Genetic diversity in oocyte donors used in in vitro bovine embryo production programs in Brazil / Diversidad genética en donadoras de ovocitos utilizadas en programas de producción de embriones bovinos in vitro en Brasil / Diversidade genética em doadoras de oócitos utilizadas em programas de produção de embriões bovinos in vitro no Brasil

Abstract Background: Current reproductive management of bovine elite populations involves the use of assisted reproductive technologies (ARTs), aiming to obtain the greatest genetic gain. However, inadequate use of ARTs may lead to loss of genetic diversity in the offspring. Objective: To assess the genetic diversity in elite female cattle populations used in commercial in vitro embryo production. Methods: Using genetic and ecological approaches for the study of populations based on microsatellite markers, we assessed the genetic diversity between and within populations of cows used in commercial in vitro embryo production programs in Brazil. Results: Endogamy within populations varied from zero to 9.1%, while heterozygosity between populations (FST) was <0.05 in the different population interactions. AMOVA showed 1% variation between populations, 8% between individuals and 91% within individuals. The dimensionality reduction method utilized indicated a lack of structure in the populations analyzed, identifying two main clusters in the three populations. Conclusions: Low genetic diversity between cow populations associated with commercial programs of in vitro embryo production in Brazil was evidenced. Variable levels of endogamy within the populations were observed. Approaches of population genetics as well as ecological diversity can be implemented to more thoroughly estimate genetic diversity in livestock populations.

Resumen Antecedentes: El actual manejo reproductivo en poblaciones de bovinos de élite incluye la utilización de tecnologías de reproducción asistida (ARTs) con el fin de obtener mayor ganancia genética. Sin embargo, el uso inadecuado de las ART puede llevar a la pérdida de diversidad genética en los descendientes. Objetivo: Evaluar la diversidad genética en poblaciones de vacas de élite utilizadas en la producción comercial de embriones bovinos in vitro. Métodos: Utilizando abordajes de la genética y ecología de poblaciones basados en marcadores microsatélites, evaluamos la diversidad genética entre y dentro de poblaciones de vacas participantes de programas comerciales de producción de embriones in vitro en Brasil. Resultados: La endogamia dentro de las poblaciones varió de cero a 9,1%, mientras que la heterocigosidad entre poblaciones (FST) fue <0,05 en las diferentes interacciones de la población. El AMOVA mostró variación del 1% entre poblaciones, 8% entre individuos y 91% dentro de individuos. El método de reducción de dimensionalidad utilizado indicó una falta de estructura en las poblaciones analizadas, identificando dos grupos principales en las tres poblaciones. Conclusiones: Se evidenció una baja diversidad genética entre las poblaciones de vacas asociadas a programas comerciales de producción de embriones in vitro en Brasil. Se evidenciaron niveles variables de endogamia entre las poblaciones. Abordajes de la genética poblacional, así como de diversidad ecológica pueden ser implementados para estimar de manera más amplia la diversidad genética en poblaciones animales de interés pecuario.

Resumo Antecedentes: O atual manejo reprodutivo das populações de elite em bovinos envolve o uso de tecnologias de reprodução assistida (ARTs), visando obter o maior ganho genético. No entanto, o uso inadequado de ARTs pode levar à perda de diversidade genética na prole. Objetivo: Avaliar a diversidade genética em populações de vacas de elite utilizadas na produção comercial de embriões bovinos in vitro. Métodos: Utilizando abordagens da genética e ecologia de populações baseadas em marcadores microssatélites, foi avaliada a diversidade genética entre e dentro das populações de vacas participantes de programas comercias de produção in vitro de embriões. Resultados: A endogamia dentro das populações variou de zero a 9,1%, enquanto a heterozigosidade entre populações (FST) foi <0,05 nas diferentes interações populacionais. AMOVA mostrou variação de 1% entre populações, 8% entre indivíduos e 91% dentro de indivíduos. O método de redução de dimensionalidade utilizado indicou uma falta de estrutura nas populações analisadas, identificando dois clusters principais nas três populações. Conclusões: Baixa diversidade genética entre populações de vacas associadas a programas de produção in vitro de embriões foi evidenciada. Níveis de endogamia variáveis dentro das populações foram observados. Abordagens da genética populacional assim como de diversidade ecológica podem ser implementadas na tentativa de estimar de maneira mais abrangente a diversidade genética em populações animais de interesse pecuário.

Variants in the CYP19A1 gene can affect in vitro embryo production traits in cattle.

PURPOSE: This study aimed to associate DNA variants in promoter and exon flanking regions of the CYP19A1 gene with in vitro embryo production traits in cattle. The role of transcription factor binding sites created or lost due to DNA sequence variation and their possible effect on gene expression was also evaluated. METHODS: We collected date from Gyr dairy oocyte donor cows (Bos taurus indicus) at a commercial in vitro embryo production farm and analyzed the genotype-phenotype association with in vitro production traits. Using Sanger sequencing and web-based software, we assessed important CYP19A1 gene regions in oocyte donor cows and analyzed the effects of variants on the transcription factor binding sites. RESULTS: Two SNP mutations significantly associated with oocyte production, oocyte viability, embryo development, and pregnancies were found (T > C in the untranslated exon 1 flanking region ([GenBank: AJ250379.1]: rs718446508 T > C), and a T > C in the 5'-upstream region (1.1 promoter) ([GenBank: AC_000167.1]: rs41651668 T > C). Six new transcription factor binding sites were created. A binding site for transcription factors associated with the development of the placenta and embryo implantation was eliminated due to variations in the DNA sequence identified. CONCLUSIONS: The CYP19A1 gene contributes to genetic variation of in vitro embryo production traits in cattle. The complexity of the physiological phenomena related to estrogen pathways and their influence on reproduction in cattle allow indication of the mutations evaluated here as possible genetic markers for embryo production traits, which should be validated in the next steps of marker-assisted selection.

Surface temperature of ewes during estrous cycle measured by infrared thermography.

The use of infrared thermography has not been used previously to assist sheep reproduction. The aim of this study was evaluate the viability of infrared thermography to identify body surface temperature patterns during ewes' estrous cycle. The estrous cycles of 20 Santa Ines ewes were synchronized through hormone treatment (progesterone implant). The ewes were submitted to measurements of body temperature (rectal and vaginal) with a digital thermometer and surface temperature of the anus, vulva, muzzle, left ear and left eye by infrared thermography. Temperature and humidity of the environment and wet-bulb globe temperature (WBGT) were measured. After the progesterone implant's removal, the follicular dynamic was evaluated daily during five days with ultrasound. The estrous cycle was separated into six phases to facilitate interpretation of the differences in body temperatures in each period. Phase 1 was the period before estrous synchronization, phase 2 was the beginning of estrous synchronization, phase 3 was the intermediate period and end of synchronization, phase 4 was the period of estrus manifestations, phase 5 was the ovulation period, and phase 6 was the post-ovulatory period. There was a difference among phases for all body regions evaluated (P < 0.05). All temperatures were higher in the ovulation phase. The rectal and vaginal temperatures measured by digital thermometer were lower during the diestrus period and increased during and after ovulation. The surface temperature of the vulva, measured by thermography, was higher from the estrus phase (4) to the ovulation and post-ovulation phases (5 and 6). The surface temperature of the muzzle was higher from the ovulation phase (5) to the post-ovulation phase (6), showing potential to detect ovulation. The thermography was efficient to detect small temperature variations during different phases of the estrous cycle, allowing the identification of different phases of the cycle in Santa Ines ewes.

Evaluation of environmental effects on reproductive characteristics of Mangalarga Marchador mares in a commercial embryo transfer program.

The objective of this study was to evaluate the environmental effects on embryo recovery rate and pregnancy rate of Mangalarga Marchador mares. The reproductive characteristics of donor and recipient mares were evaluated during five years in Brazilian tropical environment. The mares were used throughout the year and seasons were classified as: October to April (breeding season - BS); May (autumn transition out of the breeding season - ATBS); June to August (non-breeding season - nBS); and September (vernal transition into the breeding season - VTBS). Daily temperature rainfall and hours of daylight (photoperiod) were measured during all months and years of evaluation. The embryo recovery rate (ERR) and the pregnancy rate (PR) were observed and frequencies were calculated. The effect of environmental variables, day of flushing, and hormonal treatments (estradiol benzoate and progesterone) were determined for the reproductive measures using the Chi-square and Kruskal-Wallis tests. Significant effects were noted of the year, season and temperature on ERR (P < 0.05), but no significant effects were observed of the environmental parameters (year, season, hormone treatment, rainfall and photoperiod) on PR (P > 0.05). The day of uterine flush affected ERR (P < 0.05) but did not affect PR (P > 0.05). In addition, hormone treatment also supported favorable results of PR in recipient mares during nBS. The conclusion is that mares of this breed can be used in reproduction all year long, with good pregnancy rates, in Brazil's tropical environment. The hormone treatment also supported favorable pregnancy rates in recipient mares during the non-breeding season. It seems that mares can have good pregnancy rates throughout the year in Brazil´s tropical environment.

Diagnostic methods to detect uterus illnesses in mares.

Uterine illnesses are the major problem in horse reproductive programs. The diagnosis of these pathologies is often neglected or does not provide enough information about the cause. The aim of this study was to evaluate different diagnoses to provide more accurate information about endometritis and the real endometrial profile at the beginning of a breeding season. Fifty-one mares with reproductive failure were evaluated. Two different uterine cytology diagnoses, culture of uterine flush material and biopsy assay, were performed. The data were analyzed by the Chi-squared test to compare different diagnostic methods for the same sample from each mare, and sensitivity and specificity tests were performed. Most of evaluations showed no association between cytology and other diagnostic methods, except with bacterial culture. The most common bacterium found was Escherichia colli, but it was not associated with neutrophil presence in cytology. The presence of ß-hemolitic streptococcus was well characterized by positive cytology and cloudy lavage fluid aspect. Endometrial profile was affected by age and whether the mare was a donor or recipient. The most sensitive diagnostic method was gross aspect of lavage fluid. Endometritis should be diagnosed by at least two methods to increase accuracy. Endometritis is not the only uterine disease that causes embryo loss, since endometrial fibrosis can also be a cause.

Relationship of serum testosterone, sperm production, and testis traits with fertility of Mangalarga Marchador stallions / Relación entre testosterona sérica, producción espermática y características testiculares con la fertilidad en sementales Mangalarga Marchador / Relação entre testosterona sérica, produção espermática e características testiculares com fertilidade em garanhões Mangalarga Marchador

Abstract Background: Assessing breeding soundness is important to predict the potential fertility of stallions. Objective: To investigate the association of testis traits, total number of spermatozoa, and testosterone with fertility of Mangalarga Marchador breed stallions. Methods: The traits measured were testicular length, width and height, right and left testicular volume and total testicular volume. We also evaluated the total number of spermatozoa (TNS), serum testosterone concentration and fertility of stallions (by embryo recovery from donor mares) in the breeding and non-breeding seasons during 1 year. Analysis of variance was performed to verify the effects of age category (AC; young/adult) and reproductive season on the traits. Correlations and repeatability of the traits were also calculated. Results: The AC did not influence testis traits (p>0.05). Reproduction season affected left testicular length, left testicular height, right testicular width and height (p<0.05). The AC and season affected testis volume (p<0.05). There were no effects of AC and season on TNS and testosterone (p>0.05). We observed strong correlations between all testicular measurements and testicular volume (0.53 to 0.75), medium correlation between TNS and testis volume (0.32), and low correlation between testosterone and testis traits (0.20). Repeatability of the characteristics of left and right testis was medium to high (0.22 to 0.78). Embryo recovery rate was 60% and its correlation with TNS was 0.44. Conclusion: Testis size evaluation is recommended over the evaluation of serum testosterone concentration to predict sperm output. Sperm output (TNS) is a good predictor of stallion fertility.

Resumen Antecedentes: Es importante realizar examen andrológico de sementales para predecir su potencial reproductivo. Objetivo: Evaluar la asociación entre características testiculares, número total de espermatozoides y testosterona con la fertilidad de sementales de raza Mangalarga Marchador. Métodos: Las características medidas fueron: longitud testicular, ancho y altura del testículo. También fue calculado el volumen testicular derecho, izquierdo y total, así como el número total de espermatozoides (TNS), concentración sérica de testosterona y fertilidad (por recuperación embrionaria de las yeguas) de los sementales dentro y fuera de la temporada reproductiva durante un año. Se realizó análisis de varianza para evaluar el efecto de categoría de edad (AC) y estación reproductiva sobre las características. Resultados: La AC no influenció las características testiculares (p>0,05). La estación reproductiva influenció la longitud del testículo izquierdo, la altura del testículo izquierdo, y el ancho y altura del testículo derecho (p<0,05). La AC y la estación reproductiva influenciaron el volumen testicular (p<0,05). No hubo efecto de AC y estación reproductiva en el TNS y testosterona (p>0,05). Se observó una alta correlación entre todas las características testiculares y el volumen testicular (0,53 a 0,75), mediana correlación entre TNS y el volumen testicular (0,32), y una baja correlación entre las medidas testiculares y la testosterona (0,20). La repetitividad de las medidas testiculares fue media a alta (0,22 a 0,78). La tasa de recuperación embrionaria fue de 60% y la correlación con TNS fue 0,44. Conclusión: Para predecir la producción espermática se recomienda evaluar las características testiculares, sin necesidad de medir testosterona. La producción espermática es un buen predictor de la fertilidad de los sementales.

Resumo Antecedentes: Realizar o exame andrológico no garanhão, é importante para predizer seu potencial reprodutivo. Objetivo: Avaliar a associação das características testiculares, número total de espermatozoides, testosterona e fertilidade de garanhões Mangalarga Marchador. Métodos: As características mensuradas foram: comprimento do testículo, largura do testículo e altura do testículo. Também foi calculado o volume testicular, direito, esquerdo e total, assim como calculado o número total de espermatozoides (TNS), concentração sérica de testosterona e fertilidade dos garanhões (pela recuperação embrionária das éguas doadoras) durante e fora da estação reprodutiva durante um ano. Foi feita análise de variância para verificar efeito da categoria de idade (AC) e estação reprodutiva sobre as características. Resultados: A AC não influenciou nas características testiculares (p>0,05). Estação reprodutiva influenciou no comprimento testicular esquerdo, altura testicular esquerda, largura e altura testicular direita (p<0,05).A AC e estação reprodutiva influenciaram no volume testicular (p<0,05). Não houve efeito da AC e estação reprodutiva no TNS e testosterona (p>0,05). Foi observado alta correlação entre todas características testiculares e volume testicular (0,53 a 0,75), media correlação entre TNS e volume testicular (0,32), e baixa correlação entre medidas testiculares e testosterona (0,20). A repetitividade das medidas testiculares foi meia a altas (0,22-0,78). A taxa de recuperação embrionária foi 60% e a correlação com TNS foi de 0,44. Conclusão: É recomendada a avaliação das características testiculares para predizer a produção espermática sem necessidade de mensurar testosterona. A produção espermática é um bom preditor da fertilidade dos garanhões.

Evaluation of the breeding behavior of Brazilian Pony stallions.

The aim of this study was to evaluate the breeding behavior of Brazilian Pony stallions during semen collection to examine the effect of age category (AC) and month of evaluation on the characteristics of breeding behavior. Nine stallions were evaluated during the breeding and non-breeding season, 5-8 (AC1) and 9-13 years (AC2) of age. The variables evaluated were reaction time (RT), mount time one (MT1) and two (MT2), mount and ejaculation time (MEjT), mounting without erection (MWEr), mounting without ejaculation (MWEj), flehmen, kicks, bites, smells, vocalizations and serum testosterone concentration. Analyses of variance and correlation were performed. The average scores were RT: 56.2±92.9s, MT1: 115.0±122.4s, MT2: 196.4±160.7s, MEjT: 21.8±3.2s, MWEr: 0.8±1.3s, MWEj: 2.1±3.5s, flehmen: 3.8±3.0s, kicks: 0.3±1.2s, bites: 1.0±2.0s, smells: 5.7±3.5s, vocalizations: 6.4±6.3s and testosterone: 0.89±0.52ng/mL. The AC affected the RT (P<0.05) but not the other traits. Month of evaluation did not affect most variables (P>0.05). The correlations between age and RT and between age and MT1 and MT2 were significant. The correlation between testosterone and MEjT was moderate and negative. The breeding behavior of Brazilian Pony stallions was similar across different months of evaluation and different age categories. The month of evaluation and age category also did not affect testosterone concentrations, meaning that semen collection is feasible throughout the year.

Sugarcane and mulberry silage supplementation of sheep during the peripartum period.

The aim of this study was to evaluate the effects of supplementation with silage made from sugarcane alone or mulberry and sugarcane on the gastrointestinal nematodes and hematological parameters in peripartum Santa Inês ewes. Three groups, each of 15 ewes, were supplemented with either 100 % sugarcane silage or sugarcane silage and mulberry at ratios of either 75:25 or 50:50. The variables analyzed were weight, body condition, packed cell volume, total plasma protein, hemoglobin, total leukocytes, eosinophils, and number of parasite eggs per gram of feces. All variables were measured individually every 14 days, making ten samples per ewe. Diets with sugarcane added to different proportions of mulberry branches did not influence the blood and parasitological parameters of Santa Inês sheep naturally infected in the peripartum period.