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The reduction of antibiotic use in food producing animals becomes increasingly important. Therefore, suitable alternatives for mastitis treatment in dairy cows have to be considered. Oxytocin (OT) induces milk ejection and hence supports milk removal from infected mammary quarters. Beyond udder emptying, the injection of very high dosages of OT causes increased somatic cell counts (SCC) in milk and enables the transfer of immunoglobulins (Ig) from blood into milk through a reduced blood-milk barrier integrity. The aim of the present study was to investigate pathogen-specific changes of SCC, the blood derived milk components lactate dehydrogenase (LDH), serum albumin (SA), and IgG in milk of cows suffering from mastitis caused by different pathogens treated with two intravenous injections of high dosages of OT (100 IU). Milk samples from 184 dairy cows from different farms were collected on day 1 (day of clinical examination and mastitis diagnosis) and on days 2, 3, 14, and 28. Bacteriological examination (day 1) identified involved pathogens. Cows were randomly assigned to treatment (OT injections on days 1 and 2) or control group (no OT). Independently of the assigned experimental group, cows received the common therapy protocol of the veterinary practice after sample collection if the general condition was affected. Milk SCC, LDH, SA, and IgG changed specifically depending on involved pathogens. Highest values of all three parameters were measured in mastitis caused by Streptococcus uberis. Changes were less pronounced with other Streptococci spp., Staphylococci spp. or Corynebacterium bovis. Oxytocin treatment did not affect any of the studied parameters independent of the involved pathogen. Only in quarters infected with Staphylococci other than Staphylococcus aureus a decreased SCC and increased IgG concentrations in quarters, where no pathogens were detected, were observed. Thus, high dosage OT administration is obviously not suitable as a stand-alone mastitis treatment in dairy cows.Die Reduzierung des Antibiotikakonsums bei Tieren in der Lebensmittelproduktion wird immer wichtiger. Daher müssen geeignete Alternativen zur Mastitisbehandlung bei Milchkühen geprüft werden. Oxytocin (OT) induziert das Einschiessen der Milch in das Milchgangsystem und unterstützt somit das Ausmelken der Milch aus infizierten Vierteln der Milchdrüse. Über die Euterentleerung hinaus führt die Injektion sehr hoher OT-Dosierungen durch eine verringerte Integrität der Blut-Milch-Schranke zu einer erhöhten Anzahl somatischer Zellen (SCC) in der Milch und ermöglicht den Transfer von Immunglobulinen (Ig) aus der Blutbahn in die Milch. Das Ziel der vorliegenden Studie war es, bei an Mastitis erkrankten und mit zwei hohen Dosen Oxytocin (100 IE iv.) behandelten Kühen, in Abhängigkeit des Pathogens die Veränderungen von SCC, den aus Blut stammenden Milchkomponenten Lactatdehydrogenase (LDH), Serumalbumin (SA) und IgG in Milch zu untersuchen. Milchproben von 184 Milchkühen aus verschiedenen Betrieben wurden am Tag 1 (Tag der klinischen Untersuchung und Mastitisdiagnose) und an den Tagen 2, 3, 14 und 28 entnommen. Der Nachweis der Krankheitserreger erfolgte mittels einer bakteriologischen Untersuchung (Tag 1). Die Kühe wurden zufällig der Behandlungs- (OT-Injektionen an den Tagen 1 und 2) oder der Kontrollgruppe (kein OT) zugeordnet. Unabhängig von der zugewiesenen Versuchsgruppe wurden die Kühe mit reduziertem Allgemeinzustand nach der Probenentnahme gemäss dem Therapieprotokoll der Tierarztpraxis behandelt. Milch-SCC, LDH, SA und IgG änderten sich spezifisch in Abhängigkeit der beteiligten Pathogene. Die höchsten Werte aller drei Parameter wurden bei durch Streptococcus uberis verursachten Mastitiden gemessen. Die Veränderungen waren bei Streptococci spp., Staphylococci spp. oder Corynebacterium bovis weniger ausgeprägt. Unabhängig vom verursachenden Pathogen hatte die Oxytocin-Behandlung keinen Einfluss auf einen der untersuchten Parameter. Einzig bei Mastitiden verursacht durch andere Staphylokokken als Staphylococcus aureus wurden eine verminderte SCC gemessen und bei Mastitiden ohne Nachweis eines Pathogens konnte eine erhöhte IgG-Konzentration beobachtet werden. Daher ist eine hochdosierte OT-Verabreichung nicht als eigenständige Mastitisbehandlung bei Milchkühen geeignet.La réduction de l’utilisation d’antibiotiques chez les animaux destinés à l’alimentation devient de plus en plus importante. Par conséquent, des alternatives appropriées au traitement des mammites chez les vaches laitières doivent être envisagées. L’ocytocine (OT) induit l’éjection du lait et favorise donc l’élimination du lait des quartiers infectés. Au-delà de la vidange de la mamelle, l’injection de doses très élevées d’OT entraîne une augmentation du nombre de cellules somatiques (CSC) dans le lait et permet le transfert d’immunoglobulines (Ig) du sang vers le lait grâce à une réduction de l’intégrité de la barrière sang-lait. Le but de la présente étude était d’étudier les changements spécifiques aux agents pathogènes du CSC, les composants du lait dérivés du sang que sont la lactate déshydrogénase (LDH) et l’albumine sérique (SA) ainsi que les IgG dans le lait de vaches souffrant de mammites causées par différents agents pathogènes traités par deux injections intraveineuses de doses élevées d’OT (100 UI). Des échantillons de lait de 184 vaches laitières de différentes exploitations ont été prélevés au jour 1 (jour de l’examen clinique et diagnostic de mammite) et aux jours 2, 3, 14 et 28. L’examen bactériologique (jour 1) a identifié les agents pathogènes impliqués. Les vaches ont été assignées au hasard au traitement (injections d’OT les jours 1 et 2) ou au groupe témoin (pas d’OT). Indépendamment du groupe auquel elles étaient attribuées, les vaches ont reçu le protocole thérapeutique usuel du cabinet vétérinaire après le prélèvement de l’échantillon si leur état général était affecté. Le CSC, la LDH, la SA et les IgG du lait ont varié spécifiquement en fonction des agents pathogènes impliqués. Les valeurs les plus élevées des trois paramètres ont été mesurées dans les mammites causées par Streptococcus uberis. Les changements étaient moins prononcés avec d’autres Streptococci spp., Staphylococci spp. ou Corynebacterium bovis. Le traitement à l’ocytocine n’a affecté aucun des paramètres étudiés indépendamment de l’agent pathogène impliqué. On a uniquement observé, dans les mammites causées par des staphylocoques autres que Staphylococcus aureus, une diminution du CSC et, dans les mammites où aucun agent pathogène n’a été détecté, une augmentation des concentrations d’IgG dans les quartiers. Ainsi, l’administration d’OT à forte dose n’est pas appropriée comme traitement unique des mammites chez les vaches laitières.La riduzione dell’uso di antibiotici negli animali per la produzione alimentare sta assumendo un’importanza sempre maggiore. Di conseguenza, è opportuno considerare delle alternative adeguate per il trattamento della mastite nelle vacche da latte. L’ossitocina (OT) induce l’eiezione del latte e quindi aiuta la rimozione di questo dai quarti mammari infetti. L’iniezione di dosi molto elevate di OT provoca non solo lo svuotamento della mammella, ma pure un aumento del numero di cellule somatiche (SCC) nel latte permettendo quindi il trasferimento di immunoglobuline (Ig) dal sangue al latte attraverso una ridotta integrità della barriera tra sangue e latte. Lo scopo del presente studio era di esaminare i cambiamenti patogeno-dipendenti dal SCC, i componenti del latte derivati dal sangue del lattato deidrogenasi (LDH), l’albumina del siero (SA) e le IgG nel latte, delle vacche affette da mastite causata da diversi patogeni e trattate con due iniezioni intravenose ad alti dosaggi di OT (100 UI). I campioni di latte provenienti da 184 vacche da latte di diverse aziende sono stati raccolti il giorno 1 (giorno dell’esame clinico e della diagnosi di mastite) e nei giorni 2, 3, 14 e 28. L’esame batteriologico (giorno 1) ha identificato gli agenti patogeni coinvolti. Le vacche sono state assegnate in modo casuale al gruppo con il trattamento (iniezioni di OT il giorno 1 e 2) o al gruppo di controllo (nessuna OT). Indipendentemente dal gruppo sperimentale assegnato, se dopo la raccolta del campione le condizioni generali dell’animale risultavano compromesse, le vacche ricevevano un comune protocollo terapeutico dello studio veterinario. Si è rilevato che a seconda degli agenti patogeni coinvolti, l’SCC del latte, la LDH, la SA e le IgG cambiavano in modo specifico. I valori più alti fra tutti e tre i parametri sono stati misurati in caso di mastite causata da Streptococcus uberis. I cambiamenti erano meno pronunciati con gli Streptococchi spp., Staphylococci spp. o Corynebacterium bovis. Si è notato che il trattamento con ossitocina non influenzava nessuno dei parametri studiati, indipendentemente dal patogeno coinvolto. Si è osservato nei quarti infettati da stafilococchi diversi dallo Staphylococcus aureus una diminuzione della SCC mentre nei quarti dove non venivano rilevati agenti patogeni si evidenziava un aumento delle concentrazioni di IgG. Pertanto, la somministrazione di OT ad alto dosaggio non è ovviamente adatta al trattamento stand-alone della mastite nelle vacche da latte.

Embryo cryopreservation ensures that genetic biodiversity is preserved over time. This study evaluates the survival of donkey embryos subjected to slow freezing and vitrification after thawing and in vitro culture. Seven-day-old in vivo produced donkey embryos were subjected to slow freezing (SF, N = 14) or vitrification (VIT, N = 22). After one year of cryopreservation, embryos were warmed, washed and placed in incubation for in vitro culture (IVC). In order to assess the embryo viability, the quality grade and developmental stage were recorded after thawing and after 24 and 48 h of IVC. Eleven embryos (SF = 4 and VIT = 7) were incubated under a time-lapse camera, for up to 68 h, in order to determine the area and growth. The survival rate was not influenced by the procedure but by the developmental stage: after 48 h of IVC blastocyst survival rate (1/8, 12.5%) was significantly lower compared to both morulas (8/12, 66.7%) and early blastocysts (11/16, 68.7%) (P < 0.05). Embryo diameter class at recovery did not significantly influence the survival rate. In terms of the embryos that were judged to be alive after 48 h of IVC, quality grade 1 was observed in 7/8 (88%) and 4/12 (33%) of the SF and VIT embryos, respectively (P < 0.05). After time-lapse analysis, the IVC embryo area as well as growth percentage were statistically higher in the SF than the VIT embryos (P < 0.05). In conclusion, no difference in survival rates was found between the two cryopreservation procedures, although embryo quality was more negatively affected by vitrification.

Zusammenfassung Ziel Das Ziel dieser Studie war, erstmals zweidimensionale (2D) und dreidimensionale (3D) transrektale Ultraschalluntersuchungen (USU) hinsichtlich ihrer Richtigkeit und dem zeitlichen Aufwand zur Geschlechtsdiagnose bei Pferdefeten zu vergleichen. Material und Methoden Zu diesem Zweck wurden 47 Stuten zwischen den Tagen 58 und 115 der Gravidität zunächst einer 2D-USU unterzogen, um das fetale Geschlecht anhand der Lokalisation des Genitaltuberkels oder dem Vorhandensein von äußeren Geschlechtsteilen zu ermitteln. Im Folgenden wurden 3D-Bilder des Fetus angefertigt und anschließend an einem PC ausgewertet. Ergebnisse Der beste Zeitraum zur Geschlechtsbestimmung war zwischen dem 58. und 79. Graviditätstag mit 77 % (2D-USU und 3D-Bild-Auswertung) korrekten Diagnosen. Durch wiederholtes Auswerten der 3D-Bilder konnte eine Steigerung richtiger Diagnosen um 16 % erzielt werden. Der zusätzliche Zeitaufwand für die 3D-USU und die 3D-Bild-Auswertung betrug etwa 6–7 Minuten pro Stute. Schlussfolgerung und klinische Relevanz Die Ergebnisse der Studie zeigen, dass die 3D- gegenüber der 2D-Sonografie die Zuverlässigkeit der transrektalen Geschlechtsbestimmung erhöht. Erfahrenen Untersuchern bringt die 3D-Ultraschalltechnik jedoch keine entscheidenden Vorteile und ist zudem zeitaufwendiger.

The anti-Müllerian hormone (AMH) plays an inhibitory role during folliculogenesis by regulating the number of follicles entering the growing pool. Antral follicle counts (AFC) are highly correlated with serum AMH concentrations and both appear to be related to the ovarian reserve in several species. Few data on AMH and AFC in mares exist, especially with regard to fertility. Therefore, the objective of the current study was to investigate the interrelationship between antral follicle count, serum AMH concentrations and fertility outcome in mares. One hundred and twenty-seven mares were enrolled in the study and grouped according to their reproductive status. Around time of estrus, serum AMH concentrations and AFC before and after ovulation were determined. Mares were artificially inseminated and pregnancy diagnosis was performed 14 to 18 days later. A high inter-individual variability in AFC and AMH concentration and a positive relationship between AMH and AFC for follicles ≤ 30 mm in diameter were observed, with a stronger correlation in mares older than 18 years. A high correlation between AFC measurements before and after ovulation was identified. The AFC after ovulation was higher than AFC before ovulation. AMH concentrations were neither related to the mares' reproductive status nor to age, number of cycles needed for pregnancy and pregnancy outcome. Excepted for a higher AFC in the middle-aged mares (9-18 years) compared to the younger mares (9 years), no associations between AFC and age, reproductive status as well as fertility of mares were found. This study confirms the relationship between AFC and AMH and a high degree of reproducibility of AFC measurements. However, based on our findings, neither AFC nor AMH are useful predictors of fertility in mares.Das Anti-Müller-Hormon (AMH) hemmt während der Follikulogenese die Anzahl heranwachsender Follikel. Die Anzahl der antralen Follikel («antral follicle count», AFC) korreliert stark positiv mit der Serumkonzentration von AMH und beide sind bei mehreren Spezies ein Indikator für die ovarielle Reserve. Da bisher wenige Daten über AFC und AMH bei Stuten, insbesondere im Hinblick auf deren Auswirkungen auf die Fertilität vorliegen, sollte dies in der vorliegenden Arbeit un­tersucht werden. Hundertsiebenundzwanzig Stuten ­wurden in die Studie eingeschlossen und nach Reproduk­tionsstatus gruppiert. Die AFC und die AMH-Konzen­tration wurden während der Rosse vor und nach der Ovulation ermittelt. Die Stuten wurden künstlich besamt und die 14 bis 18 Tage später eine Trächtigkeitsunteruchung durchgeführt. Es bestanden hohe Variabilität im AFC und in der AMH-Konzentration zwischen den Stuten und ein positiver Zusammenhang zwischen AMH und AFC für Follikel mit einem Durchmesser ≤ 30 mm, wobei die Korrelation bei Stuten18 Jahre stärker ausgeprägt war. Die AFC-Messungen vor und nach Ovulation korrelierten sehr gut miteinander, wobei der AFC nach Ovulation grösser war als vor der Ovulation. Die AMH-Konzentrationen waren unabhängig vom Alter, dem Reproduktionsstatus, der Anzahl benötigter Zyklen bis zur Erreichung einer Trächtigkeit und dem Trächtigkeitsausgang. Mit Ausnahme der Beobachtung, dass die AFC bei mittelalten Stuten (9–18 Jahre) höher als diejenige bei AFC jüngerer Stuten (9 Jahre) war, konnten keine Assoziationen zwischen AFC und Alter, Reproduktionsstatus oder Fertilität erkannt werden. Diese Studie bestätigt den Zusammenhang zwischen AFC und AMH und eine hohe Reproduzierbarkeit der AFC-Messungen. Allerdings können anhand der vorliegenden Ergebnisse weder AFC noch AMH als brauchbare Prädiktoren für die Fruchtbarkeit von Stuten herangezogen werden.Il existe peu de données sur l’AMH et l’AFC chez les juments, particulièrement en rapport avec la fertilité. L’objectif de cette étude était donc d’examiner la relation entre le compte de follicules antraux, le taux sérique d’AMH et la fécondité chez les juments. Cent vingt-sept juments ont été inclues dans l’étude et groupées selon leur état reproducteur. Les taux sériques d’AMH et l’AFC ont été déterminés pendant l’oestrus avant et après l’ovulation. Les juments ont été inséminées artificiellement et le diagnostic de gestation réalisé 14 à 18 jours plus tard. Une grande variabilité interindividuelle de l’AFC et l’AMH et une corrélation positive entre l’AMH et l’AFC pour les follicules de diamètre ≤ 30 mm ont été observées, cette dernière étant plus forte chez les juments âgées de plus de 18 ans. L’AFC après ovulation était supérieur à l’AFC avant ovulation, et une forte corrélation entre les deux mesures a été constatée. Aucun lien entre les taux sériques d’AMH, l’état reproducteur, l’âge, le nombre de cycles œstraux par gestation et le taux de gestation n’a été observé. Hormis un AFC supérieur chez les juments d’âge moyen (9-18 ans) comparé aux juments plus jeunes (.L’ormone antimulleriano (AMH) funge da regolatore della follicologenesi inibendo il reclutamento e lo sviluppo follicolare iniziale. La conta dei follicoli antrali («antral follicle count», AFC) è altamente correlata alla concentrazione sierica di AMH ed entrambi sembrano avere valore predittivo sulla riserva ovarica in diverse specie. Pochi dati sono disponibili sull’AMH e la AFC nelle cavalle, specialmente in relazione alla fertilità. Lo scopo di questo studio era quindi quello di indagare sull’interrelazione tra la conta dei follicoli antrali, i livelli sierici di AMH e la fertilità nelle cavalle. Centoventisette cavalle sono state arruolate nello studio e divise in gruppi in base al loro stato riproduttivo. Le concentrazioni sieriche di AMH e l’AFC sono state determinate durante l’estro prima e dopo l’ovulazione. Le cavalle sono state inseminate artificialmente e la diagnosi di gravidanza è stata effettuata 14-18 giorni più tardi. Sono state osservate un’alta variabilità interindividuale ed una correlazione positiva tra l’AMH e la AFC per i follicoli con diametro ≤ 30 mm, la quale era più forte nelle cavalle di età superiore a 18 anni. La AFC dopo l’ovulazione era più alta della AFC prima dell’ovulazione, ed è stata riscontrata un’elevata correlazione tra i due conteggi. I livelli sierici di AMH non erano in relazione con lo stato riproduttivo delle cavalle, né con la loro età, il numero di cicli estrali per gravidanza e tasso di gravidanza. Non sono state identificate associazioni tra AFC, età, stato riproduttivo e fertilità delle cavalle, ad eccezione di una AFC maggiore nelle cavalle di media età (9-18 anni) rispetto a quelle più giovani (.

The objective of this prospective field study was to evaluate the effects of extending the lactation period of high-yielding dairy cows on milk production, udder health characteristics, and development of body condition. On 40 d in milk (DIM), an examination of the genital tract (transrectal palpation, sonography, vaginoscopy) was performed. Cows without signs of clinical endometritis were blocked by parity and were randomly allocated to 1 of 3 experimental groups with a voluntary waiting period of 40, 120, and 180 d, respectively (G40, n = 135; G120, n = 141; G180, n = 139). Mean daily milk and energy-corrected milk production did not differ between the 3 groups regarding the first 305 d or for the whole lactation (d 1 and up to dry off, culling, or 600 DIM). In late lactation (306 to 600 DIM), G40 had lower average productivity (23.8 kg) compared with G120 (26.5 kg), with G180 showing intermediate values (25.7 kg). The extended lactation groups showed greater persistency, as the rate of decline based on a Wilmink function was lower for G120 (c = -0.063 and -0.045 for milk and energy-corrected milk, respectively) and G180 (c = -0.061 and -0.047) compared with G40 (c = -0.071 and -0.056). We found no difference between the 3 groups regarding the evaluated udder health characteristics (somatic cell count, incidence of mastitis, and days off milk due to mastitis). More cows in G180 (7.9%) were culled due to low productivity compared with G40 (0.7%) and as a tendency compared with G120 (2.8%). Moreover, cows of G180 showed higher median body condition score at the time of dry off compared with cows of both G40 and G120 (3.50 for G180 vs. 3.25 for both G40 and G120). At the time of dry off, G180 cows also had greater backfat thickness (25.0 mm) compared with both G40 (22.2 mm) and G120 cows (21.6 mm). Based on our results, the extension of the voluntary waiting period of high-yielding cows up to 120 d has no adverse effects regarding milk production, involuntary culling, udder health, or BCS gain.

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.

Metritis is an important disorder in dairy cows during the early postpartum period. Myometrial contractility is a prerequisite for uterine involution; however, very scanty literature is available about the effect of metritis on this process and endocrine responsiveness. This study was aimed to evaluate the effect of inflammation on uterine contractility in vitro, and the inflammation was induced by incubating myometrial strips with lipopolysaccharides (LPS). Myometrial samples were collected from 17 healthy Holstein Friesian cows during caesarean section. Eight longitudinal strips from each cow were incubated in organ baths with LPS concentrations of 0 (LPS0 ), 0.1 (LPS0.1 ), 1 (LPS1 ) and 10 µg/ml (LPS10 ). Spontaneous contractility and contractility induced by increasing concentrations of oxytocin (10-10 - 10-7 mol/L) were recorded during nine 30-min intervals (T1 to T9). The minimum amplitude (minA), maximum amplitude (maxA), mean amplitude (meanA) and area under the curve (AUC) were calculated for each time interval. LPS had an effect (p ≤ .05) on maxA, meanA and AUC. In T1, myometrial strips incubated with LPS0.1 and LPS1 had higher (p ≤ .05) maxA, meanA and AUC than the strips incubated with LPS0 . In T9 without oxytocin, LPS0 led to higher (p ≤ .05) maxA, meanA and AUC than LPS0.1 and LPS1 . In T8 and T9 with oxytocin, LPS1 had lower (p ≤ .05) maxA, meanA and AUC than the other LPS concentrations. Interestingly, the results show that LPS has a transient positive effect on myometrial contractility in vitro and that this effect is dependent on LPS concentration and duration of incubation.

The aim of this study was to investigate the effects of PGF2α and oxytocin in vitro on myometrial contractility in puerperal uteri. Thirteen puerperal uteri were removed and perfused after euthanasia of cows with (n=7) and without metritis (n=6). Measurement of uterine contractility was done using four piezoelectric crystals, which were implanted into the myometrium along the greater curvature of the uterine horn where fetal implantation occurred during the previous pregnancy. After 30min of equilibration, oxytocin (5 IU) or PGF2α (2.5mg Dinoprost) was administered randomly into both uterine arteries, and 30min later, the second administration of either oxytocin or PGF2α occurred. Treatment with oxytocin induced contractions in uteri with metritis and uteri without metritis (P 0.05) result in increased contractions in the uteri without metrtitis, however, induced an initial decrease in contractions followed by an increase (P

Effects of a plasmolysed yeast product enriched with herbs, malt, honey and orange syrup on semen characteristics and oxidative status in stallions were evaluated. Twenty stallions (mean age ± standard deviation = 9.5 ± 4.5 years) were randomly divided into a treatment group (n = 10) receiving 0.06 mL/kg bodyweight of plasmolysed herbal yeast, and a control group (n = 10) receiving the same amount of placebo daily in the feed for 10 weeks. Ejaculates were collected weekly from all stallions starting at Week 0. Volume, sperm concentration, motility, and velocity were evaluated immediately, 24 and 48 h after cooled storage at 5 °C. At the two storage time points, membrane lipid peroxidation was determined using the BODIPY-C11. Additionally, blood samples were collected at Weeks 0, 1, 5 and 9, and analysed for antioxidant status, consisting of superoxide dismutase, cholesterol, thiobarbituric acid reactive substances, and non-esterified fatty acids. Due to the nature of the data, the Mann-Whitney U test was applied as preliminary analysis. The BODIPY-C11 in the semen was less at 24 h and greater at 48 h after collections in Week 1 to 3 (P < 0.01) and Week 1 to 10 (P

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

The objective of this prospective field study was to evaluate the effects of extending the lactation period on various reproductive measurements of high-yielding Holstein cows. On 40 d in milk (DIM), cows were gynecologically examined (transrectal palpation, sonography, vaginoscopy). Cows without signs of clinical endometritis were blocked by parity and were randomly allocated to 1 of 3 experimental groups with a voluntary waiting period (VWP) of 40, 120, and 180 d, respectively (G40, n = 135; G120, n = 141; G180, n = 139). Cows of G120 and G180 were reexamined at the end of the VWP. If natural estrus was detected within 46 d after the end of the VWP, an artificial insemination was performed. If no estrus was detected, the respective cows were synchronized by applying the classical Ovsynch protocol. We found no difference in the proportion of cows in which estrus was detected between 40 to 86 DIM or in the days to first estrus between the 3 groups. Estrus detection in this period was lower in cows with body condition score

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 μL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P

Oxidative stress in spermatozoa has effects on subsequent embryo development. The aim of the present study was to elucidate whether sperm oxidative stress results in increased DNA damage in the embryo. To this end, bovine spermatozoa were incubated for 1 h at 37°C without or with 100 µM H2O2, resulting in non-oxidised (NOX-S) and oxidised (OX-S) spermatozoa respectively. Non-incubated spermatozoa served as the control group (CON-S). After IVF, developmental rates 30, 46 and 60 h and 7 days after IVF were assessed. DNA damage was analysed in embryos using the comet assay and a DNA damage marker (γH2AX immunostaining); the apoptotic index was determined in blastocysts. Exposure of spermatozoa to H2O2 induced a significant amount of sperm chromatin damage. The use of OX-S in IVF resulted in significantly reduced cleavage and blastocyst rates compared with the use of CON-S and NOX-S. Furthermore, in embryos resulting from the use of OX-S, a developmental delay was evident 30 and 46 h after IVF. γH2AX immunostaining was lower in blastocysts than in early embryos. In blastocysts, the comet and apoptotic indices were significantly higher in embryos resulting from the use of OX-S than CON-S and NOX-S. In conclusion, oxidative stress in spermatozoa induces developmental abnormalities and is a source of DNA damage in the resulting embryos.

The aim of this study was to examine effects of sodium pyruvate on viability as well as on synthesis of reactive oxygen species (ROS), lipid peroxidation and DNA integrity of cryopreserved bovine sperm. In each of 23 Simmental AI bulls three ejaculates were collected. In a split sample design ejaculates were diluted by using Triladyl® extender without and with the addition of 5mM sodium pyruvate. Both aliquots were equilibrated for 24h before freezing. Frozen sperm samples were thawed, and examined immediately after thawing (0h) as well as after 3, 6, 12, and 24h incubation at 37°C. The percentages of rapidly motile sperm (RMS), plasma membrane and acrosome intact sperm (PMAI), sperm with a high mitochondrial membrane potential (HMMP), amounts of ROS synthesis (dichlorofluorescein-diacetate (DCFH), CellROX Deep Red Reagent® probe (CellROX)) and lipid peroxidation of sperm (LPO) and percentage of sperm with a high degree of DNA fragmentation (%DFI) were determined. Overall, sperm diluted with the extender containing sodium pyruvate showed higher levels of RMS, PMAI and HMMP, CellROX and lower %DFI values (P 0.05) of sodium pyruvate on LPO and DCFH. The results of this study show that the addition of sodium pyruvate to the semen extender improved the viability as well as DNA integrity of cryopreserved sperm and did not affect their lipid peroxidation, although it increased the synthesis of some ROS.

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 0.05 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.

Resumption of ovulatory activity and the timely lysis of the first CL postpartum (pp) are important determinants for the reproductive performance of dairy cows. Cystic ovarian follicles (COFs) and persistent CLs preclude normal ovarian cyclicity and increase the calving interval. The objective of this study was to investigate the effect of GnRH on the incidence of COFs and the effect of PGF2α on the incidence of a prolonged luteal phase (PLP) and on fertility parameters in dairy cows. A total of 476 cows were examined ultrasonographically for the presence of a dominant follicle (12-25 mm, without CL >10 mm; n = 237) or a functional CL (≥20 mm; n = 239) between 28 and 35 days pp and were allocated to one of four groups. Cows with a dominant follicle received 10-μg GnRH (buserelin; group F-T; n = 118) or saline (group F-C; n = 119), and cows with a functional CL received 0.5 mg of a PGF2α analogue (cloprostenol; group CL-T; n = 119) or saline (group CL-C; n = 120) on the day of initial examination, defined as Day 0. Cows were reexamined 7 and 21 days (F-T and F-C) and 3 and 24 days (CL-T and CL-C) later, and COFs were treated immediately after diagnosis in all cows. On the basis of the ovarian findings on Days 21 and 24, cows were treated according to a protocol aimed at timely breeding. The incidence of COFs by Days 7 (F-T vs. F-C; 7.6% vs. 16.8%) and 21 (11.0% vs. 21.8%) decreased (P ≤ 0.03) with GnRH; however, this did not lead to a substantial improvement of calving-to-conception interval (means ± standard error of the mean; 107.91 ± 5.70 vs. 117.94 ± 6.63 days), first-service conception rate (42.3% vs. 41.3%), and number of services per conception (2.06 ± 0.12 vs. 2.31 ± 0.15). Treatment with PGF2α decreased (P < 0.0001) the incidence of PLP by Day 24 (CL-T vs. CL-C; 1.7% vs. 17.5%), decreased calving-to-conception interval(91.28 ± 4.77 vs. 101.75 ± 5.03 days), increased first-service conception rate (63.3% vs. 38.7%), and reduced the number of services per conception (1.65 ± 0.10 vs. 2.08 ± 0.12; each P ≤ 0.01). The results indicate that strategic treatment with GnRH or PGF2α in week 5 pp to induce early ovulation and luteolysis reduces the incidence of COFs and PLP, respectively. Initial treatment with PGF2α also enhanced reproductive performance when used in conjunction with a standardized treatment protocol for all cows in week 8 pp (aimed at timely breeding). In contrast, GnRH did not improve fertility parameters of cystic cows in herds where all cows with COFs were treated as expeditiously as possible.

The objective of this study was to investigate whether confirmation of suitability for service by traditional methods, observation and transrectal palpation, is improved in cattle by additional ultrasonographic examination of the uterus and ovaries. The data from 600 lactating cows from Southern Germany submitted for the first service and alternately assigned to manual (group M, n = 301) and manual and sonographic examination (group S, n = 299) were evaluated. Cows of group M with a large, soft follicle with an estimated diameter of 12.0 to 22.5 mm, without a palpable CL (>10 mm), and with a contractile uterus without palpable content were considered suitable for service and inseminated. Cows of group S with a soft follicle measuring 12.0 to 22.5 mm, without a CL (>10 mm), and with a contractile uterus and no echoic content were inseminated. Pregnancy was determined by ultrasonography at 28 to 35 days after the first service. In group S, 10.9% fewer cows (P = 0.003) were considered suitable for service than in group M. The main reason (29.6%) for exclusion from service in group S was the presence of a CL greater than 10 mm in addition to a preovulatory follicle (12.0-22.5 mm), which was diagnosed less frequently in group M (4.5%; P < 0.0001). The first-service conception rate tended to be higher in group S than in group M (62.7% vs. 54.9%; P = 0.10), and the percentage of open cows, based on all cows submitted for service, was higher in group M than in group S (35.2% vs. 25.1%; P = 0.007). We concluded that sonographic examination of cows submitted for service facilitates the exclusion from service of proestrous and diestrous cows and reduces the number of pointless services.

The aim of this study was to compare the efficacy of two different short-term progestagen treatments for estrus synchronization in cyclic ewes. A total of 292 ewes of 3 flocks (A–C) on different farms were treated for 6 days with Eazi-breedTM CIDR® G (n = 145) or Chronogest® CR (n = 147) vaginal inserts in combination with 0.125 mg Cloprostenol and 300IU eCG at device removal. Blood samples were taken for progesterone (P4) determination at insert application and removal as well as 14 days later. One day after insert removal rams fitted with marking harnesses were joined to ewes for 35 days and marked ewes recorded daily. Lambing data were recorded and fertility to first service period and overall assessed. Results show that 24–96 h after insert removal 98.8% and 89.4% of the synchronized ewes in the flocks B and C were in estrus. Estrus response was not affected by the type of progestagen treatment and was lower (P < 0.05) in nulli- (82.1%) than in pluriparous (97.5%) ewes. Type of progestagen treatment did not affect fertility, but flock and parity influenced the percentage of ewes that lambed as well as the lambing rate. Overall more ewes (P < 0.001) lambed in flock A compared to flocks B and C (93.0 vs. 72.5 and 59.1%). In nulliparous animals the percentage of ewes that lambed was higher to the first service (P < 0.05) period but lower (P < 0.001) to the overall service period compared to pluriparous animals (51.5% vs. 49.3% and 64.7% vs. 84.2%). Lambing rates were higher in flock A than in flocks B and C to first service period and overall (1.3 ± 1.4, 0.9 ± 1.0, and 0.7 ± 1.0, P < 0.01 and 1.9 ± 1.1, 1.3 ± 0.9, and 1.0 ± 1.0, P < 0.001) and in nulliparous overall lower than in pluriparous ewes (1.1 ± 1.1 and 1.6 ± 1.1, P < 0.01). Serum P4 concentrations measured 14 days after insert removal were higher in the Chronogest® CR than in the Eazi-breedTM CIDR® G group (6.8 ± 4.1 ng/mL vs. 5.7 ± 3.3 ng/mL). Ewes of flock A had higher P4 values compared to ewes of flocks B and C (8.3 ± 4.1 ng/mL vs. 4.5 ± 1.5 ng/mL and 3.8 ± 1.7 ng/mL) and nulliparous lower P4 concentrations than pluriparous ewes (4.5 ± 2.2 ng/mL vs. 6.7 ± 4.0 ng/mL). In conclusion, a 6-day treatment with Chronogest® CR and Eazi-breedTM CIDR® G with prostaglandin and eCG at insert removal resulted in high estrus response and similar fertility in cyclic ewes. © 2012 Elsevier B.V. All rights reserved.

The aim of this study was to measure obstetrically relevant dimensions in calves manually and via computed tomography and to investigate their relationship with the dimensions of fetal body parts that are accessible to the obstetrician during early stages of vaginal delivery. Twenty Holstein-Friesian stillborn calves (Bos taurus) weighing 41.1 ± 3.7 kg (33.6-46.5 kg) were examined and the maximum height (H), width (W), circumference (C) and cross-sectional area (A) of their body was determined. The largest (P0.05) one-dimensional variable was the height of the thorax in the region of cranial sternum (H-Thorax; 29.3 ± 1.3 cm), and the largest (P0.05) two-dimensional variables were the cross-sectional area of the shoulder region at greater tubercles of the humeri (A-Shoulder; 307 ± 27 cm²) and of the thorax in the region of cranial sternum (A-Thorax; 306 ± 25 cm²). The dimensions of the front legs, which included the circumference of the canon bone and width of the fetlock joint, did not correlate (P0.05) with H-Thorax, A-Shoulder and A-Thorax. There were moderate significant correlations between the perpendicular height of the head (H-Head) and A-Thorax (r = 0.65, P0.05) and between the circumference of the head (C-Head) and A-Shoulder (r = 0.64, P0.05) and A-Thorax (r = 0.52, P0.05), but other relationships (P0.05) between the dimensions at the level of the head and H-Thorax, A-Shoulder and A-Thorax were not significant (P0.05). The results of this study show that the shoulders and thorax are the obstetrically most relevant calf dimensions, but the size of these variables cannot be reliably predicted by evaluating the dimensions of the forelimbs and head of calves during delivery.

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.

Data from 96 Holstein Friesian cows on a commercial dairy farm were used to investigate whether body condition and serum levels of nonesterified fatty acids (NEFAs) postpartum (pp) affect progesterone (P4) levels, estrous activity, and fertility in dairy cows. The examination period started 14 days before the expected calving date and ended either when a cow was inseminated or at a maximum of 90 days pp. Body condition score (BCS; 1-5 scale) and backfat thickness (BFT) were determined every 2 weeks. Blood for analysis of NEFA and P4 concentrations was sampled weekly during the first 35 days pp and then every 48 hours until an ovulation was observed. Transrectal ultrasonography of the ovaries started at 21 days pp and was performed after blood sampling. If cows were not inseminated because of silent ovulation, sampling and ultrasonography continued on Days 7, 14, and 18 after ovulation and again every 48 hours until the next ovulation. Estrous activity was continuously measured with the Heatime estrus detection system. Pregnancy controls were performed ultrasonographically 28 and 42 days after AI. Cows with increased NEFA levels at 28 days pp had an increased risk of maintaining minimum P4 levels above 0.4 ng/mL at first recognized estrus (P = 0.03). Higher NEFA levels at Day 7 were associated with lower probability for a cow to have elevated P4 levels (≥2 ng/mL) by Day 35 pp, indicating delayed commencement of luteal activity (C-LA). Estrous activity was not influenced (P > 0.10) by minimum P4 concentrations at estrus, but more animals with C-LA until Day 35 pp showed estrous activity compared to cows without C-LA throughout this period (P = 0.006). Estrous activity was lower in cows with a low BCS 14 days pp (P = 0.02) and with a low BFT 42 days pp (P = 0.03). Moreover, the probability to exhibit estrus was reduced with higher NEFA levels at 21 days pp (P = 0.01). Eighty-five cows were inseminated and 37 (44%) got pregnant after insemination. Higher NEFA levels, especially 14, 28, and 42 days pp, were associated with lower probability to inseminate a cow. Regarding the cows that were inseminated, no influence of BCS, BFT, or NEFA levels on pregnancy per artificial insemination was found. In conclusion, cows with higher loss of body condition and elevated NEFA concentrations had higher P4 concentrations during estrus, a reduced estrous activity and insemination rate, but no depression in pregnancy per artificial insemination.

The objective of this study was to investigate the effect of time of first postpartum ovulation on endometrial inflammation in dairy cows with and without uterine disease during the early puerperal period. Transvaginal follicular puncture (FP) was carried out to suppress postpartum ovulation and formation of a CL until Day 42. Fifty-three lactating Holstein Friesian cows were divided into four groups on the basis of presence (UD+) or absence (UD-) of uterine disease, which was defined as retained fetal membranes and/or metritis, and whether FP had (FP+) or had not been (FP-) carried out. This resulted in the following groups: UD-FP- (n = 15), UD-FP+ (n = 13), UD+FP- (n = 13), and UD+FP+ (n = 12). Cloprostenol was given on Days 55 to 60 postpartum, and GnRH was administered 2 days later for synchronization of ovulation. In the FP- groups, endometrial swab and biopsy samples were collected during the second estrus (approximately Day 40) and during the estrus after synchronization. In the FP+ groups, the same samples were collected during the first estrus (approximately Day 49) and during the estrus after synchronization. The prevalence of positive bacteriologic cultures of the endometrium was not affected by FP (P > 0.05). Histologic signs of endometritis were more severe in UD+FP- cows at second sampling than in UD+FP+ cows (P ≤ 0.05). Endometrial expression of IL1α (in UD- after first sampling and in UD+ after second sampling) and IL1β (in UD- and UD+ after first sampling) was higher (P ≤ 0.05) in FP- cows than in FP+ cows. Regardless of group, cows with histopathologic evidence of endometritis had higher expression (P ≤ 0.05) of IL1α, IL1β, IL6, and TNFα than cows without endometritis. In conclusion, suppression of early ovulation by transvaginal FP enhances clearance of uterine inflammation in postpartum cows.

A study involving a small number of cows found that the concentrations of insulin-like growth hormone 1 (IGF1) may be a useful predictor of metabolic disease. Further, IGF1 may provide also a pathophysiological link to metabolic diseases such as ketosis. The objective of the current study was to test whether the low antepartal total IGF1 or IGF1 binding protein (IGFBP) concentrations might predict ketosis under field conditions. Clinical examinations and blood sampling were performed antepartum (262-270 d after artificial insemination) on 377 pluriparous pregnant Holstein Friesian cows. The presence of postpartum diseases were recorded (ketosis, fatty liver, displacement of the abomasum, hypocalcemia, mastitis, retention of fetal membranes, and clinical metritis or endometritis), and the concentrations of IGF1, IGFBP2, IGFBP3, and nonesterified fatty acids were measured. Cows with postpartum clinical ketosis had lower IGF1 concentrations antepartum than healthy cows. The sensitivity of antepartal IGF1 as a marker for postpartum ketosis was 0.87, and the specificity was 0.43; a positive predictive value of 0.91 and a negative predictive value of 0.35 were calculated. The cows with ketosis and retained fetal membranes had lower IGFBP2 concentrations compared with the healthy cows. It can be speculated that lower IGF1 production in the liver during late pregnancy may increase growth hormone secretions and lipolysis, thereby increasing the risk of ketosis. Lower IGFBP2 concentrations may reflect the suppression of IGFBP2 levels through higher growth hormone secretion. In conclusion, compared with nonesterified fatty acids as a predictive parameter, IGF1 and IGFBP2 may represent earlier biomarkers of inadequate metabolic adaptation to the high energy demand required postpartum.

In this study, the effect of various unilamellar liposomes on cryopreservation of bovine spermatozoa has been investigated. Liposomes were composed of saturated lipids with various acyl chain lengths: DSPC (18:0), DPPC (16:0), DMPC (14:0), or DLPC (12:0). Alternatively, liposomes were prepared using unsaturated egg phosphatidylcholine (EPC) or DOPC (18:1, neutral), alone or in combination with lipids with various head groups: DOPS (negatively charged), DOPG (negatively charged), and DOPE (neutral). Fourier transform infrared spectroscopy studies showed that bovine sperm membranes display a gradual phase transition from 10 to 24 (o)C. Phase transition temperatures of the liposomes varied from -20 to +53 (o)C. Sperm was incubated in the presence of liposomes for either 6 or 24 h at 4 °C prior to freezing. Postfreeze survival rates were determined based on the percentage of progressively motile cells as well as the percentage of acrosome- and plasma membrane-intact cells. With DOPC liposomes a postthaw progressive motility of 43% was obtained compared with 59% using standard egg yolk freezing extender. Postthaw progressive motility increased up to 52% using DOPC:DOPG (9:1) liposomes, whereas DOPC:DOPS or DOPC:DOPE liposomes did not increase survival compared with DOPC liposomes. Among the saturated lipids, only DMPC was found to increase cryosurvival, up to 44% based on progressive motility. DLPC liposomes caused a complete loss in cell viability, already prior to freezing, whereas DPPC and DSPC liposomes neither positively nor negatively affected cryosurvival. Taken together, the higher postthaw survival obtained with DOPC:DOPG liposomes as compared with DOPC liposomes can likely be attributed to increased liposome-sperm interactions between the charged phosphatidylglycerol groups and charged regions in the sperm membranes. Interestingly, the lipid phase state of the liposomes during preincubation is not the decisive factor for their cryoprotective action.

Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements. Additionally, AI centers use computer-assessed sperm concentration in the sample as a basis for calculating the number of insemination doses available from a given ejaculate. The reliability of data is often limited and results can vary even when the same CASA systems with identical settings are used. The objective of the present study was to develop a computer-based training module for standardized measurements with a CASA system and to evaluate its training effect on the quality of the assessment of sperm motility and concentration. A digital versatile disc (DVD) has been produced showing the standardization of sample preparation and analysis with the CASA system SpermVision™ version 3.0 (Minitube, Verona, WI, USA) in words, pictures, and videos, as well as the most probable sources of error. Eight test persons educated in spermatology, but with different levels of experience with the CASA system, prepared and assessed 10 aliquots from one prediluted bull ejaculate using the same CASA system and laboratory equipment before and after electronic learning (e-learning). After using the e-learning application, the coefficient of variation was reduced on average for the sperm concentration from 26.1% to 11.3% (P ≤ 0.01), and for motility from 5.8% to 3.1% (P ≤ 0.05). For five test persons, the difference in the coefficient of variation before and after use of the e-learning application was significant (P ≤ 0.05). Individual deviations of means from the group mean before e-learning were reduced compared with individual deviations from the group mean after e-learning. According to a survey, the e-learning application was highly accepted by users. In conclusion, e-learning presents an effective, efficient, and accepted tool for improvement of the precision of CASA measurements. This study provides a model for the standardization of other laboratory procedures using e-learning.

Impaired function of polymorphonuclear neutrophilic leukocyte (PMNL) during the peripartal period is a major reason for increased susceptibility of dairy cows to infections in this critical interval. Factors dysregulating PMNL function are widely unknown. Insulin-like growth factor (IGF-I) enhanced PMNL functions in vitro. The objective of this study was to investigate the influence of IGF-I and, additionally, β-hydroxybutyrate and nonesterified fatty acid concentrations on phagocytic activity (PA, percentage of viable PMNL) and phagocytic capacity (PC, mean fluorescence intensity of phagocytic PMNL) assessed by flow cytometry. Antepartum (i.e., wk -3, -2, -1; before calving), plasma concentrations of IGF-I were high (80-110 ng/mL) without significant differences between primiparous and pluriparous cows (n=18 and n=41, respectively). Concentrations of IGF-I declined toward the week of calving (wk 1). Postpartum (i.e., wk 2, 3, and 4; after calving), IGF-I remained lower than before parturition, with concentrations higher in primiparous compared with those of pluriparous cows. The PA was constant in primiparous cows throughout the study period. Conversely, PMNL of pluriparous cows had a significantly increased and higher PA in wk 2 and 3 postpartum compared with that of primiparous cows. The PC decreased significantly only in primiparous cows the week of calving, whereas the number of PMNL in primiparous cows exceeded that of pluriparous cows significantly. The phagocytic power (PP, a product of PA by PC), but not the phagocytic overall performance (POP, a product of PA, PC, and PMNL number), differed between primiparous and pluriparous cows in wk 3 postpartum. No significant differences in POP were found, except in wk 4 after calving between the primi- and pluriparous cows. In both groups, POP increased in the week of calving (wk 1). In contrast to β-hydroxybutyrate, which was weakly positive correlated with PA and PP in pluriparous cows in the transition period (wk -3 antepartum to wk 4 postpartum), pluriparous animals had weak negative correlations of PMNL number, PA, PP, POP, and IGF-I concentration in this period. In primiparous animals, only PP and PC were weakly negatively correlated with IGF-I in the transition period. Increased plasma IGF-I concentrations were not associated with enhanced phagocytosis function of bovine blood PMNL ex vivo and, thus, can not be regarded as a suitable predictor for this function.

The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples each). The pregnancy rate per oestrus cycle (PRC) served as an estimation of fertility. An adverse effect on stallion fertility caused by low dietary selenium intake was excluded, as all stallions had sufficient selenium levels in their blood. Interestingly, no significant correlations (P > 0.05) between the selenium level in blood and the selenium level in seminal plasma or spermatozoa were found, suggesting that the selenium level in blood is no indicator of an adequate selenium supply for spermatogenesis. The selenium level in spermatozoa (nmol billion–1) was correlated with PMI, PMS and PAS (r = 0.40, r = 0.31 and r = –0.42, respectively; P ≤ 0.05), and the selenium concentration in spermatozoa (nmol g–1) was correlated with PRC (r = 0.40, P < 0.03). The results of the present study show that the determination of an adequate selenium status for the male equine reproduction requires the analysis of selenium in spermatozoa. Furthermore, selenium is associated with improved sperm quality and fertility in the stallion.

The aim of this study was to use computed tomography for the evaluation of the suitability of external pelvimetry to determine obstetrically relevant parameters. External pelvimetric measurements obtained in vivo using calipers and internal pelvimetric measurements obtained in vitro using computed tomography were taken in 30 German Holstein-Friesian cows (Bos taurus). All measurements were highly reproducible with intraclass correlation coefficientsor=98%. Hip width was the external variable with the highest correlation to internal variables, particularly pelvic inlet area and circumference, pelvic volume, medial horizontal diameter of the pelvic inlet, and the narrowest horizontal diameter of the midpelvis (r(2)0.60, P0.0001). The pelvic inlet area and circumference, the pelvic volume, and the diagonal diameter of the pelvis were sufficiently predicted with the aid of external pelvic measurements and age (r(2)or=0.80, P0.0001). The results of this study show that external pelvimetry yields reliable information about the size of the pelvis when the age of the cow is considered.

Whether extraction of a calf in longitudinal anterior presentation should be carried out by simultaneous or alternate traction on the forelimbs remains controversial. Because most recommendations are based on empirical observations rather than on scientific studies, the aim of this study was to develop an in vitro model to objectively compare the forces occurring during alternate and simultaneous traction. In a biomechanical in vitro model, 12 dead Holstein-Friesian (Bos taurus) calves were pulled through the prepared pelvic specimen of a cow at a controlled speed using two electric motors. Traction was applied simultaneously (ST) to both legs or alternately (AT) to one leg at a time to advance the calf 5 cm (AT 5) or 10 cm (AT 10). Forces on each limb were measured digitally using load cells. In all cases, two peaks of maximum force occurred during the extraction of the cranial part of the body. The first peak was observed when the elbows were pulled into the pelvis, and the second peak occurred when the chest emerged from the pelvis. Up to and including entry of the elbows into the pelvis, the maximum force on a single limb (341+/-106 N) was lowest (P0.01) using AT10. The maximum traction forces acting on a single limb using AT5 (411+/-86 N) and ST (431+/-127 N) did not differ (P0.05). During extraction of the thorax, the maximum force acting on a single limb was lower (P0.0001) using ST (352+/-98 N) compared with AT5 (432+/-79 N) and AT10 (547+/-115 N). Based on these findings, alternate-limb traction, 10 cm at a time, should be used until both elbows have entered the pelvis. Simultaneous traction should then be applied to both forelimbs to complete extraction of the chest.

The development of Doppler processing extended the scope of sonographic imaging from an anatomical to a physiological basis. This technique became established as a clinical tool in human gynaecology. For example, it has been discussed that the implantation of an embryo is influenced by the uterine blood flow. In cows, this uterine blood flow was investigated, using surgically implanted Doppler ultrasonic or electromagnetic blood flow probes prior to the introduction of colour Doppler sonography in bovine medicine. Therefore, the aims of our studies were to use transrectal Doppler sonography for the non-invasive measurement of uterine and ovarian blood flow in cows and to determine changes in genital perfusion during the oestrous cycle, pregnancy and puerperium, respectively. The results of our studies show that transrectal flow imaging can be used to obtain blood flow velocity waveforms from the uterine arteries at any time during the oestrous cycle, pregnancy and puerperium. During all these phases, characteristic changes in the uterine blood flow could be observed. This uterine blood flow was low during diestrus and high during proestrus and oestrus. During pregnancy, an exponential rise in uterine blood supply could be detected. There was a positive relationship between the uterine blood flow volume (BFV) at the end of gestation and the birth weight of calves. During puerperium, the uterine BFV declined tremendously, especially during the first week after birth. In cows, with pathological disturbances of the pueperium a delayed decrease in the uterine BFV was observed. Characteristic alterations occurred also in the luteal blood flow during the oestrous cycle, which were highly related to those of the progesterone levels. Furthermore, it has been detected by the colour Doppler technique that there is no decrease, but an increase of the luteal blood flow at the beginning of luteolysis in cows. Another group has found that there are close relationships between the LH-surge and the follicular blood flow before ovulation. In conclusion, these studies show that transrectal colour Doppler sonography is a useful technique for the investigation of the genital blood flow and provides new information about physiological changes of the genital organs during, all reproductive phases. The influence of the genital blood flow on fertility in cows needs to be examined further in future studies.

BACKGROUND: The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring. METHODS: Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay. RESULTS: FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay. CONCLUSIONS: The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

Transrectal Doppler ultrasound was used for the noninvasive investigation of uterine blood flow in three cows during pregnancy. The uterine arteries ipsi and contralateral to the conceptus were scanned monthly. Blood flow was reflected by the following parameters: resistance index (RI), time-averaged maximum velocity (TAMV), diameter of the vessel (D) and the volume of blood flow (VOL). RI values were negatively correlated to all other blood flow parameters (P0.01). Positive correlations occurred between TAMV, D and VOL (P0.0001). While blood flow parameters did not differ between cows (P0.05), the month of gestation showed a positive effect on RI and negative effects on TAMV, D and VOL (P0.0001). The RI was lower and TAMV, D and VOL higher in the uterine artery ipsilateral to the conceptus (P0.05). RI values decreased continuously during the first 8 months of gestation and remained from then until birth at a relatively constant level. While TAMV increased especially in two-thirds of pregnancy, a relatively uniform rise of D was noticed. VOL increased exponentially with stage of gestation. The results show that transrectal Doppler sonography is a suitable, noninvasive method for the examination of uterine blood flow during pregnancy in cows. Using this technique it might be possible in the future to determine the role of uterine blood flow in cows at the risk of abortion.

The objective was to examine if there are relationships between alterations in sperm viability, reactive oxygen species (ROS) synthesis, and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 hours at 37 °C, and the other frozen, thawed, and incubated for 24 hours at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 hours of incubation. Progressive motile sperm was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm, sperm with a high mitochondrial membrane potential, sperm showing a high degree of DNA fragmentation (%DFI), and their reactive oxygen species content were assessed with dichlorofluorescein-diacetate, dihydrorhodamine, diaminofluorescein diacetate, and mitochondrial superoxide indicator using flow cytometry. Although all other sperm parameters showed alterations (P 0.05) during the 24-hour incubation time, %DFI stayed constant (P 0.05, 0.91 ± 0.23) in nonfrozen sperm. Cryopreservation induced changes of all sperm parameters (P 0.05). In contrast to all other sperm parameters, dichlorofluorescein-diacetate-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2 but not to sperm viability and synthesis of other reactive oxygen species.