Seasonal variation in equine follicular fluid proteome.

BACKGROUND: Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth. METHODS: This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory networks and associated key proteins. RESULTS: Regardless of season, a total of 90 proteins were identified in FF, corresponding to 63, 72, 69, and 78 proteins detected in the SAN, SOV, SUM, and FOV seasons, respectively. Fifty-two proteins were common to all seasons, a total of 13 were unique to either season, and 25 were shared between two seasons or more. Protein-to-protein interaction (PPI) analysis indicated the likely critical roles of plasminogen in the SAN season, the prothrombin/plasminogen combination in SUM, and plasminogen/complement C3 in both SOV and FOV seasons. The apolipoprotein A1 appeared crucial in all seasons. The present findings show that FF proteome of SUM differs from other seasons, with FF having high fluidity (low viscosity). CONCLUSIONS: The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM) and may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season.

Early pregnancy diagnosis at 21 days post artificial insemination using corpus luteum vascular perfusion compared to corpus luteum diameter and/or echogenicity in Nelore heifers.

The objective was to compare the use of corpus luteum (CL) vascular perfusion to CL diameter and/or echogenicity to diagnose pregnancy at 21 d after timed-AI. Ovaries of Nelore heifers were assessed using ultrasonography in B-mode and color Doppler simultaneously 21 d after timed-AI (n = 113). Objective evaluations were performed using an image processing software to extract the number of colored pixels (ColorPix), diameter (mm) and echogenicity/mm² (EchoPix) of the CL. Subjective evaluations of the CL were performed by five evaluators using scores of estimated vascular perfusion area of color Doppler scan videos and estimated CL size and qualitative echogenicity of B-mode scan videos. The reference pregnancy diagnosis was performed 33 d after timed-AI using an ultrasonic device. Corpus luteum ColorPix, diameter and EchoPix were highly correlated (P < 0.001) with pregnancy. Pregnancy diagnosis accuracy, sensitivity, and negative predictive value were not different for CL ColorPix and diameter and was less with use of EchoPix compared to the other parameters. Size and vascular perfusion scores were correlated to the greatest extent (0.88-0.94) with the respective objective values within evaluator. The results from the ROC curve analysis indicated a smaller area under the curve for qualitative echogenicity compared to CL size and vascular perfusion. Corpus luteum vascular perfusion was the only subjective evaluation that when assessed there were no false negative pregnancy diagnoses. In conclusion, the use of the objective CL diameter resulted in the same efficacy as CL vascular perfusion evaluations for early pregnancy diagnosis in Nelore heifers.

Reproductive system development in male and female horse embryos and fetuses: Gonadal hyperplasia revisited.

In horses, pregnancy is characterized by high levels of maternal estrogens that are produced largely by the interstitial tissue inside the gonads of the offspring, associated with a physiological gonadal hyperplasia, that is uncommon in other species. However, a detailed structural-functional understanding of the early stages of gonadal development and hyperplasia has remained elusive in horse pregnancy because of the lack of substantial data. The goal of this study was to describe the genital organs' development in 19 early horse embryos and fetuses (days 20-140 of gestation) of both sexes by means of anatomy, histology, stereology, and immunohistochemistry, with a specific focus on gonadal hyperplasia and interstitial tissue development. Gonadal hyperplasia with similar amounts of interstitial cells was observed in both sexes, but only during the early stage of development (days 40-90). Surprisingly, a higher degree of hyperplasia, characterized by larger amounts of interstitial cell-rich areas, was seen in fetal ovaries from 90 days of gestation onwards. Another novel aspect was that parallel to the hyperplasia of the interstitial cells, a much more precocious and pronounced differentiation of germinal cells was seen in the ovary, characterized by an earlier peak and decrease of DAZL and OCT protein immune markers. In conclusion, a reduced degree of hyperplasia and interstitial tissue in the fetal testis after 90 days of gestation suggests the existence of a more efficient mechanism regarding the synthesis of estrogen precursors as a structural or physiological difference between both fetal sexes, which warrants further investigation.

Ultrassonografia testicular no modo color Doppler∕ espectral e avaliação andrológica de garanhões tratados com gonadotrofina coriônica humana (hCG) em diferentes estações do ano / [Testicular ultrasonography in the doppler / spectral color mode and andrological evaluation of stallions treated with human corionic gonadotrofin (hCG) in different seasons of the year]

Utilizaram-se quatro garanhões nos meses de janeiro, abril, julho e outubro de 2016, em dois protocolos, em que: GI (n=4; 5mL de solução salina, i.v.) e GII (n=4; 5000UI de hCG, i.v.) e subdividiram-se esses protocolos em ciclos (C1 e C2), seguindo o esquema crossover, sendo: CI=animais 1 (GI) e 2 (GII), avaliados nos dias D1, D3 e D5, e animais 3 (GI) e 4 (GII), em D2, D4 e D6; CII= animais 1 (GII) e 2 (GI), em D1, D3, D5, e animais 3 (GII) e 4 (GI), em D2, D4 e D6. Realizou-se o tratamento em D1 e D2 de cada ciclo e a ultrassonografia testicular no modo color Doppler e Doppler espectral, uma hora antes de cada coleta de sêmen e imediatamente após. Avaliou-se: número de reflexo de Flehmen, de montas sem ereção, início da monta, tempo de reação à ereção e total da monta e análises de qualidade seminal. Estatisticamente foram utilizados os testes de qui-quadrado e ANOVA. Não houve diferenças estatísticas (P>0,05) entre os parâmetros analisados. Concluiu-se que uma única dose de 5000UI de hCG em garanhões não causou alterações significativas nos parâmetros avaliados em diferentes estações do ano.(AU)

Four stallions were used in January, April, July and October 2016 in two protocols: GI (n=4; 5ml saline, iv) and GII (n=4; 5000 hCG, iv), and these protocols were subdivided into cycles (C1 and C2) following the cross over scheme, as follows: CI=animal 1 (GI) and 2 (GII) evaluated on days D1, D3 and D5 and animal 3 (GI) and 4 (GII) at D 2, D 4 and D 6; CII=animal 1 (GII) and 2 (GI) at D1, D3, D5 and animal 3 (GII) and 4 (GI) at D2, D4 and D6. Treatment was performed on D1 and D2 of each cycle and testicular ultrasound in color Doppler and spectral Doppler mode, one hour before each semen collection and immediately after. We evaluated: Flehmen's reflex number, mounts without erection, start of the mount, reaction time to erection and total mount and seminal quality analyzes. Statistically, the Chi-square and ANOVA tests were used. There were no statistical differences (P>0.05) between the analyzed parameters. It was concluded that a single dose of 5000IU hCG in stallions did not cause significant changes in the parameters evaluated in different seasons of the year.(AU)

Acute endometritis induced by Escherichia coli in mares evaluated through color doppler ultrasonography / [Endometrite aguda induzida por Escherichia coli em éguas avaliadas por ultrassonografia com Doppler colorido]

The objectives of this study were to characterize the endometritis induced in mares using color Doppler ultrasonography and traditional exams. Experiment 1. Mares (n=20) were submitted to intrauterine inoculation with Escherichia coli. Uterine evaluation was performed at M0 and M1. Experiment 2. Animals were divided into two groups: control group (n=10), and treated group (n=10) using phytotherapeutic solution. In both groups, the uterine evaluation was performed at time T1, T2, and T3. Experiment 3: Uterine evaluation was compared after antibiotic therapy, phytotherapy, and M0. For statistical analysis, the Tukey test, t Student, and Anova test were applied. Experiment 1. The mean values of vascularization at M1 were significantly higher than those obtained at M0 (P<0.05). Bacterial growth was observed in all samples collected. Experiment 2. The mean value of vascularization at time T1 in both groups was significantly higher (P<0.05) compared to M2 and M3. Experiment 3. After antibiotic therapy, the vascularization of the body and uterine horns was not equivalent to the vascularization presented at M0. We can conclude that it was not possible to correlate results obtained by color Doppler ultrasonography with the traditional findings for the diagnosis of endometritis.(AU)

Os objetivos deste estudo foram caracterizar a endometrite induzida em éguas utilizando-se a ultrassonografia com Doppler colorido e exames tradicionais. Experimento 1: as éguas (n=20) foram submetidas à inoculação intrauterina com Escherichia coli. A avaliação uterina foi realizada em M0 e M1. Experimento 2: os animais foram divididos em dois grupos: grupo controle (n=10) e grupo tratado (n=10), sendo usada solução fitoterápica. Nos dois grupos, a avaliação uterina ocorreu nos momentos T1, T2 e T3. Experimento 3: a avaliação uterina foi comparada após antibioticoterapia, fitoterapia e M0. Para análise estatística, foram aplicados os testes de Tukey, t de Student e ANOVA. Experimento 1: os valores médios de vascularização em M1 foram significativamente maiores que os obtidos no M0 (P<0,05). Houve crescimento bacteriano em todas as amostras coletadas. Experimento 2: o valor médio da vascularização no tempo T1 nos dois grupos foi significativamente maior (P<0,05) do que o obtido em M2 e M3. Experimento 3: após antibioticoterapia, a vascularização do corpo e dos cornos uterinos não era equivalente à vascularização apresentada em M0. Pode-se concluir que não foi possível correlacionar os resultados obtidos pela ultrassonografia com Doppler colorido com os achados tradicionais para o diagnóstico de endometrite.(AU)

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program.

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to -6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), -1 (68%), -2 (63%), -3 (66%), -4 (76%), -5 (61%), and -6 (27%). The -6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to -5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.

Use of different pressures for transvaginal follicular aspiration in mares / Uso de diferentes pressões para aspiração folicular transvaginal em éguas

The success of transvaginal follicular aspiration in mares can be influenced by several factors, such as vacuum pump pressure levels. The present study aimed to investigate the effect of different negative pressures (150, 280 and 400mmHg) of the vacuum pump on the oocyte recovery in the mares. The mares (n=10) were undergoing follicular aspiration using three different negative pressures for three consecutive estrous cycles as follows: G150 = 150mmHg (n = 10); G280 = 280mmHg (n = 10); G400 = 400mmHg (n = 10). Every estrous cycle, the group that the mare would participate was drawn, and each animal participated once in each group. Only preovulatory follicle was used, about 30 to 36 hours after application of hCG. To compare the results, the chi-square test was used (5% significance) and Fisher exact test, when recommended. Thirty preovulatory follicles (diameter 36.1±1.80mm) were aspirated and ten oocytes were recovered (33.3%). There was no statistical difference between the experimental groups (p=0.59). Thus, accord to the results observed in this study, we could conclude that the negative pressure of the vacuum pump used was not efficient to increase oocyte recovery.(AU)

O sucesso da técnica de aspiração folicular transvaginal em éguas pode ser influenciado de maneira determinante por diversos fatores, tais como níveis de pressão da bomba de vácuo. Diante disso, o presente experimento visou investigar o efeito de diferentes pressões negativas (150, 280 e 400mmHg) da bomba de vácuo sobre a taxa de recuperação de oócitos em éguas. As éguas (n=10) foram submetidas à aspiração folicular utilizando-se três diferentes pressões negativas por três ciclos estrais consecutivos, da seguinte maneira: G150= 150mmHg (n=10); G280= 280mmHg (n=10); G400= 400mmHg (n=10). A cada ciclo estral, sorteava-se o grupo do qual a égua participaria, sendo que cada animal integrou um grupo somente uma vez. Foi puncionado somente folículo pré-ovulatório, em torno de 30 a 36 horas após a aplicação do hCG. Os resultados foram comparados utilizando-se o teste qui-quadrado (a 5% de significância) e o Fisher Exato, quando recomendados. Foram aspirados 30 folículos pré-ovulatórios (diâmetro 36,1±1,80mm) e recuperados 10 oócitos (33,3%). Não houve diferença estatística entre os grupos experimentais (P=0,59). Dessa forma, mediante os resultados obtidos no presente estudo, foi possível concluir que a pressão negativa da bomba de vácuo utilizada não se mostrou determinante para elevar a recuperação oocitária.(AU)

Efeito do cloprostenol (ICI-80.996) sobre a involuçäo uterina em vacas mestiças Holandês-Zebu / Effect of cloprostenol (ICI-80.996) on the uterine involution in crossbred cows

Setenta vacas leiteiras mestiças Holandês-Zebu com 71,4 meses de idade e 3,3 lactaçöes, foram divididas em sete grupos de dez animais cada. Um grupo serviu como testemunha (G-I) e nos outros seis aplicou-se 500µg de cloprostenol (ICI-80.996), via intramuscular, em diferentes períodos pós-parto (PP). A involuçäo uterina (IU) foi acompanhada por palpaçäo retal do sexto ao 60§ dia PP, com intervalo de três dias. O período médio de IU para o G-I e para os grupos que receberam o cloprostenol no sexto dia PP (G-II), no 12§ dia (G-III), 18§ dia (G-IV), no 6§ e 12§ dia (G-V), 12§ e 18§ dia (G-VI) e no 6§, 12§ e 18§ dias (G-VII) foram, respectivamente: 31,5, 29,1, 29,1, 24,6, 29,4, 24,0 e 25,2 dias. O período de involuçäo clínica do útero foi significativamente menor (P<0,05) no G-IV, G-VI e G-VII. Recomendar esta terapêutica como rotina, entretanto, requer estudo dos aspectos econômicos