M. F. A. Balaro

Outbreak of Bluetongue virus serotype 4 in dairy sheep in Rio de Janeiro, Brazil

In late January 2013, 10 nonpregnant Lacaune dairy ewes raised under extensive husbandry management on a farm in Rio de Janeiro, Brazil, presented with the general clinical signs of lethargy, hyporexia, edema of the face, hyperemia of the exposed parts of the skin, mouth lesions, pyrexia, and lameness. Additionally, 2 pregnant ewes died suddenly after the onset of respiratory signs. The complete blood counts and biochemistry analyses showed neutrophilic leukocytosis with monocytosis and reactive lymphocytes, normocytic normochromic anemia and increased aspartate aminotransferase levels. Postmortem examination revealed erosions on the lingual mucosa, bilateral submandibular ganglia infarctions, yellow foamy fluid accumulation in the trachea and bronchial bifurcation, pulmonary congestion, and edema associated with hemorrhagic lesions on the pulmonary artery and heart. The clinical and pathological findings were suggestive of bluetongue. For a molecular and virological diagnosis, tissue samples were analyzed by Bluetongue virus–specific real-time reverse transcription polymerase chain reaction (qRT-PCR), and viral isolation was performed in embryonated chicken eggs. For viral typing, positive tissue and egg-isolated samples were analyzed by qRT-PCR using primers and probes specific for the structural VP2 gene in genome segment 2 of all 26 serotypes. There are still no contingency plans for responding to an outbreak of bluetongue disease in Brazil, and this episode emphasizes the need for continuing serological and entomological surveillance programs. Additionally, this report describes the isolation of Bluetongue virus serotype 4 in sheep in the Americas.

Effect of natural mating or laparoscopic artificial insemination in superovulated Santa Inês ewes on superovulatory response, fertility and embryo viability

This study evaluated the effect of two mating methods (GNM: natural mating or GAI: laparoscopic artificial insemination) on superovulatory response, fertility and embryo yield in superovulated ewes. Fifteen non-pregnant Santa Ines ewes were superovulated and either mated by GNM or GAI in a crossover design. Oestrus was synchronised using intravaginal progestagen sponges for 6 days and on Day 5, 300 IU eCG and 0.0375 mg d-cloprostenol were given. Twelve hours after sponge removal, 0.025 mg gonadotropin-releasing hormone was administered. Superovulation started 48 h after gonadotropin-releasing hormone treatment, using 5 IU/kg follicle-stimulating hormone (pFSH). At the first pFSH dose, new sponges were inserted. At the fifth dose, 0.0375 mg cloprostenol was administered and the sponges were removed. The GNM was mated with rams every 12 h, until the end of oestrus. The ewes of GAI were laparoscopic inseminated with frozen–thawed semen 36 and 48 h after sponge removal. Ultrasonography was performed every 24 h from the beginning of oestrus synchronisation treatment and every 12 h from the second sponge removal to 2 days after the last pFSH dose. Six to seven days after mating, the number of corpora lutea (CL) was evaluated by laparoscopy and the females with > 4 CL were subjected to embryo collection. The interval from sponge removal to ovulation was shorter (P 0.05). The number of recovered structures (6.4 ± 2.4 vs 4.5 ± 3.0), recovery rate (74.0 ± 16.0 vs 52.3 ± 26.5%), number of transferable embryos (3.0 ± 2.9 vs 3.6 ± 2.0) and viability rate (47.2 ± 45.3 vs 77.4 ± 37.1%) did not differ between GAI and GNM (P > 0.05). However, the GAI group showed a higher (P < 0.05) number of unfertilised oocytes (3.1 ± 3.1) and a higher non-fertilisation rate (47.1 ± 45.3%) than the GNM (0.9 ± 2.1 and 11.5 ± 21.5%). The mating method did not affect the superovulatory response, and production of viable embryos although the non-fertilisation rate has been inferior for the AI group.

Reproductive parameters of dairy goats after receiving two doses of d-cloprostenol at different intervals

This study evaluated the efficiency of two d-cloprostenol injections at different intervals on the reproductive parameters of dairy goats. Trial 1 comprised 54 goats allocated to receive two 37.5μg d-cloprostenol doses at intervals of seven (T7, n=19), 10 (T10, n=18), and 11.5 (T11.5, n=17) days. Trial 2 comprised 62 goats allocated to receive injections at T7 (n=30) and T11.5 (n=32). Ultrasonography was done and blood was collected just before d-cloprostenol injections. After the second dose, goats were artificially inseminated (AI) with frozen-thawed semen at 18-24h (Trial 1) or at 10-24h (adjusted according to the time of estrus onset in Trial 2) after estrus detection. Estrus response rate did not differ (P>0.05) among groups in Trials 1 (T7=94.7%; T10=88.9%; T11.5=88.2%) and 2 (T7=90.0%; T11.5=96.9). All females showed progesterone concentrations >1ng/mL before both d-cloprostenol injections. The largest follicle diameter present on ovaries was similar (P>0.05) among treatments at the first and second dose. The second largest follicle diameter was superior (P<0.05) to T7 than to T10 and T11.5 goats at first dose only. This possibly resulted in lower interval to estrus (P<0.05) in T7-treated goats than other treated goats in both trials. The conception rate was similar among treatment groups in Trials 1 (T7=55.6%; T10=18.8%; T11.5=26.7%) and 2 (T7=85.2%; T11.5=93.6%). The three treatments efficiently synchronized estrus. T7 and T11.5 protocols resulted in high estrus synchrony and conception rates when adjusting the AI time according to interval of estrus.

Accuracy of assessment of luteal morphology and luteal blood flow for prediction of early pregnancy in goats

This study aims to determine the best post-breeding time for an early pregnancy diagnosis in dairy goats, when using luteal morphology and vascularization assessment by B-Mode and color Doppler ultrasonography (US) in association or not with the anechoic uterine content. A total of 131 Saanen goats (2.0 ± 0.5 years old) were used in the two studies. In the first study, pregnancy prediction was daily performed from Day 15-23 post-breeding in 51 does. This was based on a subjective assessment of luteal morphology (B-Mode US), luteal blood flow (color Doppler US), and the presence of anechoic uterine content (B-Mode US). In the second study, pregnancy predictions were performed in 71 does on the best post-breeding day, as determined in the first study (Day 21 using luteal blood-flow assessment and Day 23 using luteal morphological assessment and overall analysis). In both studies, pregnancy diagnosis was confirmed on Day 30 (gold standard method by B-Mode transrectal ultrasonography). The B-Mode and color Doppler US performance in reaching an early pregnancy diagnosis was evaluated by calculating sensitivity (Sens), specificity (Spec), negative and positive predictive values (NPV and PPV), accuracy (Acc), the Kappa index (κ), and the Younden index (J). In Study 1, the pregnancy prediction accuracy progressively increased from Day 17-23 using B-Mode US (D17: 50.98%; D18: 52.94%; D19: 62.75%; D20: 74.51%; D21: 86.27%; D22: 90.20%; D23: 96.08%), and from Day 17-21 using color Doppler US (D17: 49.02%; D18: 54.90%; D19: 70.59%; D20: 86.27%; D21: 96.08%). In the second study, color Doppler, B-Mode, and overall assessments at Day 21 and Day 23, respectively, presented a similar pattern of sensitivity, specificity, and accuracy as was found in the first study. Therefore, both color Doppler and B-Mode assessments can be judged as effective tools for reaching a pregnancy diagnosis in goats as early as on Day 21 and Day 23, respectively, post-breeding.

Hydrometra in dairy goats: Ultrasonic variables and therapeutic protocols evaluated during the reproductive season

Hydrometra is characterized by the accumulation of fluid within the uterus due to the persistence of corpus luteum. The diagnosis of this disorder occurs with an ultrasonic exam. This study evaluated uterine drainage and fertility rates in goats after the use of d-cloprostenol in association or not with Gonadotropin-releasing hormone (GnRH) treatment. Twenty Saanen goats, diagnosed with hydrometra, received three 37.5-μg doses of d-cloprostenol laterovulvarly at 10-day intervals. On D5, the goats were assigned into two groups receiving 1 mL of GnRH or saline solution intramuscularly. Ultrasonography (US) was performed from D0 to D25. An US approach was used to rank hydrometra in scores. The pregnancy rate was assessed 45 and 90 days after the end of treatment. The uterine fluid was totally drained after the first and second administration of d-cloprostenol in 50% and 95% of the goats, respectively. In one female, full emptying of the uterus occurred only after D20. US performed at 45 and 90 days after the end of treatment indicated there was a pregnancy rate of 45.0% and 55.0%, respectively. Fertility did not differ between the GnRH-treated and control goats. Those goats not pregnant at 45 days had a follicular cyst, hydrosalpinx or hydrometra. At 90 days, no change was observed in the hydrosalpinx, and four goats had hydrometra. The use of three doses of d-cloprostenol 10 days apart was efficient for induction of draining the contents of the uterus, resulting in a relatively acceptable pregnancy rate. This treatment associated with the US approach can be important when applied in the field.

Comparison of different sperm selection techniques in ram frozen-thawed sperm.

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation. Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Ines rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37°C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05), regardless of interval incubation. However, the latter three techniques presented more (P 0.05). Discussion: The present study evaluated the sperm cell and its preparation for receiving the oocyte under optimal conditions. evaluate the sperm cell and its preparation for receiving the oocyte under optimal conditions. Although the swim-up technique promoted higher rates for some of the sperm parameters evaluated, Percoll protocols are the most widely used procedures for selection during the sperm preparation in many species, possibly because of its greater speed, practical method and convenience compared to the swim-up technique. Percoll and mini-Percoll recovered approximately 10 times more cells than swim-up, which is an important feature to be considered during sperm preparation for in vitro fertilization (IVF), being possible to use only one semen straw. The high capacitated and acrosome reaction rates observed after the treatments in the current study, are probably reinforced by changes in sperm cells caused by the cryopreservation process. In order to strength the evidence that frozen-thawed sperm, even after selection, is sensible and reactive to capacitation-like events, we demonstrated the capacitated and acrosome-reacted cell values immediately after the selection treatments behaved differently than when authors used ram fresh sperm. Possibly, this capacitation-like changes observed in frozen-thawed sperm occurs regardless of the selection treatment used. Analyzing the motility parameters immediately after the selection, all treatments maintained or increased the rates compared to the control group. The swim-up, mini-Percoll and Percoll did not differ in any parameters. Given that Percoll and mini-Percoll did not show differences in relation to swim-up for motility parameters, such techniques can be used to replace the latter, obtaining similar sperm samples with good quality. However, swim-up technique involves a procedure that recovers a clean fraction without debris and other types of cells, with high rate of mobile sperm with excellent quality, reason why it can justify the higher recovery of intact spermatozoa after the technique.