with that in T03. In T02, 98% (235 of 239) of the samples were rated negative for boar taint by the cooking test, whereas in T03, 94% (48 of 51) were rated positive. In the fat melting test, 97% of T02 were rated negative and 3% (7 pigs) were rated positive, including the pigs tested positive in the cold cooking test. In T03, 94% were rated positive. All pigs (7 of 239) in T02 that were positive for boar taint in the cooking or melting test and that were tested had androstenone and skatole concentrations in backfat below threshold levels of 1 microg/g and 0.2 microg/g, respectively.