Supplementation with long-acting injectable progesterone 3 days after TAI impaired luteal function in buffaloes.

Three experiments were conducted to evaluate the effects of long-acting injectable progesterone (iP4) in buffalo cows. In Experiment 1, ovariectomized buffaloes received 300 mg (iP300) or 600 mg (iP600) of iP4, and serum P4 concentrations were evaluated. In experiment 2, three groups were compared: control or administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after timed artificial insemination (TAI). On day 16, reproductive tract was recovered for conceptus, endometrium, and corpus luteum (CL) analysis. In experiment 3, pregnancy per AI (P/TAI) and proportion of pregnancy losses were evaluated after administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after TAI in lactating buffaloes. In experiment 1, serum P4 concentrations remained over 1 ng/mL for ~ 3 days in both groups. The 300 mg dose was used in subsequent experiments. In experiment 2, CL weight and endometrial glands density were decreased, and conceptus length was increased in iP4-D3 compared to control and to iP4-D6 (P < 0.05). Transcript abundance of Prostaglandin F Receptor (FP) and ISG15 in CL and of ISG15 and MX1 in endometrium was greater in iP4-D3 when compared to control and to iP4-D6 (P < 0.05). In experiment 3, there was no difference among experimental groups for P/TAI at D30 and pregnancy losses (P > 0.1); however, iP4-D3 presented a lower P/TAI at day 60 (41.7%) when compared to control (56.8%) and iP4-D6 (57.7%; P = 0.07). In conclusion, administration iP4 at 3 days after TAI affects CL development and consequently decreases final pregnancy outcome in buffaloes.

Relationship between sperm ubiquitination and equine semen freezability.

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very low ubiquitin intensity and IV = very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p ≤ .05 was considered significant. We observed that PF stallions showed higher percentages (p < .05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p < .05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p < .05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects.

An agent-based simulation model to compare different reproductive strategies in cow-calf operations: Technical performance.

The objective of this study was to create a stochastic, agent-based simulation model of a synthetic population of beef cattle, and then use it to compare the technical performance of different reproductive strategies. The model was parameterized using data from a real beef cattle herd and from the peer-reviewed scientific literature to represent a Nelore cattle herd in the state of São Paulo, Brazil. Ten scenarios were evaluated: natural mating (NM) only (ONM); one timed artificial insemination (TAI) plus NM (1TAI + NM); two TAI plus NM, with 24, 32, and 40 days between inseminations (2TAI/24 + NM, 2TAI/32 + NM, and 2TAI/40 + NM, respectively); three TAI without NM, with 24, 32, and 40 days between TAI (3TAI/24, 3TAI/32, and 3TAI/40, respectively); and three TAI plus NM, with 24 and 32 days (3TAI/24 + NM and 3TAI/32 + NM, respectively). NM began 10 days after the last TAI and was performed until the end of the breeding season. The size of the female herd was set to contain up to 400 individuals. The bull population was established at 0, 7, or 15 bulls depending on the used scenario. Simulation was performed for 5000 days. The outcomes for each scenario are means ± S.E. assessed on 32 farms at 1-day time intervals and on an animal-by-animal basis after steady state was reached (1825 days). The 3TAI/24 + NM scenario resulted in a greater number of births (279.85 ± 0.47 births), while the ONM scenario had the least value (202.38 ± 0.43 births). The heaviest males and females at weaning belonged to 3TAI/24, with 190.85 ± 0.17 kg for males and 173.89 ± 0.13 kg for females. The ONM scenario had the lightest males (166.84 ± 0.18 kg) and females (151.75 ± 0.16 kg). The greatest and least total pregnancy rates were found in 3TAI/24 + NM (0.91 ± 0.00) and ONM (0.62 ± 0.00), respectively. The ONM scenario required 52.5 days more than scenarios that included TAI to reach 50% of pregnancy. The greatest ages at culling for cows was 3TAI/24 + NM (3658.88 ± 10.41 days). In contrast, the lowest age at culling was found in ONM (2823.93 ± 8.28 days). We concluded that the proposed model represents the main interactions of a real beef cattle herd. It has all the advantages of a physical experiment, but does not require incurring significant expenses nor altering the real system. This study offers evidence that the scenarios that present the best technical performance are those that used TAI with a 24-day interval between inseminations.

Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers.

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

Heat loss efficiency and HSPs gene expression of Nellore cows in tropical climate conditions.

Adaptation is a relevant characteristic to be understood in livestock animals in order to maintain and raise productivity. In Brazil, the Nellore beef cattle are widely disseminated and well-adapted breed that present good thermoregulatory characteristics for tropical environment conditions. Conversely, the physiological and cellular mechanisms required for thermoregulation and thermotolerance in this breed are still limited. The aim of this study was to comprehend the heat loss efficiency at the whole animal level and heat shock response at the cellular level of Nellore cows in tropical climate conditions. Healthy purebred Nellore cows were classified according to their capacity to lose body heat as Efficient or Inefficient based on vaginal temperature which was continuously monitored by data-loggers. Rectal, tail, and ocular temperatures, sweating rate, and respiratory frequency were collected to assess other thermoregulatory responses. Peripheral mononuclear cells were used for gene expression of heat shock proteins 60, 70, and 90 induced by in vitro heat treatments at 38, 40, and 42 °C. In our findings, the Efficient cows presented higher sweating rates compared to Inefficient cows that presented higher rectal temperature with greater amplitude of vaginal temperature profile. Transcription of the HSP genes was stable at 38 and 40 °C and decreased for all HSP genes at 42 °C. In conclusion, the Nellore efficiency to lose heat was mainly associated with their sweating capacity and cellular thermotolerance confirmed by the maintenance of heat shock proteins transcripts under heat stress. Taken together, this knowledge contributes as a future key for genetic selection of adapted animals.

Sex steroids drive the remodeling of oviductal extracellular matrix in cattle.

The extracellular matrix (ECM) is a group of molecules that offer structural and biochemical support to cells and interact with them to regulate their function. Also, growth factors (GFs) stored in the ECM can be locally released during ECM remodeling. Here, we hypothesize that the balance between ECM components and remodelers is regulated according to the ovarian steroid milieu to which the oviduct is exposed during the periovulatory period. Follicular growth was manipulated to generate cows that ovulated small follicles (SF-small corpus luteum [SCL]; n = 20) or large follicles (LF-large corpus luteum [LCL]; n = 21) and possess corresponding Estradiol (E2) and Progesterone (P4) plasmatic concentrations. Ampulla and isthmus samples were collected on day 4 (day 0 = ovulation induction) and immediately frozen or fixed. The transcriptional profile (n = 3/group) was evaluated by RNA sequencing. MMP Antibody Array was used to quantify ECM remodelers' protein abundance and immunohistochemistry to quantify type I collagen. Transcriptome analysis revealed the over-representation of ECM organization and remodeling pathways in the LF-LCL group. Transcription of ECM components (collagens), remodelers (ADAMs and MMPs), and related GFs were upregulated in LF-LCL. Protein intensities for MMP3, MMP8, MMP9, MMP13, and TIMP4 were greater for the LF-LCL group. Type I collagen content in the mucosa was greater in SF-SCL group. In conclusion, that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates ECM remodeling. We speculate that differential ECM regulation may contribute to oviductal receptivity to the embryo.

Sex steroids modulate morphological and functional features of the bovine oviduct.

In cattle, the oviduct plays a major role in the reproductive process; however, molecular control of oviduct receptivity to the embryo is poorly understood. A model for receptivity based on size of the pre-ovulatory follicle (POF) was used to compare oviductal morphology, cellular proliferation, and candidate transcript abundance. Growth of the POF of Nelore (Bos indicus) cows was manipulated to produce two groups: a large POF-large corpus luteum (CL) group (LF-LCL; greater receptivity) and a small POF-small CL group (SF-SCL). Samples of the ampulla and isthmus ipsilateral and contralateral to CL were collected 4 days after GnRH-induced ovulation. Tissues were either embedded in paraffin for Harris-Hematoxylin and Eosin and periodic acid-Schiff staining and KI67 immunostaining, followed by morphological analyses, or stored at -80 °C for RNA extraction, cDNA synthesis, and qPCR analyses. The effects of group (LF-LCL and SF-SCL), region (ampulla and isthmus), and side (ipsilateral and contralateral) were analyzed using three-way nested ANOVA. The ipsilateral ampulla of the LF-LCL group presented more primary mucosal folds, a greater mucosal-folding grade and luminal perimeter, and more secretory cells and proliferating cells when compared with the ampulla of the SF-SCL group and with the contralateral ampulla of both groups. There were no morphological differences in the isthmus between groups and sides. Changes in transcript abundance are suggestive of LF-LCL-stimulated secretory activity. In summary, ovulation of a larger POF generates a periovulatory endocrine milieu that modulates morphological and functional features of the bovine oviduct which may support embryo survival and development.

Oviductal transcriptional profiling of a bovine fertility model by next-generation sequencing.

In cattle, the oviduct plays a fundamental role in the reproductive process. Oviductal functions are controlled by the ovarian sex steroids: estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex steroid milieus differentially impacts the oviductal transcriptional profile. We manipulated growth of the pre-ovulatory follicle to obtain cows that ovulated a larger (LF group) or a smaller (SF group) follicle. The LF group presented greater proestrus/estrus concentrations of estradiol and metaestrus concentrations of progesterone (Gonella-Diaza et al. 2015 [1], Mesquita et al. 2014 [2]). Also, the LF group was associated with greater fertility in timed-artificial insemination programs (Pugliesi et al. 2016 [3]). Cows were slaughtered on day 4 of the estrous cycle and total RNA was extracted from ampulla and isthmus fragments and analyzed by RNAseq. The resulting reads were mapped to the bovine genome (Bos taurus UMD 3.1, NCBI). The differential expression analyses revealed that 325 and 367 genes in ampulla and 274 and 316 genes in the isthmus were up-regulated and down-regulated in LF samples, respectively. To validate the RNAseq results, transcript abundance of 23 genes was assessed by qPCR and expression patterns were consistent between the two techniques. A functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Processes enriched in the LF group included tissue morphology changes (extracellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters). An overview of the gene expression data was deposited in the NCBI's Gene Expression Omnibus (GEO) and is accessible through the accession number GSE65681. In conclusion, differences in the peri-ovulatory sex steroid milieu modify the oviductal gene expression profiles. Such differences may be associated with the greater fertility of the LF cows. This dataset is useful for further investigations of the oviductal biology and the impact of sex-steroid on the female reproductive tract.

Size of the Ovulatory Follicle Dictates Spatial Differences in the Oviductal Transcriptome in Cattle.

In cattle, molecular control of oviduct receptivity to the embryo is poorly understood. Here, we used a bovine model for receptivity based on size of the pre-ovulatory follicle to compare oviductal global and candidate gene transcript abundance on day 4 of the estrous cycle. Growth of the pre-ovulatory follicle (POF) of Nelore (Bos indicus) cows was manipulated to produce two groups: large POF large corpus luteum (CL) group (LF-LCL; greater receptivity) and small POF-small CL group (SF-SCL). Oviductal samples were collected four days after GnRH-induced ovulation. Ampulla and isthmus transcriptome was obtained by RNA-seq, regional gene expression was assessed by qPCR, and PGR and ERa protein distribution was evaluated by immunohistochemistry. There was a greater abundance of PGR and ERa in the oviduct of LF-LCL animals thus indicating a greater availability of receptors and possibly sex steroids stimulated signaling in both regions. Transcriptomic profiles indicated a series of genes associated with functional characteristics of the oviduct that are regulated by the periovulatory sex steroid milieu and that potentially affect oviductal receptivity and early embryo development. They include tissue morphology changes (extra cellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters), and were enriched for the genes with increased expression in the LF-LCL group. In conclusion, differences in the periovulatory sex steroid milieu lead to different oviductal gene expression profiles that could modify the oviductal environment to affect embryo survival and development.

Regulation of the polyamine metabolic pathway in the endometrium of cows during early diestrus.

The timing and magnitude of exposure to preovulatory estradiol followed by post-ovulatory progesterone (periovulatory endocrine milieu) in cattle modulate endometrial gene expression, histotroph composition, and conceptus development, but the mechanisms underlying this regulation remain unknown. Using an experimental model based on the modulation of follicle growth, this work aimed to evaluate if the polyamine metabolic pathway is regulated by the periovulatory endocrine milieu. Nelore cows were manipulated to ovulate small (n = 15) or large (n = 15) follicles, then the profiles of polyamines and their synthetic enzymes were compared between groups. Transcripts for the enzymes of this pathway, ornithine decarboxylase 1 (ODC1; the rate-limiting enzyme in polyamine biosynthesis) protein quantification, adenosylmethionine decarboxylase 1 (AMD1) protein immunolocalization, and concentrations of the different polyamines (putrescine, spermidine, and spermine) were respectively quantified by quantitative reverse-transcriptase PCR, immunoblotting, immunohistochemistry, and gas chromatography-mass spectrometry in both the endometrium and uterine flushing. No differences in gene and protein expression or concentration of polyamines were observed between groups. There were significant correlations between the relative abundance of ODC1 and spermidine/spermine N1-acetyltransferase 1 (SAT1) transcripts as well as between antizyme inhibitor 1 (AZIN1) and adenosylmethionine decarboxylase 1 (AMD1) transcripts. In conclusion, our results show that the polyamine metabolic pathway is present and functional, but not regulated by the periovulatory endocrine milieu in the bovine endometrium.

Morfometría macroscópica del ovario y cuerpo lúteo de yeguas criollas de Colombia / Macroscopic Morphometry of the Ovary and Corpus Luteum of Native Mares in Colombia / Morfometria macroscópica do ovário e corpo lúteo de éguas criollas da Colômbia

El objetivo del presente estudio era realizar una descripción de parámetros morfométricos del ovario y cuerpo lúteo (CL) de yeguas criollas de Colombia. Se utilizaron cincuenta ovarios provenientes de yeguas adultas. Todos los animales se encontraban clínicamente sanos. Los tejidos se obtuvieron después del sacrificio y se fijaron inmediatamente en formalina tamponada. Se pesó y se midió el diámetro mayor y el menor del ovario. Se realizó una incisión longitudinal con la finalidad de observar el parénquima del órgano. Se removió el CL y se registró su peso y diámetro. Los datos se analizaron a través de estadística descriptiva y el grado de asociación de las variables se calculó a través de un modelo de regresión simple. Se transformaron los datos con logaritmo en base natural cuando se requirió. El diámetro mayor del ovario varió desde 2 hasta 6,2 cm. El diámetro del CL varió desde 1,1 hasta 3,6 cm. Se encontró una relación lineal entre el peso y el diámetro del ovario (R² = 0,41; p < 0,01) y entre el peso y el diámetro del CL (R² = 0,48; p < 0,01). Aunque la relación entre el peso del ovario y el cuerpo lúteo es lineal, el coeficiente de determinación fue muy bajo. La yegua criolla colombiana tiene características similares en la morfología ovárica y luteal a las reportadas en la literatura para yeguas de otras razas livianas. Los valores aquí reportados podrían ser el punto de partida para establecer valores de referencia de utilidad clínica.

The purpose of this study was to describe the morphometric parameters of the ovary and corpus luteum (CL) of native mares in Colombia. Fifty ovaries from adult mares were used. All animals were clinically healthy. The tissues were collected after slaughter and immediately fixed in buffered formalin. The large and small diameters of the ovary were weighted and measured. A longitudinal incision was made in order to observe organ parenchyma. The CL was removed and its weight and diameter were recorded. Data were analyzed with descriptive statistics, and the degree of association between the variables was calculated through a simple regression model. When required, data were transformed with natural base logarithm. The large diameter of the ovary ranged from 2 to 6.2 cm. The diameter of the CL ranged from 1.1 to 3.6 cm. A linear relationship was found between the weight and diameter of the ovary (R² = 0.41; p < 0.01) and between the weight and diameter of the CL (R² = 0.48; p < 0.01). On the other hand, although the relationship between the weight of the ovary and corpus luteum is linear, the determination coefficient was very low. Luteal and ovarian morphology of native mares in Colombia has similar characteristics to those reported in literature for other light breed mares. The values reported herein could be the starting point to establish reference values for clinical utility.

O objetivo do presente estudo era realizar uma descrição de parâmetros morfométricos do ovário e corpo lúteo (CL) de éguas criollas da Colômbia. Utilizaram-se cinquenta ovários provenientes de éguas adultas. Todos os animais se encontravam clinicamente saudáveis. Os tecidos foram obtidos depois do sacrifício e se fixaram imediatamente em formalina tamponada. Foi pesado e medido o diâmetro maior e o menor do ovário. Realizou-se uma incisão longitudinal com a finalidade de observar o parênquima do órgão. Removeu-se o CL e se registrou seu peso e diâmetro. Os dados se analisaram com estatística descritiva, e o grau de associação das variáveis se calculou através de um modelo de regressão simples. Transformaram-se os dados com logaritmo em base natural quando se requerido. O diâmetro maior do ovário variou desde 2 até 6,2 cm. O diâmetro do CL variou desde 1,1 até 3,6 cm. Encontrouse uma relação linear entre o peso e o diâmetro do ovário (R² = 0,41; p < 0,01) e entre o peso e o diâmetro do CL (R² = 0,48; p < 0,01). Enquanto que, ainda sendo linear a relação entre o peso do ovário e o corpo lúteo, o coeficiente de determinação foi muito baixo. A égua criolla colombiana tem características similares na morfologia ovárica e lútea a as relatadas na literatura para éguas de outras raças leves. Os valores aqui reportados poderiam ser o ponto de partida para estabelecer valores de referencia de utilidade clínica.