Effect of honey supplementation on Egyptian buffalo semen

This study testified the effect of honey supplementation (0.5-4.0%) in milk on the quality of chilled and frozen buffalo spermatozoa. Semen was chilled with/without honey and examined for motility, viability, plasma membranes integrity by hypo-osmotic swelling test (HOS) at 0, 1, 2 and 4 h. Frozen-thawed semen was examined for the same criteria beside the viability index and in vitro cleavage rate. The motility, livability and HOS of chilled semen upsurge with honey supplementation 1.0-2.0%. The normality of chilled spermatozoa was improved in the presence of 2.0-4.0% of honey at 4 h. Tail abnormalities decreased with milk honey 0.5, 1.0 and 2.0% at 2, 1 and 4 h, respectively. Incorporation of honey in milk extenders at levels of 0.5-2.0% was associated with an enhanced post equilibration motility. The post-thawing motility showed a steady increase with honey levels. The viability index increased (P < 0.001) with milk-honey 2.0% (109.00 ± 9.91) and 4.0% (112.00 ± 14.41). In vitro cleavage rate was clearly (P 0.001) enhanced in the co-existence of milk-honey 2.0% compared with control (74.00 vs. 45.83). In the meantime, a reasonable high cleavage rate (67.00%) was encountered with milk honey 0.5%. In conclusion, incorporation of honey in skim milk extenders is promising to enhance the characteristics and fertilizing potential of stored buffalo’s semen due to its nutritive and protective properties.

Effect of honey supplementation on Egyptian buffalo semen

This study testified the effect of honey supplementation (0.5-4.0%) in milk on the quality of chilled and frozen buffalo spermatozoa. Semen was chilled with/without honey and examined for motility, viability, plasma membranes integrity by hypo-osmotic swelling test (HOS) at 0, 1, 2 and 4 h. Frozen-thawed semen was examined for the same criteria beside the viability index and in vitro cleavage rate. The motility, livability and HOS of chilled semen upsurge with honey supplementation 1.0-2.0%. The normality of chilled spermatozoa was improved in the presence of 2.0-4.0% of honey at 4 h. Tail abnormalities decreased with milk honey 0.5, 1.0 and 2.0% at 2, 1 and 4 h, respectively. Incorporation of honey in milk extenders at levels of 0.5-2.0% was associated with an enhanced post equilibration motility. The post-thawing motility showed a steady increase with honey levels. The viability index increased (P < 0.001) with milk-honey 2.0% (109.00 ± 9.91) and 4.0% (112.00 ± 14.41). In vitro cleavage rate was clearly (P 0.001) enhanced in the co-existence of milk-honey 2.0% compared with control (74.00 vs. 45.83). In the meantime, a reasonable high cleavage rate (67.00%) was encountered with milk honey 0.5%. In conclusion, incorporation of honey in skim milk extenders is promising to enhance the characteristics and fertilizing potential of stored buffalos semen due to its nutritive and protective properties.(AU)

Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle / Efeito do diâmetro folicular, momento da primeira clivagem e metilação da H3K4 na produção embrionária de vacas Bos indicus

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryorates are obtained from embryos that cleave within 34 h after fertilization and from those produced fromfollicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage ofembryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattleembryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a keyrole in maintaining embryo viability at preimplantation stage.

Este estudo teve como objetivo investigar a relação entre a epigenética, o diâmetro folicular e a velocidade de clivagem, avaliando o potencial de desenvolvimento e a ocorrência de monometilação da H3K4 em embriões Bos indicus de clivagem precoce, intermediária e tardia produzidos a partir de oócitos fertilizados in vitro oriundos de folículos de até 2 mm de diâmetro ou entre 4 e 8 mm de diâmetro. Oócitos (n = 699) de folículos pequenos (? 2 mm) e 639 oócitos de folículos grandes (4-8 mm) foram puncionados de 1982 ovários de vacas Bos indicus de abatedouro. Após a maturação e fertilização in vitro (FIV), os embriões cultivados foram separados nos grupos de clivagem precoce (? 28 h pós-FIV), intermediária (> 28 h e ? 34 h pós-FIV) e tardia (> 34 h e ? 54 h pós-FIV). Os blastocistos foram submetidos à imunofluorescência para investigação de H3K4me. A taxa de blastocisto para embriões provenientes de folículos grandes (36,3%) foi maior que de folículos pequenos (22,9%; p 0,05). Ainda, as taxas de blastocisto para os grupos de clivagem precoce e intermediária (45,3% e 33,8%, respectivamente) foram maiores que para o grupo de clivagem tardia (13,5%; p 0,05). Blastocistos de todos os grupos mostraram marcação para H3K4me à imunofluorescência, particularmente intensa no que pareciam ser células do trofectoderma e fraca ou ausente em células semelhantes às da massa massa celular interna. Pela primeira vez em embriões indicus, os dados deste estudo demonstram que maiores taxas deblastocisto são obtidas de embriões que clivam em até 34 h pós-fertilização e dos oriundos de folículos de 4 a 8 mm de diâmetro, indicando uma maior habilidade desses embriões de se desenvolverem até o estágio de pré-implantação embrionária.

Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle / Efeito do diâmetro folicular, momento da primeira clivagem e metilação da H3K4 na produção embrionária de vacas Bos indicus

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryorates are obtained from embryos that cleave within 34 h after fertilization and from those produced fromfollicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage ofembryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattleembryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a keyrole in maintaining embryo viability at preimplantation stage.(AU)

Este estudo teve como objetivo investigar a relação entre a epigenética, o diâmetro folicular e a velocidade de clivagem, avaliando o potencial de desenvolvimento e a ocorrência de monometilação da H3K4 em embriões Bos indicus de clivagem precoce, intermediária e tardia produzidos a partir de oócitos fertilizados in vitro oriundos de folículos de até 2 mm de diâmetro ou entre 4 e 8 mm de diâmetro. Oócitos (n = 699) de folículos pequenos (? 2 mm) e 639 oócitos de folículos grandes (4-8 mm) foram puncionados de 1982 ovários de vacas Bos indicus de abatedouro. Após a maturação e fertilização in vitro (FIV), os embriões cultivados foram separados nos grupos de clivagem precoce (? 28 h pós-FIV), intermediária (> 28 h e ? 34 h pós-FIV) e tardia (> 34 h e ? 54 h pós-FIV). Os blastocistos foram submetidos à imunofluorescência para investigação de H3K4me. A taxa de blastocisto para embriões provenientes de folículos grandes (36,3%) foi maior que de folículos pequenos (22,9%; p 0,05). Ainda, as taxas de blastocisto para os grupos de clivagem precoce e intermediária (45,3% e 33,8%, respectivamente) foram maiores que para o grupo de clivagem tardia (13,5%; p 0,05). Blastocistos de todos os grupos mostraram marcação para H3K4me à imunofluorescência, particularmente intensa no que pareciam ser células do trofectoderma e fraca ou ausente em células semelhantes às da massa massa celular interna. Pela primeira vez em embriões indicus, os dados deste estudo demonstram que maiores taxas deblastocisto são obtidas de embriões que clivam em até 34 h pós-fertilização e dos oriundos de folículos de 4 a 8 mm de diâmetro, indicando uma maior habilidade desses embriões de se desenvolverem até o estágio de pré-implantação embrionária. (AU)

Cryotop and development of vitrified immature bovine oocytes / Cryotop no desenvolvimento de oócitos bovinos imaturos vitrificados

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10 percent DMSO + 10 percent ethylene glycol (EG) for 30-45sec and 2: 20 percent DMSO + 20 percent EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58 percent respectively. Vitrified oocytes using OPS and EMG showed 26 and 32 percent; and 35 and 46 percent of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes.(AU)

Avaliou-se a eficácia de diferentes dispositivos de congelamento (envasamento em palhetas (EP), microscopia eletrônica de grade (MEG) e Cryotop) para vitrificação de ovócitos imaturos de bovinos. Para tal, foram determinados o corpo polar, a metáfase II (MII), a viabilidade e as subsequentes taxas de desenvolvimento. Foram utilizados somente ovócitos com quatro ou cinco camadas de células do cumulus. Os ovócitos foram equilibrados em duas soluções de vitrificação - 1: DMSO (10 por cento) + etilenoglicol (EG; 10 por cento) por 30 a 45 segundos e 2: DMSO (20 por cento) + EG (20 por cento) + sacarose (0,5M) por 25 segundos -, transferidos para os dispositivos de congelamento e mantidos, por 10 dias, em nitrogênio líquido. Imediatamente após serem retirados do nitrogênio, os ovócitos foram removidos dos dispositivos e processados para maturação, fertilização e cultivo in vitro. Os ovócitos vitrificados com o Cryotop apresentaram as maiores taxas de extrusão do corpo polar (CP) e de maturidade nuclear (MII), 41 e 58 por cento, respectivamente. Para os ovócitos vitrificados com EP e MEG, as taxas de CP e as de MII foram, respectivamente, de 26 e 32 por cento e de 35 e 46 por cento. As taxas de viabilidade não diferiram entre os grupos Cryotop e EMG. Os ovócitos vitrificados com Cryotop apresentaram as maiores taxas de clivagem e de blastocisto. Para todas as variáveis estudadas, as taxas para os ovócitos vitrificados foram significativamente menores do que as do grupo-controle (P<0,05). Os resultados deste estudo mostraram a superioridade do dispositivo Cryotop para vitrificação de ovócitos imaturos de bovinos.(AU)

Improving in vitro maturation and cleavage rates of buffalo oocytes

The aim of the present study was to improve in vitro maturation and cleavage rates of buffalo oocytes. Good quality oocytes were divided into two experiments. In Experiment 1 oocytes were cultured for 24 h in a CO2 incubator at 38.5oC either in TCM-199, Hams F-10, MEM or FertiCult medium supplemented with either 10% FCS or 0.3% BSA. Experiment 2 was carried out to investigate the effect of different hormones (either 50 µg/ml eCG, 50 µg/ml FSH or 1 mg/ml E2) added to four of the afore mentioned media enriched with 10% FCS at the same culture conditions. Matured oocytes were fertilized in vitro using frozen thawed semen capacitated with heparin and caffeine. The sperm-oocytes were co-cultured for 22 h in BO or TALP medium. The fertilized oocytes were cultured in either BO or TALP medium for an additional five days at the same culture conditions and checked daily for cleavage. Addition of FCS to all media led to a higher maturation rate, without any significant variation, than BSA did (75.6 vs. 71.3%). Although no influence on the maturation rate was observed, addition of eCG to TCM-199, Hams F-10 or MEM media resulted in a non-significant increase of in vitro maturation rate of buffalo oocytes compared to other hormonal additives to the same media. Furthermore, supplementation of maturation media with eCG resulted in a non-significant higher in vitro maturation rate of buffalo oocytes compared to FSH and E2 (80.4% for eCG supplemented media vs. 74.0% and 73.0% for FSH and E2, respectively). There was a non-significant difference in the cleavage rate of buffalo oocytes matured in TCM-199 supplemented with either sera or hormones and fertilized either in BO or TALP medium. However, the highest non-significant cleavage rate was achieved when oocytes were matured in TCM-199 supplemented with eCG and fertilized in TALP medium (50%). The overall cleavage rate was not significantly greater in TALP than in BO medium (33.7 vs. 15.5%). It could be concluded that supplementation of maturation media with FCS and/or eCG could successfully improve IVM rate of buffalo oocytes. Furthermore, high cleavage rate could be achieved when oocytes were matured in TCM-199 supplemented with FCS and eCG and fertilized in TALP medium.(AU)

Improving in vitro maturation and cleavage rates of buffalo oocytes

The aim of the present study was to improve in vitro maturation and cleavage rates of buffalo oocytes. Good quality oocytes were divided into two experiments. In Experiment 1 oocytes were cultured for 24 h in a CO2 incubator at 38.5oC either in TCM-199, Ham’s F-10, MEM or FertiCult medium supplemented with either 10% FCS or 0.3% BSA. Experiment 2 was carried out to investigate the effect of different hormones (either 50 µg/ml eCG, 50 µg/ml FSH or 1 mg/ml E2) added to four of the afore mentioned media enriched with 10% FCS at the same culture conditions. Matured oocytes were fertilized in vitro using frozen thawed semen capacitated with heparin and caffeine. The sperm-oocytes were co-cultured for 22 h in BO or TALP medium. The fertilized oocytes were cultured in either BO or TALP medium for an additional five days at the same culture conditions and checked daily for cleavage. Addition of FCS to all media led to a higher maturation rate, without any significant variation, than BSA did (75.6 vs. 71.3%). Although no influence on the maturation rate was observed, addition of eCG to TCM-199, Ham’s F-10 or MEM media resulted in a non-significant increase of in vitro maturation rate of buffalo oocytes compared to other hormonal additives to the same media. Furthermore, supplementation of maturation media with eCG resulted in a non-significant higher in vitro maturation rate of buffalo oocytes compared to FSH and E2 (80.4% for eCG supplemented media vs. 74.0% and 73.0% for FSH and E2, respectively). There was a non-significant difference in the cleavage rate of buffalo oocytes matured in TCM-199 supplemented with either sera or hormones and fertilized either in BO or TALP medium. However, the highest non-significant cleavage rate was achieved when oocytes were matured in TCM-199 supplemented with eCG and fertilized in TALP medium (50%). The overall cleavage rate was not significantly greater in TALP than in BO medium (33.7 vs. 15.5%). It could be concluded that supplementation of maturation media with FCS and/or eCG could successfully improve IVM rate of buffalo oocytes. Furthermore, high cleavage rate could be achieved when oocytes were matured in TCM-199 supplemented with FCS and eCG and fertilized in TALP medium.