Intrauterine infusion of killed semen adversely affects uterine blood flow and endometrial gene expression of inflammatory cytokines in mares susceptible to persistent breeding-induced endometritis.

Persistent breeding-induced endometritis (PBIE) is a leading cause of infertility in mares. The objective of the study was to assess genital perfusion and endometrial gene expression of inflammatory cytokines in mares classified as susceptible (n = 5) or resistant (n = 5) to PBIE. Ten mares were examined daily during estrus until 6 d after hCG-induced ovulation for two estrous cycles. Twenty-four hours after application of 1500 IU hCG, 4 mL of killed (by repeated freezing in liquid nitrogen and thawing at 50 °C) deep-frozen semen or sterile saline was instilled into the uterine body and examinations were carried out immediately before and 3, 6, and 12 h after intrauterine infusion. Examinations included blood sampling to determine plasma progesterone (P4) concentrations, and transrectal ultrasonography in B- and color Doppler mode to determine follicular and luteal size and blood flow, the extent of intrauterine fluid, as well as time-averaged maximum velocity (TAMV), blood flow volume (BFV), and blood flow resistance (expressed as pulsatility index, PI) of the uterine arteries. Additionally, endometrial biopsies were obtained at 24 h before, and 2 and 7 d after infusion, and mRNA expressions of IL1B, IL6, IL8, IL10, TNF, CASP3, and COX2 were determined by qRT-PCR. Statistical analyses were performed with mixed models. Intrauterine fluid retention (diameter >20 mm for at least 3 d) was found after infusion of killed semen in five susceptible mares. There was no treatment effect (semen vs saline; P > 0.05) on genital blood flow, plasma P4 concentration, and endometrial gene expression. In comparison to resistant mares, susceptible mares had an increased (P = 0.04) BFV of the uterine arteries at 24 h before intrauterine infusion of killed semen, and an increased (P = 0.03) PI at 2 d after infusion. The TAMV, plasma P4 concentrations, and follicular and luteal size and blood flow did not differ (P > 0.05) between resistant and susceptible mares. Endometrial mRNA expression of IL1B increased (P = 0.05) at 2 d after the infusion of killed semen in the susceptible mares, and the expression of IL10 increased (P = 0.003) at 7 d after the infusion within the resistant mares. Interleukin 6 mRNA was increased (P = 0.05) in susceptible compared to resistant mares at 2 d after infusion. In summary, an intrauterine infusion of killed semen increases uterine blood flow resistance and alters endometrial gene expression of inflammatory cytokines for at least 7 d but does not affect ovarian blood supply and luteal function in mares susceptible to PBIE.

Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility.

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; nHF = 10 bulls) or low fertility bulls (LF; nLF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC1(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (Cpos), intact plasma membrane (PIneg), unstained acrosome (PNAneg), low intracellular Ca2+ levels (Fneg), and high mitochondrial membrane potential (Mpos). In both fertility groups, Mpos cells comprised more than 90 and 84% of PInegPNAneg sperm at 0 and 3 h, respectively. The percentage of CposPInegPNAnegFnegMpos sperm did not differ between HF and LF ejaculates; however, the percentage of Fneg cells within the PInegPNAneg and PInegMpos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of Fneg sperm within the PInegMpos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI.

[Evaluation of three-dimensional sonography for antral follicle count in cycling dairy cows]. / Beurteilung der Follikelzahl mittels transrektaler dreidimensionaler Sonographie bei zyklischen Milchkühen.

INTRODUCTION: The objective of the present study was to investigate reliability of transrectal three-dimensional ultrasound (3D-S) for antral follicle count (AFC) in dairy cows. Furthermore individual differences of AFC between cows and the fluctuation of AFC within and between different cycles were evaluated. To test the reliability of 3D-S, AFC was determined on the ovaries of 10 cows in vivo and compared with counts obtained after slaughter using computer tomography. To evaluate cyclic follicle dynamics, six cows were repeatedly examined with 3D-S over a period of two cycles. Using 3D-S, follicles with a mean diameter greater than 2 mm could be recorded. AFC determined with 3D-S and computer tomography showed a significant correlation (r ≥ 0.86, p < 0.05) and values were similar (p ≥ 0.05). AFC differed between individuals (p < 0.0001) and a moderate fluctuation within and between two cycles within the same cow was apparent (p < 0.05). In conclusion, 3D-S is a suitable method for determination of AFC in cattle. AFC differs between cows and cyclic fluctuations are apparent in a lesser extent.

INTRODUCTION: L'objectif de la présente étude était d'étudier la fiabilité de l'échographie tridimensionnelle transrectale (3D-S) pour le comptage des follicules (FC) chez les vaches laitières. Sur la base du nombre de follicules, on a contrôlé s'il y avait des variations individuelles au sein et entre les différents cycles. Pour tester la fiabilité de la 3D-S, le FC a été déterminée in vivo sur les ovaires de 10 vaches et comparé aux résultats obtenus après l'abattage par tomodensitométrie. Pour évaluer les variations cycliques, six vaches ont été examinées à plusieurs reprises par 3D-S sur une période de deux cycles. En utilisant 3D-S, les follicules avec un diamètre moyen supérieur à 2 mm peuvent être visualisés. Le FC déterminé avec 3D-S et la tomodensitométrie ont montré une significatif corrélation (r ≥ 0,86, p.

Stallion semen quality depends on major histocompatibility complex matching to teaser mare.

The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm numbers as compared to exposure to an MHC-similar mare. Sperm velocity seemed not affected by the treatment. Overall genetic similarity between stallions and mares (determined from polymorphic microsatellites on 20 different chromosomes) played no significant role here. The MHC type of the teaser mare also affected characteristics of cold-stored sperm after 24 and 48 hr. As expected from ejaculate economics, sperm viability was elevated after exposure to an MHC-dissimilar mare. However, oxidative stress and the percentage of sperm with a high DNA fragmentation were mostly increased after exposure to an MHC-dissimilar mare, depending also on whether the teaser mare was in oestrous or not. We conclude that males can quickly adjust ejaculate quality relative to a female's MHC, and that this male reaction to the social environment can also affect important characteristics of cold-stored semen.

Evaluation of different dose rate combinations of ketamine, romifidine and azaperone for castration of 3-4 and 5-6 weeks old piglets.

INTRODUCTION: In this study dose rate combinations of ketamine, azaperone and romifidine were tested in 3-4 (G3-4) and 5-6 weeks (G5-6) old piglets according to a dose rate decision tree (DRT) to provide calm anaesthesia induction, reaction-free anaesthesia for castration and smooth recovery within 2 hours. When a combination failed to meet those criteria in 2 piglets, the next dosage of the DRT was tested. In G3-4 four combinations were tested in 14 piglets with dose rates of 10-20 mg/kg ketamine, 3 mg/kg azaperone and 0.15-0.20 mg/kg romifidine IM. Induction was smooth in all piglets, but depth of anaesthesia or recovery (11 and 6 piglets) were insufficient. In G5-6 five combinations were tested in 37 piglets with dose rates of 10-20 mg/kg ketamine, 3-4 mg/kg azaperone and 0.15-0.20 mg/kg romifidine IM. Induction was smooth in all piglets but 2. Depth of anaesthesia or recovery (17 and 7 piglets, respectively) were insufficient. In the present study with 3-4 and 5-6 weeks old piglets, acceptable quality of anaesthesia could not be achieved despite using high drug dose rates.

INTRODUCTION: Des combinaisons de doses de kétamine, azapérone et romifidine ont été testées sur la base d'un arbre de décision de dose afin de définir la dose qui assurait, chez des porcelets de 3-4 (G3-4) et de 5-6 (G5-6) semaines une induction tranquille, une anesthésie sans réaction lors de la castration et une phase de réveil calme, terminée en deux heures. Lorsqu'une combinaison de ces critères n'était pas obtenue chez 2 porcelets, on passait à la dose suivante proposée par l'arbre de décision. Dans le G3-4, 4 combinaisons avec des doses de 10-20 mg/kg de kétamine, 3 mg/kg d'azapérone et 0.15-0.20 mg/kg de romifidine IM ont été testées chez 14 porcelets. L'induction était calme chez tous les porcelets mais la profondeur de l'anesthésie ou la phase de réveil étaient insuffisantes chez 11 respectivement 6 porcelets. Dans le G5-6, 5 combinaisons avec des doses de 10-20 mg/kg de kétamine, 3-4 mg/kg d'azapérone et 0.15-0.20 mg/kg de romifidine IM ont été testées chez 37 porcelets. L'induction était calme à deux exceptions près mais la profondeur de l'anesthésie ou la phase de réveil étaient insuffisantes chez 17 respectivement 7 porcelets. Lors de la présente étude, il n'a pas été possible, malgré l'utilisation de doses élevées, d'obtenir chez des porcelets de 3-4 respectivement de 5-6 semaines une qualité d'anesthésie acceptable.

Seasonal changes of DNA fragmentation and quality of raw and cold-stored stallion spermatozoa.

In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca+2 level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca2+ level of sperm.

Quality of seminal fluids varies with type of stimulus at ejaculation.

The theory of ejaculate economics was mainly built around different sperm competition scenarios but also predicts that investments into ejaculates depend on female fecundity. Previous tests of this prediction focused on invertebrates and lower vertebrate, and on species with high female reproductive potential. It remains unclear whether the prediction also holds for polygynous mammals with low female reproductive potential (due to low litter size and long inter-birth intervals). We used horses (Equus caballus) to experimentally test whether semen characteristics are adjusted to the oestrous cycle of the mare a stallion is exposed to during few moments before ejaculation. We analysed 122 weekly semen samples collected from 16 stallions during exposure to either an oestrous or a dioestrous mare. Semen volume and the rate of motile sperm were higher when stallions were exposed to an oestrous than to a diestrous mare, while total sperm counts and sperm velocity remained unchanged. Sperm collected after exposure to an oestrous mare also showed reduced oxidative degeneration of cell membranes over a period of 48 hours. We conclude that stallions invest more into their seminal fluids when the chance of fertilization is elevated, and that this adjustment of ejaculate quality can happen very quickly.

Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen.

This study was designed to investigate the effects of an equilibration period up to 96 hours and three extenders (AndroMed, OPTIXcell, and Triladyl) on the quality of cryopreserved bull semen and to evaluate, whether an extension of the equilibration time to 72 hours does affect fertility in the field. One ejaculate of 17 bulls was collected and divided into three equal aliquots and diluted, respectively, with the three extenders. Each aliquot was again divided into five parts and equilibrated for 4, 24, 48, 72, and 96 hours before freezing in an automatic freezer. Sperm motility, plasma membrane and acrosome integrity (PMAI), and DNA fragmentation index (% DFI) were measured during equilibration. In addition to the parameters measured during equilibration, the percentage of viable sperm cells with high mitochondrial membrane potential (HMMP) was measured immediately after thawing, and after 3 hours of incubation at 37 °C. Sperm motility was assessed using CASA, and PMAI, HMMP, and % DFI were measured using flow cytometry. Equilibration time did affect all parameters before freezing (P < 0.01), and also the extender affected all parameters except HMMP (P < 0.05). After thawing, all parameters except HMMP immediately after thawing were influenced by the equilibration period (P < 0.001), whereas all parameters except % DFI immediately after thawing were influenced by the extender (P < 0.001). The changes of semen characteristics during 3 hours of incubation were also dependent on the equilibration time and the extender used in all parameters (P < 0.01). In the field study, semen of nine bulls was collected thrice weekly, processed using Triladyl egg yolk extender, and frozen in 0.25 mL straws with 15 × 106 spermatozoa per straw. In total, the nonreturn rates on Day 90 after insemination (NRR90) of 263,816 inseminations in two periods were evaluated. Whereas semen collected on Mondays and Wednesdays was equilibrated for 24 hours in both periods, semen collected on Fridays was equilibrated for 4 hours in period one and equilibrated for 72 hours in period 2. No differences in NRR90 could be found (P > 0.05). In conclusion, extension of the equilibration time from 4 hours to 24-72 hours can improve motility and viability of cryopreserved semen after thawing. The extent of improvement in semen quality is dependent on the extender used. Prolongation of the equilibration period from 4 hours to 72 hours had no effect on fertility in the field.

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality.

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at -196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing-thawing.

Impact of nutritional programming on the growth, health, and sexual development of bull calves.

The growth, health, and reproductive performance of bull calves are important prerequisites for a successful cattle breeding program. Therefore, several attempts have been made to improve these parameters via nutritional programming. Although an increase in energy uptake during the postweaning period (7-8 mo of age) of the calves leads to a faster growing rate, it has no positive effects on sexual development. In contrast, a high-nutrition diet during the prepubertal period (8-20 wk of age) reduced the age at puberty of the bulls and increased the size and/or weight of the testis and the epididymal sperm reserves. This faster sexual development is associated with an increased transient LH peak, which seems to be mediated by an increase in serum IGF-I concentrations. However, the exact mechanisms responsible for the interaction between nutrition and the subsequent development of the calves are not clear. The sexual development of bull calves depends not only on the nutrition of the calves after birth but also on the feed intake of their mothers during pregnancy. In contrast to the effects of the feed intake of the bull calves, a high-nutrition diet fed to the mother during the first trimester has negative effects on the reproductive performance of their offspring. In conclusion, it has been clearly demonstrated that growth, health, and reproductive performance can be improved by nutritional programming, but further studies are necessary to obtain a better understanding about the mechanisms responsible for this phenomenon.

[Sonographic diagnosis of pregnancy and determination of fetal number and gestational age in ewes]. / Sonographische Trächtigkeitsunter- suchung und Bestimmung von Anzahl und Alter der Feten beim Schaf.

The aim of the present study was to evaluate accuracy of transabdominal ultrasonography for pregnancy diagnosis, determination of fetal number and estimation of gestational age in ewes. A total of 1068 ewes from 8 different flocks (Swiss White Alpine n = 379, Swiss Black-Brown Mountain n = 189, Oxford-type n = 164, Ostfriesian n = 154, Texel n = 104, Lacaune x Osfriesian crossbred n = 78) was investigated using an Ovi-Scan™ ultrasound scanner with a 3.5 MHz 170° mechanical axial probe (BCF Technology Ltd., BelIshill, Great Britain). Scanning was performed transabdominally at the right inguinal region in ewes restrained in standing position. Sonographic findings were compared with lambing data recorded by the farmers. Included in the analysis were all ewes that, at the time of examination, were not pregnant and those that were pregnant between 26 and 110 days with a known lambing date and number of lambs born (n = 882). The sensitivity of pregnancy diagnosis was 97.8% and the specificity 97.6% (overall accuracy 97.8%, n = 853). Sensitivity and specificity for discrimination between single and multiple pregnancies was 95.8% and 90.5% (overall accuracy 93.9%, n = 752), respectively, when multiples were defined as positive. Discrimination between twins and triplets reached a sensitivity of 86.0% and a specificity of 99.5% (overall accuracy 98.0%, n = 458) when triplets were defined as positive. Considering a gestation period of 150 days, the mean deviation of the estimated to the effective day of gestation at examination was 5.6 ± 5.0 days (n = 781). The correlation between estimated and true gestational age was very high (r=0.936, P<0.0001). In conclusion, a highly accurate and efficient diagnosis of pregnancy with reliable estimation of fetal number and age is possible using an Ovi-Scan™ ultrasound scanner by an experienced examiner.

A new laboratory model using bull and boar spermatozoa and fluorescent beads to assess a membrane's occlusive potential.

OBJECTIVES: The objective of the present study is to assess the potential of bull and boar spermatozoa and fluorescent beads to be used as a surrogate cell model to determine the cell occlusive potential in vitro using membranes of standardized porosities. MATERIALS AND METHODS: A two-chamber model system consisting of upper and lower chambers, which could be separated by membranes, was constructed. Isopore polycarbonate membranes with different standardized pore diameters were used to assess the mobile cellular penetration behavior of spermatozoa or the more passive non-cellular permeability of fluorescent particles (beads) of different diameter and color. In a first experiment, spermatozoa were placed in the lower chamber, whereas semen extender only was placed in the upper chamber. After 10 min of incubation at 37 °C, the sperm number was assessed in the latter. In a second experiment, a bead solution was drawn through resorbable collagen membranes from the upper into the lower chamber by vacuum using a syringe and bead number and size was analyzed by flow cytometry. All experiments were carried out in triplicates. A non-porous polyester membrane was used as negative control to assess the overall tightness of the setup. RESULTS: Boar and bull spermatozoa had average cell body lengths and widths of 9 × 5 µm and were unable to pass through pores ≤2 µm, whereas they were detectable at pore sizes ≥3 µm. Their number increased with increasing pore diameters, i.e., from minimal concentrations of 0.1 × 106/ml for boar and 0.5 × 106/ml for bull spermatozoa at 3 µm to maximal concentrations of 2.1 × 106/ml for boar and 13.1 × 106/ml for bull spermatozoa at 8 µm. The fluorescent beads followed the expected pattern of permeability reliably correlating bead and pore diameter. CONCLUSIONS: Within the limitations of this laboratory study and the xenogeneic cell surrogate material, the model allows to easily assess cell and particle penetration through porous structures like membranes. We hope to further assess, improve, and validate this model, which we aim to use for the screening of dental membranes after being exposed to different degradation methods. CLINICAL RELEVANCE: Convenient and rapid test procedures to evaluate membranes for regenerative procedures are still warranted.

Short communication: Transmission of border disease virus to seronegative cows inseminated with infected semen.

The goal of this study was to investigate the transmissibility of border disease (BD) virus to seronegative cows via artificial insemination with cryopreserved semen from a bull persistently infected with BD virus. Five pestivirus naive cows were inseminated with BD virus-infected semen. Blood was collected for detection of pestivirus antibody by means of an ELISA on day 0 (day of insemination) and then every 7 days until day 56, at which time a serum neutralisation test (SNT) for differentiation of BD and BVD virus was carried out. Seroconversion was first noticed in two cows on day 14, in two cows on day 21 and in one cow on day 28. In the SNT, all cows had distinctly positive titres against BD virus. Therefore, BD virus is readily transmitted by infected semen, but none of the cows conceived, most likely because of poor semen quality.

Reproductive performance of Lacaune dairy sheep exposed to artificial long days followed by natural photoperiod without and with additional progestagen treatment during the nonbreeding season.

This study compared the reproductive performance of Lacaune dairy ewes exposed to a light program and subsequent male introduction without (n = 36) or with (n = 36) an additional 6-day progestagen treatment during the nonbreeding season. All ewes were exposed to extended day length (16 hours light and 8 hours darkness) for 77 days during winter (December 15 until March 2) followed by increasing natural photoperiod. At the end of the photoperiodic treatment, three blood samples were collected 6 days apart for progesterone (P4) analysis to determine cyclic activity. One half of the ewes were additionally subjected to a 6-day progestagen treatment in combination with PGF2α and eCG at insert withdrawal. Rams fitted with marking harnesses were introduced to females for 45 days and marked ewes recorded. Ewes exposed to the light program only were joined 40 days after the end of photoperiodic treatment, and ewes with additional progestagen treatment were joined 1 day after insert removal (40-44 days after the end of photostimulation). Lambing data were recorded and fertility (percentage of ewes lambing, lambing rate, and litter size) assessed to the first service period and overall. Mean serum P4 concentrations were similarly (P > 0.05) low in both groups (0.4-0.7 ng/mL vs. 0.4-0.6 ng/mL). On the basis of elevated P4 levels (>1 ng/mL), evidence of luteal activity was found in 27.8% of the ewes at the end of the light program. Estrus response was equally high (97.2%) and estrus distribution highly synchronized in progestagen-treated ewes (91.7% within 4 days). In ewes exposed to the light program only, estrous activity was recorded within 4 days (six ewes), from Day 8 to Day 17 (17 ewes) and from Day 19 to Day 25 (12 ewes) after joining. The percentage of ewes that lambed to the first service period was higher (P < 0.05) in ewes exposed to the light program only than that in the group additionally treated with progestagen/PGF2α/eCG (94.4% vs. 69.4%). Overall, the percentage of lambing ewes was similar in both groups (97.2% and 94.4%), and lambing rates (1.4-1.9) and litter sizes (1.9-2.1) were high and not influenced (P > 0.05) by the treatment. In conclusion, this study reports that exposition of Lacaune ewes to artificial long days followed by natural day length and male introduction is highly effective to induce fertile estrous activity during the nonbreeding season and offers a reliable and practical alternative to hormonal manipulation for out-of-season breeding in sheep.

Immunization against gonadotropin-releasing hormone in dairy cattle: antibody titers, ovarian function, hormonal levels, and reversibility.

Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle not intended for breeding. A cattle-specific anti-GnRH vaccination (Bopriva, Zoetis Australia Ltd., West Ryde, Australia) is approved for use in heifers and bulls in New Zealand, Australia, Mexico, Brazil, Argentina, Turkey, and Peru. Eleven healthy, cyclic Swiss Fleckvieh cows were included in the study and vaccinated twice with Bopriva 4wk apart. Injection site, rectal body temperature, and heart and respiratory rates were recorded before and 3d following each vaccination. Blood samples were taken weekly for progesterone and estrogen analysis and to determine GnRH antibody titer. Ovaries were examined weekly, using ultrasound to count the number of follicles and identify the presence of a corpus luteum. Thirty weeks after the first vaccination, the cows were subjected to a controlled internal drug-releasing device-based Select-Synch treatment. The GnRH antibody titers increased after the second vaccination and peaked 2wk later. Estrogen levels were not influenced by vaccination, and progesterone level decreased in 7 of 11 cows up to 3wk after the second vaccination and remained low for 10 to 15wk following the second vaccination. The number of class I follicles (diameter ≤5mm) was not influenced by vaccination, whereas the number of class II follicles (diameter 6-9mm) decreased between 7 and 16wk after the first vaccination. Class III follicles (diameter >9mm) were totally absent during this period in most cows. The median period until recurrence of class III follicles was 78d from the day of the second vaccination (95% confidence interval: 60-92d). After vaccination, all cows showed swelling and pain at the injection site, and these reactions subsided within 2wk. Body temperature and heart and respiratory rates increased after the first and second vaccinations and returned to normal values within 2d of each vaccination. The cows in our study were not observed to display estrus behavior until 30wk after the first vaccination. Therefore, a Select-Synch protocol was initiated at that time. Ten cows became pregnant after the first insemination (the remaining cow was reinseminated once until confirmed pregnancy). Bopriva induced a reliable and reversible suppression of reproductive cyclicity for more than 2mo. The best practical predictor for the length of the anestrus period was the absence of class III follicles.

Tritrichomonas foetus: prevalence study in naturally mating bulls in Switzerland.

Switzerland is officially free from bovine Tritrichomonas foetus. While bulls used for artificial insemination (AI) are routinely examined for this pathogen, bulls engaged in natural mating, as well as aborted fetuses, are only very sporadically investigated, indicating that the disease awareness for bovine tritrichomoniasis is low. Natural mating in cattle is becoming increasingly popular in Switzerland. Accordingly, a re-introduction/re-occurrence of T. foetus in cattle seems possible either via resurgence from a yet unknown bovine reservoir, or via importation of infected cattle. The low disease awareness for bovine tritrichomoniasis might favor an unnoticed re-establishment of T. foetus in the Swiss cattle population. The aim of our study was thus to search for the parasite, and if found, to assess the prevalence of bovine T. foetus in Switzerland. We included (1) bulls over two years of age used in natural mating and sent to slaughter, (2) bulls used for natural service in herds with or without fertility problems and (3) aborted fetuses. Furthermore, the routinely examined bulls used for AI (4) were included in this study. In total, 1362 preputial samples from bulls and 60 abomasal fluid samples of aborted fetuses were analyzed for the presence of T. foetus by both in vitro cultivation and molecular analyses. The parasite could not be detected in any of the samples, indicating that the maximal prevalence possibly missed was about 0.3% (95% confidence). Interestingly, in preputial samples of three bulls of category 1, apathogenic Tetratrichomonas sp. was identified, documenting a proof-of-principle for the methodology used in this study.

[Castration in the bull calf and ram lamb]. / Die Kastration beim männlichen Kalb und Lamm.

This paper aims to provide an overview of the accepted techniques of pain relief and castration and guidelines of how to best perform these painful interventions in an animal-friendly way under Swiss conditions. Calves should be castrated at the age of 14 days or less, at least 10 minutes after local anesthesia with lidocaine, applying a single rubber ring. Concurrently, a NSAID should be administered intravenously (ketoprofen, 3 mg/kg of bodyweight) and Tetanus-serum subcutaneously (off label use). If possible, ketoprofen(4.5 mg/kg BW) should be orally administered for 3 - 5 days postoperatively. At 10 days after applying the rubber ring, the dried-off scrotum including the rubber ring should be removed with a clean knife or a scalpel. Local anesthesia is not necessary for this procedure. Ram lambs should be castrated at the age of 14 days or less, at least 10 minutes after local anesthesia with lidocaine, applying a rubber ring. The toxic dose of 4 mg lidocaine/kg BW (corresponds to 1 ml lidocaine 2 % per lamb of 5 kg BW) should not be exceeded. Concurrently, a NSAID (off label use) and Tetanus-serum should be administered systemically. Immunization against GnRH represents an animal-friendly and economically feasible alternative to rubber ring castration. With two immunizations at an interval of 3 - 4 weeks testicular development can be inhibited for at least 3 months and the onset of puberty clearly delayed. However, a specific vaccine for use in ruminants is currently registered neither in Switzerland nor in Europe.

Le présent travail donne un aperçu des méthodes praticables pour l'anesthésie et la castration ainsi que des recommandations pour une réalisation de cette intervention dans le respect du bien-être animal dans les conditions suisses. Les veaux devraient être castrés dans les 14 premiers jours de vie au moyen d'un élastique posé au plus tôt 10 minutes après la réalisation de l'anesthésie locale avec de la lidocaïne. Il convient d'administrer simultanément un AINS (kétoprofène, 3 mg/kg PC) en i/v lente ainsi qu'un sérum antitétanique (reconversion). Dans la mesure du possible, il convient d'administrer oralement du kétoprofène (4.5 mg/kg PC) par voie orale pendant les 3 à 5 jours suivants. Le scrotum desséché y compris l'élastique doit être supprimé sans anesthésie au moyen d'un couteau propre ou d'un scalpel stérile 10 jours après la pose de l'élastique. Les agneaux doivent être castrés dans leurs 14 premiers jours de vie, comme les veaux avec un élastique posé au minimum 10 minutes après la réalisation d'une anesthésie à la lidocaïne. On prendra garde à ne pas dépasser la dose maximale de 4 mg/kg de lidocaïne (ce qui représente 1 ml de lidocaïne 2 % pour un agneau de 5 kg). Simultanément à l'anesthésie, on appliquera un AINS (reconversion) et un sérum antitétanique. La vaccination anti-GnRH représente une alternative pratique et économique ménageant les animaux pour la castration des veaux et des agneaux mâles. Une double vaccination à 3 à 4 semaines d'intervalle permet de bloquer le développement testiculaire pendant au moins 3 mois et de retarder ainsi la maturité sexuelle. Toutefois il n'existe actuellement pas en Suisse ni en Europe de vaccin spécifique enregistré pour l'application chez les ruminants.

[Fertility after treatment with Eazi-breedTM CIDR G for 6 or 12 days outside the breeding season in Lacaune dairy sheep]. / Fruchtbarkeit nach 6- und 12-tägiger Behandlung mit Eazi-breed™ CIDR® G ausserhalb der Zuchtsaison beim Lacaune Milchschaf.

This study compares the fertility after short- and long-term synchronization using a progesterone intravaginal device in Lacaune dairy sheep outside the breeding season. For the experiment 108 Lacaune sheep were treated with Eazi-breed™ CIDR® G intravaginal devices (Pfizer Animal Health, Zürich) for 12 days (Group L, n = 60) or 6 days (Group K, n = 48) in combination with eCG (Group L) or with eCG and 125 µg Cloprostenol (Group K) at device removal. Thereafter the ewes were kept together with rams for 60 days, ewes in estrus were recorded and the fertility was assessed after lambing. Blood progesterone concentration was measured at device application, withdrawal and 14 days later. Results show that neither treatment nor parity had an influence on estrus rate (Group L 91.7 %, Group K 93.8 %, nulli- and pluriparous animals 96.9 % and 90.8 %, respectively). Group L showed a tendency towards a better first cycle lambing rate and a significantly (P < 0.05) higher overall lambing rate compared to sheep of Group K (71.7 % vs. 60.4 % and 83.3 % vs. 72.9 %). Pluriparous ewes had higher (P < 0.05) lambing rates and greater (P < 0.05) numbers of lambs born per synchronized ewe than nulliparous sheep for the first cycle (75.0 % vs. 46.9 % and 1.4 ± 1.0 vs. 0.9 ± 1.1) as well as for the overall service period (92.1 % vs. 46.9 % and 1.7 ± 0.8 vs. 0.9 ± 1.1). Fourteen days after insert withdrawal progesterone concentrations were higher (P < 0.05) in Group L than in Group K (7.7 ± 4.3 vs. 5.6 ± 2.7 ng/mL) and in nulli- compared to pluriparous (9.1 ± 5.6 vs. 5.7 ± 2.1 ng/mL) ewes. In conclusion, the overall lambing rate was higher after long-term compared to short progesterone treatment and nulliparous ewes were less suitable for estrus induction outside the breeding season.

Dans ce travail, on étudie la fertilité chez de brebis de race Lacaune lait après une synchronisation des chaleurs de courte et de longue durée au moyen d'un pessaire intra vaginal à la progestérone. Pour cela, 108 brebis Lacaune lait ont été traitées pendant 12 jours (groupe L, n = 60) ou 6 jours (groupe K, N = 48) avec un pessaire vaginal Eazi-breed™ CIDR® G (Pfizer Animal Health, Zürich) en combinaison avec 500 IE d'eCG (groupe L) respectivement 500 IE d'eCG et 125 µg de Cloprostenol (groupe K) au moment du retrait du pessaire. Par la suite, les brebis ont été détenues pendant 60 jours avec des béliers, les chaleurs ont été relevées ainsi que la fertilité après l'agnelage. Le taux sanguin de progestérone a été mesuré lors de la mise en place et du retrait du pessaire ainsi que 14 jours plus tard. Les résultats montrent que ni le traitement ni le nombre de gestations antérieures n'avaient d'influence sur le taux de chaleurs (groupe L 91.7 %, groupe K 93.8 %, brebis nulli- et pluripares 96.9 % respectivement 90.8 %). Les brebis du groupe L montraient, un taux de mise bas tendentiellement meilleur lors du premier cycle et au total significativement plus haut (P < 0.05) que celles du groupe K (71.7 % par rapport à. 60.4 % et 83.3 % par rapport à 72.9 %). Les pluripares avaient, lors du premier cycle et en général, des taux de mise-bas plus élevés que les nullipares (75.0 % contre 46.9 % respectivement 92.1 % contre 46.9 %, P < 0.05) ainsi qu'un nombre d'agneaux plus élevé par brebis synchronisée (1.4 ± 1.0 contre 0.9 ± 1.1 respectivement 1.7 ± 0.8 contre 0.9 ± 1.1, P < 0.05). Quatorze jours après le retrait du pessaire, les taux de progestérone étaient plus élevés dans le groupe L que dans le groupe K (7.7 ± 4.3 contre 5.6 ± 2.7 ng/mL) aussi bien chez les nulli- que chez les pluripares (9.1 ± 5.6 contre 5.7 ± 2.1 ng/mL). En résumé on constate que le taux de mise-bas était meilleur après un traitement long qu'après un traitement court et que les brebis nullipares étaient moins adaptées à la synchronisation des chaleurs hors de la saison de reproduction.

[Clinical evaluation of the new coat colour macchiato in a male Franches-Montagnes horse]. / Klinische und genetische Befunde bei einem männlichen Freiberger mit der neuen Fellfarbe Macchiato.

In April 2008 a Franches-Montagnes colt was born with an unusual coat colour phenotype which had never been observed in that population before. The foal showed extended white markings on body and legs, a white head and blue eyes. As both parents have an unremarkable bay coat colour phenotype, a de novo mutation was expected in the offspring and a candidate gene approach revealed a spontaneous mutation in the microphthalmia associated transcription factor gene (MITF). A detailed clinical examination in 2010 indicated an impaired hearing capacity. As in the American Paint Horse large white facial markings in combination with blue eyes are associated with deafness, the hearing capacity of the stallion was closer examined performing brainstem auditory-evoked responses (BAER). The BAER confirmed bilateral deafness in the Franches-Montagnes colt. It is assumed that the deafness is caused by a melanocyte deficiency caused by the MITF gene mutation. Unfortunately, due to castration of the horse, the causal association between the mutation in the MITF gene and clinical findings cannot be confirmed by experimental matings.

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.