Genital route in experimental infection, a promising approach to study genital leptospirosis in ruminants.

Infection by Leptospira sp., mainly strains from the Sejroe serogroup, impairs the reproductive efficiency of ruminants leading to economic losses. Although the majority of experimental studies use the intraperitoneal route of leptospiral infection, it has been suggested that natural infection occurs frequently by sexual transmission. Thus, we assessed the genital route of infection to study genital leptospirosis in the sheep model. A strain of L. borgpetersenii serogroup Sejroe, serovar Hardjobovis was inoculated in 18 ewes, divided into three groups for inoculation: intraperitoneal (n=6; Gip), cervical superficial (genital) (n=6; Ggen) and conjunctival (n=6; Gconj). Monthly, for 90 days, blood samples were collected for serology (MAT) and PCR was performed on urine, cervical-vaginal mucus, and uterine fragments. All ewes were successfully infected, independently of the infection route. Gip and Ggen did not differ throughout the experiment, either on seroconversion or on PCR positivity on urine or genital samples. In contrast, Gconj presented fewer seroreactive animals (P<0.05) and fewer PCR-pos on genital samples than the other groups. The results obtained demonstrated that, although all groups presented both urinary and genital infections, the genital route was more efficient and did not differ from the traditional intraperitoneal. It indicates that genital via, besides being a naturally occurring transmission via, represents a promising and interesting route regarding future studies related to genital leptospirosis in ruminants, and its use should be encouraged.

Proposal of an iELISA for Mycoplasma bovis diagnosis in dairy cattle and associated risk factors / [Proposta de um iELISA para diagnóstico de Mycoplasma bovis em bovinos leiteiros e fatores de risco associados]

Mycoplasma bovis is a highly contagious agent associated with several pathologies in cattle. The detection of reactive antibodies to M. bovis by Indirect Enzyme-Linked Immunosorbent Assay (iELISA) identifies if there was an exposure to the microorganism. The current study aimed to optimize an iELISA from M. bovis total cell antigen, applying it to bovine serum samples, and to evaluate risk factors. Serum samples were obtained from 400 cows from 17 herds from Southeast Brazil. In the optimization of iELISA, the following was established: 2 µg/mL of antigen, sera dilution 1:300, and conjugate dilution 1:15000. The frequency was 62.3% (249/400) of reactive animals and 100% (17/17) of reactive herds. Risk factors were: herds with more than 100 animals (OR= 3.1; CI= 95%); Holstein breed (OR= 72.5; CI= 95%); cows (OR= 29.7; CI= 95%); intensive breeding system (OR= 3.3; CI= 95%); associated small ruminant production (OR= 4.4; CI= 95%); milk production above 500L (OR= 2.9; CI= 95%); no quarantine (OR= 1.5; CI= 95%); mechanical milking (OR= 5.5; CI= 95%) and cases of mastitis (OR= 5.5; CI= 95%). The proposed iELISA was able to detect antibodies reactive to M. bovis in bovine serum. Knowledge of these risk factors can assist in the implementation of prophylactic measures.(AU)

Mycoplasma bovis é um agente altamente contagioso relacionado a várias patologias em bovinos. A detecção de anticorpos reativos a M. bovis por Ensaio de Imunoadsorção Enzimática Indireto (iELISA) identifica se houve exposição ao microrganismo. O presente estudo teve como objetivo otimizar um iELISA de antígeno celular total de M. bovis, aplicando-o a amostras de soro bovino, bem como avaliar fatores de risco. Amostras de soro foram obtidas de 400 vacas de 17 rebanhos da Região Sudeste do Brasil. Na otimização do iELISA foram obtidos: 2µg/mL de antígeno, diluição dos soros 1:300 e do conjugado 1:15000. A frequência de animais reativos foi de 62,3% (249/400) e de 100% (17/17) para os rebanhos. Os fatores de risco foram: rebanhos com mais de 100 animais (OR= 3,1; IC= 95%); raça Holandesa (OR= 72,5;IC= 95%); vacas (OR= 29,7;IC= 95%); sistema intensivo (OR= 3,3; C= 95%); produção de pequenos ruminantes (OR= 4,4;IC=95%); produção de leite acima de 500L (OR= 2,9;IC= 95%); sem quarentena (OR= 1,5;IC= 95%); ordenha mecânica (OR= 5,5;IC= 95%) e casos de mastite (OR= 5,5;IC= 95%). O iELISA proposto foi capaz de detectar anticorpos reativos a M. bovis no soro bovino. O conhecimento desses fatores de risco pode auxiliar na implementação de medidas profiláticas.(AU)

Mini-percoll gradient may be used for frozen-thawed sperm selection in sheep / Gradiente de mini-Percoll pode ser utilizado na seleção espermática de sêmen congelado de ovinos

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)

O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)

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.

Transrectal ultrasound evaluation in tropical dairy goats: an indispensable tool for the diagnosis of reproductive disorders.

In Brazil, great milk productivity was achieved after the implementation of a genetic improvement program. However, reproductive efficiency is still far from optimal, possibly due to the high number of undiagnosed disorders that may affect fertility. The aim of this study was to evaluate occurrences of the main reproductive disorders in dairy goats in southeastern Brazil. Data were collected between January 2015 and May 2017 from 23 commercial herds of different breeds, with goats ranging from 8 months to 12 years of age. Transrectal ultrasound exams were performed in 2680 goats. A total of 14.8% of the does showed a disorder in the reproductive tract: hydrometra (10.0%), ovarian follicular cysts (2.3%), gestational loss (1.5%), and hydrosalpinx (1.1%). This was the first study evaluating reproductive disorders in live animals that used a high number of Brazilian dairy goats. Considering that all these diseases affect fertility to different degrees, the performance of transrectal ultrasonography exams twice a year is strongly suggested, in order to guide precocious treatment or discard the animal as soon as possible, thus reducing economic losses in dairy goat farming.

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.

Potential role for GnRH in the synchronization of follicular emergence before the superovulatory Day 0 protocol.

The ability of gonadotropin-releasing hormone (GnRH) to synchronize ovulation and new follicular wave emergence before a "superovulatory Day 0" protocol was assessed in Santa Inês ewes. For estrus synchronization, a 60-mg medroxyprogesterone acetate sponge was inserted for 6 d. One day before sponge removal, 37.5-µg d-cloprostenol and 300 IU equine chorionic gonadotropin were injected intramuscularly (i.m.). After sponge removal, ewes were assigned to the following 3 groups: (1) GC-1 mL saline at 12 h (n = 10); (2) G24h-0.025-mg lecirelin (GnRH agonist) i.m. at 24 h (n = 10); or (3) G36h-0.025-mg lecirelin i.m. at 36 h (n = 9). Ovarian ultrasonography was conducted to assess follicular dynamics. Blood was collected to determine plasma concentrations of progesterone and estradiol. Females from G36h and GC had a greater (P < 0.05) estrous response than those from the G24h group (78.0 and 90.0 vs 0.0%, respectively). Ewes from G24h and G36h had earlier (P < 0.05) ovulation (48.0 ± 10.2 and 56.7 ± 5.7 h) compared with those from Gc (64.1 ± 9.7 h). The mean number of ovulations per ewe was greater (P < 0.05) in Gc (1.9 ± 0.6) and G36h (2.0 ± 1.0) than G24h (1.2 ± 0.4). Plasma concentrations of progesterone and estradiol differed over time. Follicular growth during the postovulatory day was affected (P < 0.05) by day of the estrus cycle as well as by the interaction (P < 0.05) of treatment and day of the estrus cycle. There was a larger (P < 0.05) population of medium follicles during the first 24 h after the ovulation in G24h compared with Gc, and there was an absence of large follicles in G36h between 36 and 72 h after ovulation. In conclusion, the use of GnRH agonist at 36 h more efficiently synchronized ovulation and promoted the absence of dominant follicles during early diestrus and may be used at the start of superovulatory treatment at 80 h in Santa Inês ewes.