Successful xenotransplantation of testicular cells following fractionated chemotherapy of recipient birds.

An essential step in the success of germ cell transplantation is the preparation of the recipient's testicular environment to increase the availability of stem cell niches. However, most methods for this purpose in birds face serious limitations such as partial germ cell depletion, high toxicity and mortality, or the need to use expensive technologies. Here, we validated a simple and practical technique of transferring quail testicular cells into chicken testes depleted of endogenous spermatozoa by fractioned chemotherapy (20 mg/kg/week busulfan for 5 weeks). This protocol resulted in a very low mortality of the treated day-old chicks and, despite maintenance of androgenic activity, sperm production was decreased by 84.3% at 25 weeks of age. NANOG immunostaining revealed that very few to no germ cells were present following treatment with 20 and 40 mg/kg, respectively. RT-qPCR data also showed that c-MYC and NANOG expression declined in these treatments, but GRFα1 and BID expressions remained unaltered among groups. After xenotransplantation, quail germ cells were immunodetected in chicken testes using a species-specific antibody (QCPN), and quail ovalbumin DNA was found in seminal samples collected from chicken recipients. Together, these data confirm that fractionated administration of busulfan in hatchlings is a practical, effective, and safe protocol to prepare recipient male birds capable of supporting xenogeneic spermatogenesis.

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos.

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Inhibition of FGF receptor impairs primitive endoderm differentiation in bovine embryos.

The first cellular differentiation event in the pre-implantation embryo results in the trophectoderm (TE) and the inner cell mass (ICM). A second event occurs in the latter, resulting in the epiblast and the primitive endoderm (PE). This second differentiation is still not fully characterized in bovine development, although it is likely to involve FGF signalling. Thus, in this study, we tested the hypothesis that stimulation or inhibition of the FGF pathway during bovine embryo in vitro culture would only interfere with PE differentiation if maintained until later blastocyst stages. At first, we characterized the expression of PE marker SOX17 at different blastocyst stages. Then, we treated in vitro produced embryos during different windows of time: days 5.0-7.0 (D5-D7), D7-D9, and D5-D9 with 1 µg/ml FGF4 and 1 µg/ml heparin or 1 mM FGFR inhibitor, AZD4547. We observed that the SOX17-positive cell number only increases in late-stage blastocysts compared to early stages. Treatment of embryos with FGF4 did not change the number of SOX17-positive cells, while inhibition of FGFR signalling reduced SOX17-positive cells from D5-D7 and completely ablated SOX17 expression when kept until D9. In conclusion, FGFR inhibition repressed PE differentiation in bovine embryos at all time points, although stimulation with FGF4 did not interfere with PE cell numbers.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

Paternal age impairs in vitro embryo and in vivo fetal development in murine.

The association between advanced paternal age and impaired reproductive outcomes is still controversial. Several studies relate decrease in semen quality, impaired embryo/fetal development and offspring health to increased paternal age. However, some retrospective studies observed no alterations on both seminal status and reproductive outcomes in older men. Such inconsistency may be due to the influence of intrinsic and external factors, such as genetics, race, diet, social class, lifestyle and obvious ethical issues that may bias the assessment of reproductive status in humans. The use of the murine model enables prospective study and owes the establishment of homogeneous and controlled groups. This study aimed to evaluate the effect of paternal age on in vitro embryo development at 4.5 day post conception and on in vivo fetal development at 16 days of gestation. Murine females (2-4 months of age) were mated with young (4-6 months of age) or senile (18-24 months of age) males. We observed decreased in vitro cleavage, blastocyst, and embryo development rates; lighter and shorter fetuses in the senile compared to the young group. This study indicated that advanced paternal age negatively impacts subsequent embryo and fetal development.

Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Cell differentiation events in pre-implantation mouse and bovine embryos.

Early mammal embryogenesis starts with oocyte fertilization, giving rise to the zygote. The events that the newly formed zygote surpasses are crucial to the embryo developmental success. Shortly after activation of its genome, cells of the embryo segregate into the inner cell mass (ICM) or the trophectoderm (TE). The first will give rise to the embryo while the latter will become the placenta. This first segregation involves cellular and molecular processes that include cell polarity linked to intracellular pathway activation, which will regulate the transcription of trophectoderm-related genes. Then, cells of the ICM undergo the second event of mammalian cell differentiation, which consists of the separation between epiblast (EPI) and hypoblast or primitive endoderm (PrE). This second segregation involves paracrine signaling, leading to differential expression of key genes that will dictate the fate of the cell. Although these processes are described in detail in the mouse, recent studies suggest that the bovine embryo could also be an interesting model for early development, since there are differences to the mouse and similarities with early human embryogenesis. In this review, we gathered the main data available in the literature upon bovine and mouse early development events, suggesting that both models should be analyzed and studied in a complementary way, to better model early events occurring in human development.

Endometrial prostaglandin F2α in vitro production and its modulation regarding dominant follicle position in cattle / Produção in vitro de PGF F2α endometrial e sua modulação referente ao folículo dominante em bovinos

Prostaglandin F2α (PGF2α) determines luteolysis in cattle, and the ability to manipulate its endogenous synthesis is indispensible for large-scale animal breeding. Estradiol (E2) and progesterone (P4) modulate several molecular pathways in endometrial cells, including the synthesis of PGF2α; however, its specific mechanisms are still not totally known. This study investigated the production in vitro and possible modulation of endometrial PGF2α due to a local effect of endogenous E2 in the ipsilateral uterine horn (UH) containing the dominant follicle (DF) or from P4 in ipsilateral horn containing the corpus luteum (CL). The PGF2α stimulators oxytocin (OT) and phorbol 12,13-dibutyrate (PDBu) were incubated with endometrial explants, and PGF2α content was measured. For that, cycling cows were synchronized, the development of DF and CL was examined by ultrasonography and on the seventh day of the estrous cycle, endometrial explants were collected and cultured in medium supplemented with 10-6 M PDBu or 10-6 M OT or non-supplemented. Media samples were collected immediately after treatment and 60 min later. Radioimmunoassay showed that the PGF2α content of the UH ipsilateral to the DF was 49% less than that of the contralateral UH (8.22 ± 0.95 vs. 12.24 ± 0.95 pg/mL/mg tissue, respectively; P < 0.01). However, the PGF2α levels did not differ between the UHs as a function of the CL position (9.46 ± 0.95 vs. 11 ± 0.95 pg/mL/mg; P > 0.05). The cellular stimulators promoted an increase in PGF2α synthesis (P < 0.02), and the effects differed among the animals (P < 0.04). The PGF2a production was higher in the explants treated with PDBu rather than OT (13.68 ± 1.16 vs. 10.01 ± 1.16 pg/mL/mg tissue, respectively; P < 0.05). In conclusion, PGF2α synthesis is modulated by the presence of the DF (local E2) but not the CL (local P4), and both PDBu and OT stimulated PGF2a synthesis.(AU)

A prostaglandina F2α (PGF2α) determina a luteólise em bovinos. A capacidade de manipular sua síntese endógena é indispensável para a produção animal em grande escala. O estradiol (E2) e a progesterona (P4) modulam diversas vias moleculares das células endometriais, incluindo a síntese de PGF2α; no entanto, pouco se sabe sobre seus mecanismos específicos. Este trabalho investigou a produção in vitro e a possível modulação da PGF2α endometrial devido a um efeito local do E2 endógeno no corno uterino ipsilateral ao folículo dominante (FD) ou da P4 no corno ipsilateral ao corpo lúteo (CL). Os estimuladores de PGF2α oxitocina (OT) e 12,23-dibutirato de forbol (PDBu) foram incubados com explantes endometriais, e o conteúdo de PGF2α foi mensurado. Para tal, vacas cíclicas foram sincronizadas, o desenvolvimento de FD e CL foi examinado por ultrassonografia, e no 17º dia do ciclo estral os explantes endometriais foram coletados e cultivados em meio ou suplementados com PDBu 10-6M ou 10-6M OT. As amostras de meio foram coletadas imediatamente após o tratamento e sessenta minutos depois. O radioimunoensaio mostrou que o conteúdo de PGF2α do corno ipsilateral ao FD foi 49% menor que o do corno contralateral (8,22 ± 0,95 vs. 12,24 ± 0,95 pg/mL/mg de tecido, respectivamente, P < 0,01). No entanto, os níveis de PGF2α não diferiram entre os cornos em função da posição do CL (9,46 ± 0,95 versus 11 ± 0,95 pg/mL/mg; P > 0,05). Os estimuladores celulares promoveram um aumento na síntese de PGF2α (P < 0,02), e os efeitos diferiram entre os animais (P < 0,04). A produção de PGF2α foi maior nos explantes tratados com PDBu em comparação à OT (13,68 ± 1,16 versus 10,01 ± 1,16 pg/mL/mg de tecido, respectivamente, P < 0,05). A conclusão obtida foi que a síntese de PGF2α é: modulada pela presença do FD (E2 local), mas não do CL (P4 local); e estimulada por PDBu e OT.(AU)

Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

Impact of hormonal modulation at proestrus on ovarian responses and uterine gene expression of suckled anestrous beef cows.

BACKGROUND: This study evaluated the impact of hormonal modulation at the onset of proestrus on ovarian response and uterine gene expression of beef cows. METHODS: A total of 172 anestrous beef cows were assigned to one of four groups according to the treatment with estradiol cypionate (ECP) and/or equine chorionic gonadotropin (eCG) [CON (n = 43), ECP (n = 43), eCG (n = 44) and ECP + eCG (n = 42)]. RESULTS: ECP-treated cows (ECP and ECP + eCG groups) presented greater occurrence of estrus (44.6% vs. 65.4%; P = 0.01) and pregnancy per AI [47.1% vs. 33.3%; P = 0.07], but similar progesterone (P4) concentration at subsequent diestrus than cows not treated with ECP (CON and eCG groups). Nonetheless, eCG-treated cows (eCG and ECP + eCG groups) presented larger follicle at timed AI (12.6 ± 0.3 vs. 13.5 ± 0.3 mm; P = 0.03), greater ovulation rate (96.5% vs. 82.6%; P = 0.008) and greater P4 concentration at d 6 (3.9 ± 0.2 vs. 4.8 ± 0.2 ng/mL; P = 0.001) than cows not treated with eCG (CON and ECP groups). Next, cows with a new corpus luteum 6 d after TAI were submitted to uterine biopsy procedure. Uterine fragments [CON (n = 6), ECP (n = 6)] were analyzed by RNA-Seq and a total of 135 transcripts were differentially expressed between groups (73 genes up-regulated by ECP treatment). Subsequently, uterine samples were analyzed by qPCR (genes associated with cell proliferation). ECP treatment induced greater abundance of PTCH2 (P = 0.07) and COL4A1 (P = 0.02), whereas suppressed EGFR (P = 0.09) expression. Conversely, eCG treatment increased abundance of HB-EGF (P = 0.06), ESR2 (P = 0.09), and ITGB3 (P = 0.05), whereas it reduced transcription of ESR1 (P = 0.05). Collectively, supplementation with ECP or eCG at the onset of proestrous of anestrous beef cows influenced ovarian responses, global and specific endometrial gene expression. CONCLUSION: Proestrus estradiol regulate the endometrial transcriptome, particularly stimulating proliferative activity in the endometrium.

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein / Células da glândula mamária ovina cultivadas in vitro expressam beta-lactoglobulina e beta-caseina

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe serum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine serum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins' mRNA expression by ovine mammary epithelial cells in vitro.(AU)

A expressão in vitro de proteínas do leite é essencial para explorar as células da glândula mamária como um modelo biológico. A desagregação tecidual via enzimática é amplamente utilizada para o estabelecimento cultivo de células mamárias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamária ovina ainda não é bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamária ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseína e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamárias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseína. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamárias ovinas in vitro.(AU)

Effect of age on expression of spermatogonial markers in bovine testis and isolated cells.

Spermatogonial stem cells (SSC) are the most undifferentiated germ cell present in adult male testes and, it is responsible to maintain the spermatogenesis. Age has a negative effect over stem cell, but the aging effect on SSC is not elucidated for bovine. The present study aim to evaluate the effect of age on the expression of undifferentiated spermatogonial markers in testis and in enriched testicular cells from prepubertal calves and adult bulls. In this matter, testicular parenchyma from calves (3-5 months) (n=5) and bulls with 3 years of age (n=5) were minced and, isolated cells were obtained after two enzymatic digestions. Differential platting was performed for two hours onto BSA coated dish. Cell viability was assessed by Trypan Blue solution exclusion method and testicular cells enriched for SSC was evaluated by expression of specific molecular markers by qRT-PCR (POU5F1, GDNF, CXCR4, UCHL1, ST3GAL, SELP, ICAM1 and ITGA6) and flow cytometry (GFRA1, CXCR4 and ITGA6). CXCR4 and UCHL1 expression was evaluated in fixated testes by immunohistochemistry. We observed that age just affected the expression of selective genes [SELP (Fold Change=5.61; p=0.0023) and UCHL1 (Fold Change=4.98; p=0.0127)]. By flow cytometry, age affected only the proportion of ITGA6+ cells (P<0.001), which was higher in prepubertal calves when compared to adult bulls. In situ, we observed an effect of age on the number of UCHL1+ (p=0.0006) and CXCR4+ (p=0.0139) cells per seminiferous tubule. At conclusion, age affects gene expression and the population of cells expressing specific spermatogonial markers in the bovine testis.

Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm.

Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.

Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts.

Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro-produced early- and late-dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo-produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast- and slow-dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast- and slow-dividing embryo groups, whereas blastocysts from the slow-dividing group were more similar to in vivo-produced blastocysts. Gene ontology enrichment analysis showed that the slow-dividing and in vivo-produced blastocysts shared biological processes related to groups of up- or down-regulated genes when compared to the fast-dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro-produced bovine embryos. Embryos from fast- and slow-dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post-in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage-stage and morulae groups, but was higher in the blastocysts of the slow-dividing group. On the other hand, blastocysts of the fast-dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324-336, 2016. © 2016 Wiley Periodicals, Inc.

Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

System for evaluation of oxidative stress on in-vitro-produced bovine embryos.

This study proposed a quantitative evaluation of oxidative status (OS) in bovine embryos. Sixteen-cell stage embryos, cultured under 5% O2, were treated with oxidative stress inducer menadione (0, 1, 2.5 and 5 µmol/l) for 24 h. Blastocyst rate (BLR) was recorded and expanded blastocysts were stained with CellROX®Green (CRG; OS evaluation) and evaluated under epifluorescence microscopy (ratio of pixel/blastomere). A significant effect of menadione was observed for BLR (P = 0.0039), number of blastomeres/embryo (P < 0.0001) and OS (P < 0.001). Strong negative correlations were found between BLR and the number of blastomeres with OS evaluation, demonstrating the efficacy of this analysis to evaluate OS levels of IVF bovine embryos.

Effects of the utilization of homeopathic elements in commercial diluent on swine sperm viability.

It has been speculated that the homeopathic treatment of sperm cells in order to improve semen quality could be promising. However, few data is available and its use in spermatozoa requires investigation. It is well established that mitochondrial membrane potential is an important viability parameter of spermatozoa and it is intimately related to reproductive efficiency. In this manner, new technologies in order to improve the activity of sperm cells and, finally, the fecundity of swine herds are of extremely importance. Due to the lack of knowledge of homeopathic treatment effect on spermatozoa, the aim of the present study was to verify the effect of three different homeopathic treatments on viability of boar sperm cells. Three homeopathic treatments composed by Pulsatila CH6, Pulsatila and Avena CH6, Avena CH6 and one control treatment (sucrose) were added to diluted boar semen, which were cooled for 24 or 48 h. Interestingly, no positive effect of homeopathic treatments was observed over semen viability. However, it was demonstrated that the 24 h of cooling storage provided more viable sperm cells when compared to the 48-h period. This effect of storage period on sperm viability was assessed by intact plasmatic membrane, intact acrosome and mitochondrial membrane potential evaluation.

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Reproductive performance of sows inseminated with diluted semen treated with homeopathic medicine

Aims: this study sought to assess the reproductive performance of sows inseminated with sperm treated with homeopathic medicines. Materials and methods: the semen of 2 sexually mature boars age 18 months Pietrain and Duroc cross-bred with similar genetic and reproductive performance were chosen, as well as 125 sows. Sixteen samples of semen were collected and standardized through semen evaluation. Three homeopathic preparations and a placebo (control) were tested on the sperm (n=31/32 per group): Avena sativa 6cH, Pulsatilla nigricans 6cH and Avena sativa 6cH + Pulsatilla nigricans 6cH. Sows were inseminated 3 times with the same estrous diagnostic procedures. Results: there was significant difference (p<0.05) between Avena sativa 6cH and the other 3 groups regarding the return to estrus and parturition rate. There was no significant difference among the groups regarding the number of newborn piglets. Conclusion: these data suggest that homeopathic preparation Avena sativa may be used directly on sperm cells to improve the parturition rate in technified swine farms.

Objetivos: este estudo buscou avaliar o rendimento reprodutivo de suínos inseminados com esperma tratado com medicamentos homeopáticos. Materiais e métodos: o esperma de dois suínos sexualmente maduros de 18 meses de idade híbridos Pietrain e Duroc, com rendimento genético e reprodutivo similares foi selecionado, assim como 125 fêmeas. Dezesseis amostras de esperma foram coletadas e estandarizadas através de avaliação do esperma. Três preparações homeopáticas e um controle (placebo) foram testados no esperma (n=31/32 por grupo): Avena sativa 6cH, Pulsatilla nigricans 6cH and Avena sativa 6cH + Pulsatilla nigricans 6cH. As fêmeas foram inseminadas 3 vezes com os mesmos procedimentos de diagnóstico do estro. Resultados: houve diferença significativa (p< 0,05) entre Avena sativa 6cH e os outros 3 grupos no retorno ao estro e taxa de partos. Não houve diferença significativa entre os grupos no número de recém-nascidos. Conclusão: os dados sugerem que a preparação homeopática Avena sativa pode ser utilizada diretamente nas células espermáticas para melhorar a taxa de partos em fazendas tecnificadas de criação de suínos.

Objetivos; este estudio busco evaluar el rendimiento reproductivo de porcinos inseminados com esperma tratado com medicamentos homeopáticos. Materiales y métodos: el esperma de 2 porcinos sexualmente maduros de 18 meses de edad híbridos Pietrain y Duroc, com rendimiento genético y reproductivo similares fue seleccionado, así como 125 hembras. Dieciséis muestras de esperma fueron recogidas y estandarizadas mediante evaluación del esperma. Tres preparados homeopáticos y un control (placebo) fueron testeados en el esperma (n=31/32 por grupo): Avena sativa 6cH, Pulsatilla nigricans 6cH and Avena sativa 6cH + Pulsatilla nigricans 6cH. Las hembras fueron inseminadas 3 veces con los mismos procedimientos de diagnóstico del estro. Resultados: hubo diferencia significativa (p< 0,05) para Avena sativa 6cH en comparación con los demás grupos en el retorno al estro y tasa de partos. No hubo diferencia significativa entre ninguno de los grupos en el número de recién nacidos. Conclusión: estos datos sugieren que el preparado homeopático Avena sativa puede ser directamente utilizado en las células espermáticas para mejorar la tasa de partos en granjas tecnificadas de porcinos.Palabras llave: porcinos; rendimiento reproductivo; esperma; homeopatía; estudio ciego controlado.

Elevated progesterone concentrations enhance prostaglandin F2alpha synthesis in dairy cows.

The objective was to evaluate the influence of varying plasma progesterone (P(4)) concentrations throughout the luteal phase in dairy cows on PGF(2alpha) production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF(2alpha); PGFM) following treatment with estradiol-17beta (E(2)) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n=9), received 20mg LH (n=7), or had ovarian follicles larger than 6mm aspirated (n=8). In Experiment 2, cows on Day 6 were untreated (n=9) or received 5000 IU hCG (n=10). In Experiments 1 and 2, all cows received 3mg E(2) on Day 17, and blood samples were collected every 30 min from 2h before to 10h after E(2). Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n=4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n=7) received the alternate sequence. Blood was collected daily for measurement of P(4) 4h after concentrate feeding. On Day 17, blood was collected from 1h before to 1h after a 100 IU OT injection. In Experiment 1, both plasma P(4) and release of PGF(2alpha) were similar between LH-treated and control cows (P>0.10). In Experiment 2, plasma P(4) was elevated to a greater extent on Day 17 in cows treated with hCG (P<0.05) and plasma PGFM was also greater in hCG-treated animals (treatment x time interaction; P<0.05). In Experiment 3, there was a group x period interaction (P<0.01) for plasma P(4), indicating that less concentrate feeding was associated with greater plasma P(4). Release of PGF(2alpha) in response to OT was greater for cows receiving less concentrate (group x period interaction; P<0.05). In conclusion, dairy cows with more elevated blood P(4) concentrations released more PGF(2alpha) in response to E(2) or OT.