The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.
Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient [Validação comparativa utilizando PCR quantitativo em tempo real (qPCR) ABSTRACTThe objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR) of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05). The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.
The effectiveness of induction of the acrosome reaction (AR) test as a parameter to in vitro estimate embryo production (IVP) in Nelore breed and the AR pattern by the Trypan Blue/Giemsa (TB) stain were evaluated. Frozen semen samples from ten Nelore bulls were submitted to AR induction and were also evaluated for cleavage and blastocyst rates. The treatments utilized for AR induction were: control (TALP medium), TH (TALP medium + 10μg heparin), TL (TALP medium + 100μg lysophosphatidylcholine) and THL (TALP medium + 10μg heparin + 100μg lysophosphatidylcholine). Sperm acrosomal status and viability were evaluated by TB staining at 0 and after 4h incubation at 38°C. The results obtained for AR presented a significant difference (P<0.05) in the percentage of acrosome reacted live sperm after 4h of incubation in the treatments that received heparin. The cleavage and blastocyst rates were 60% and 38% respectively and a significant difference was observed among bulls (P<0.05). It was founded a satisfactory model to estimate the cleavage and blastocyst rates by AR induction test. Therefore, it can be concluded that the induction of the AR test is a valuable tool to predict the IVP in Nelore breed.Keywords: cattle, fertility, semen, acrosome reaction, fertilization Palavras-chave: bovino, fertilidade, sêmen, reação acrossomal, fertilização RESUMO Avaliou-se a eficiência da técnica de indução da reação acrossomal (RA) como parâmetro para estimar a produção in vitro (PIV) de embriões Nelore e analisou-se o padrão de RA pela técnica de coloração Azul de Tripan/Giemsa (TB). Amostras de sêmen congelado de dez touros foram submetidas à indução da RA e avaliadas quanto a taxa de clivagem e blastocisto. Os tratamentos utilizados para indução da RA foram: controle (meio TALP), TH (meio TALP + 10μg heparina), TL (meio TALP + 100μg lisofosfatidilcolina) e THL (meio TALP + 10μg heparina + 100μg lisofosfatidilcolina
The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y-specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.Keywords: density gradient centrifugation, embryo sex ratio, Percoll™, swim-up RESUMO
The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvement programs can increase the profitability of cattle production, in particular when it does not decrease reproductive efficiency. The aim of this work was to evaluate the sex ratio deviation of thawed bovine semen processed by density gradient centrifugation and swim-up. Semen doses were collected from ten bulls of different breeds, and each experimental group was replicated ten times.A Percoll™ gradient was prepared by mixing Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) isotonic solutions with Percoll™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) stock solution with 0.3% BSA, resulting in densities ranging from 1.110 to 1.123 g mL-1. The layers of discontinuous density gradient were disposed from the larger density (bottom of the tube) to the smaller into 15-mL conical centrifuge tubes. About 40 million sperm were overlaid on Percoll™ gradient and were centrifuged at 500 g for 15 min, at 22°C.The thawed semen samples were deposited in 15-mL conical centrifuge tubes containing 5 mLof DMEM and centrifuged twice at 300 g for 5 min for extender removal. After the second centrifugation, the supernatant was discarded and the sediment diluted in 1.0 mL of DMEM supplemented with 0.3% BSA. The tube was maintained in an incubator at 38.5°C and 5% CO2 for 1 h. One milliliter of supernatant was recovered and evaluated for the sperm motility and vigor. Eighty million sperm were submitted to the swim-up method. For the association of density gradient and swim-up, the swim-up supernatant was recovered and overlaid on Percoll™ gradient. The gradients were centrifuged and the sperm pellet recovered. The recovered sperm were submitted to DNA extraction with phenol-chloroform. Quantitative Real Time PCR (Parati et al. 2006 Theriogenology 66, 2202-2209) was used for the determination of the proportion of X-chromosome-bearing sperm, after centrifugation through density gradient and after swim-up. The results of amplification were analyzed in 7500 Sequence Detection System Software (Applied Biosystems, Foster City, CA, USA), using Relative Quantification (Ct) Study assay. The results of X-sorted sperm samples analyzed by multiple pairwise comparisons (Tukey) were not different (P > 0.05). The percentage of X-chromosome-bearing sperm after the density gradient comprised 50.3% of the sample, after the swim-up was 49.9% and after swim-up combined with Percoll™ gradient centrifugation was 56.6%. Therefore, sperm sex selection using Percoll™ gradient centrifugation and swim-up was not effective.
Recently, ultrasonography has been used to study reproductive tract and testes development in the bull. The testicular ultrasound allows identification of subtle changes in the echotexture of the testicular parenchyma in the different stages of reproductive development. The aim of the present study was to characterize the pixel intensity (echotexture) of testicular ultrasonograms of Guzerat bulls in the peripubertal period to attempt to raise efficiency of sire selection programs and puberty identification. Seventy-five animals from 9 to 30 months of age were evaluated monthly for 6 consecutive months. The testes were examined using a 5-MHz linear array transducer connected to a B-mode ultrasound scanner (Fukuda®, Tokyo, Japan). Images were frozen on the monitor of the ultrasound scanner and recorded on VHS tape. The images were digitalized and the average pixel intensity (PI) of the testes was determined by the J1.58 (National Institutes of Health, Bethesda, MD, USA) software image package. Additionally, all of the bulls were submitted to a complete andrological examination and seminal collection was attempted when males had a scrotal perimeter > 20 cm. Onset of puberty was considered to have occurred when at least one motile sperm cell was detected in the ejaculate. Statistical analysis was performed using the SAS statistical package (SAS Institute Inc., Cary, NC, USA; Student-Newman-Keuls test and Pearson correlation). The PI in pubertal animals (95.0 ± 39.01 cm3) was higher (P < 0.05) than non-pubertal animals (56.5 ± 43.1 cm3). Changes in patterns of testicular echotexture during the peripubertal stage reflect the gradual and significant increase (P < 0.05) of the intensity of pixels observed in animals from 9 months (36.7 ± 22.2 cm3) up to 30 months of age (127.4 ± 46.2 cm3). The correlation between age and intensity of pixels was significant (P < 0.05; r = 0.534). The increase of PI is an important indicator of puberty in bovine males. Nevertheless, it was not possible to establish a pattern of testicular echotexture that characterized the precise time of onset of puberty. Thanks to Fazenda do Rosário, CAPES, and CNPq.
The aims of this study were to separate X-chromosome-bearing bovine sperm by discontinuous Percoll ™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradients, validate the sexing of resultant IVF embryos by PCR, replace the bovine fetal serum (BFS) with BSA in the culture medium, to decrease male development advantage, and verify whether the gradient can be used in an IVF laboratory routine. The gradient was prepared by mixing Dubelcco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) with Percoll™ isotonic solution with 0.3% BSA, for different densities obtained ranging from 1.110 to 1.123 g mL-1, disposed in 3 layers into 15-mL conical tubes. For sexing, 40 million thawed sperm were overlaid on density gradients. The tubes were centrifuged at 500 g, for 15 min, at 22°C. After centrifugation, sperm sediment was used for IVF. For the control group, a Percoll™ 45, 90% gradient was used. The oocytes were selected from ovaries from slaughterhouse and maturated for 24 h in TCM-199 medium. After fertilization, oocytes and sperm were incubated for 20 h in 5% CO2, in humidified air at 38.5°C. Presumptive zygotes were denuded of cumulus cells, and washed in modified SOF medium and then transferred to 500 μL SOF in four well dishes. Embryo culture was carried out under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5°C, and the cleavage assessed at 46 h and development to the blastocyst stage at Day 7. To obtain embryonic cell DNA for sex determination by PCR, 115 embryos of the sexed and 82 of the control group were used. Two pairs of primers of Y-specific sequences were split in two distinct samples. The first pair detected a sequence of 210 bp, and the second one 196 bp of the bovine Y-chromosome. A third one detected an autosomal sequence of 280 bp, indicating the presence of bovine genomic DNA. PCR multiplex was carried out in the same tube with first and third primers and the PCR of the second one was carried out in another tube. The results were analyzed by X2. Of the sexed group, from a total of 373 oocytes, the cleavage rate was 58.2% (n = 217); 35.6% (n = 133) produced embryos; 36.5% (n = 42) were male embryos and the female embryo rate was 63.5% (n = 73). From a total of 268 control oocytes, the cleavage rate was 63.8% (n = 171); produced embryos 37.3% (n = 100); 57.3% (n = 47) were male embryos and the female rate was 42.7% (n = 35). The Percoll™ density gradient for sperm sexing altered the proportion of IVF embryos toward more females. Because of fast and easy preparation, the gradient can be used routinely in an IVF laboratory and also, BSA can replace FBS for the IVF. FAPESP process number 59357-9 and CAPES
This study was designed to investigate if the time of onset of FSH treatment [near the emergence of first or last follicular wave on progesterone (P4) protocol] influenced the superovulatory response and embryo yield in Santa Ines ewes during breeding season. Days of emergence of the follicular waves were defined in a previous study that evaluated the follicular dynamic in oestrus synchronization treatments (Oliveira et al. 2011 Acta Sci. Vet. 40). We observed emergence of the first and last follicular wave on 5.69 ± 0.42 and 11.25 ± 0.39 days of protocol, respectively. Twenty Santa Ines ewes were submitted to 2 superovulatory protocols according to the time that FSH treatments were initiated (G-first wave, n = 10; G-last wave, n = 10). On Day 0, all ewes received a P4 device (CIDR®; Pfizer Animal Health, New York, NY, USA) and injection of 37.5 µg of d-cloprostenol, IM. The FSH treatments started on Day 6 and Day 11 of protocol for G-first and G-last, respectively. The superovulatory regimen consisted of 8 IM injections of pFSH administrated twice daily (40, 40, 30, 30, 20, 20, 10, and 10 mg of pFSH). The P4 device was removed on Day 8 and Day 13 for G-first and G-last, respectively. At these times, all ewes received another injection of 37.5 µg of d-cloprostenol and a dose of 200 IU of eCG. During 4 days after the P4 device removal, ewes were mated by a fertile ram. Embryo collections were accomplished 7 days after CIDR withdrawal. The ovaries were evaluated by ultrasonography (3 times daily, during the mating period) and laparotomy (concomitantly with embryo collection). The superovulatory response was observed by classified by score: 0 = 4 or fewer corpora lutea (CL); 1 = between 5 and 10 CL; and 2 = 11 or more CL. Data were analysed by GLIMMIX using SAS software (SAS Institute Inc., Cary, NC, USA). All donors from G-first had superovulatory response classified as score 2, whereas 60% of ewes from G-last were classified as score 2, 20% as score 1, and 20% as score 0 (P < 0.05). There were effects between treatments (P < 0.05) for ovulation rate (G-first: 97.9 ± 1.4% v. G-last: 88.5 ± 4.4%) and number of ovulations (G-first: 17.0 ± 2.3 v. G-last: 12.5 ± 2.6). The numbers of luteinized unovulated follicles were 0.7 ± 0.5 for G-first and 1.2 ± 0.4 for G-last (P > 0.05). There was no difference between G-first and G-last (P > 0.05) in the rate of ova/embryos recovered (54.9 ± 5.7% v. 49.3 ± 8.5%), mean number of ova/embryos recovered (9.0 ± 1.4 v. 6.3 ± 1.1), number of viable embryos (3.8 ± 1.5 v. 3.4 ± 0.8), or viability rate (40.3 ± 10.8 v. 53.4 ± 12.1). In conclusion, the FSH treatment started near the emergence of the first follicular wave of progesterone protocol in Santa Ines ewes during the breeding season resulted in a higher superovulatory response than that started near the last follicular wave; however, no improvements in embryo yield were observed. Financial support: FAPESP.
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