Effect of different concentrations of inulin on ram sperm quality during cryopreservation.

BACKGROUND: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation. OBJECTIVES: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing. MATERIALS AND METHODS: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196 degree C. RESULTS: The total motility, viability, and DNA integrity were significantly improved after freeze-thawing with 28 mM inulin, compared to other treatment groups (P < 0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p <0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P < 0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P < 0.05). CONCLUSION: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm. Doi.org/10.54680/fr24510110512.

The effect of different concentrations of laminarin on the quality of cryopreserved ram semen.

BACKGROUND: Increasingly, sheep breeders are using artificial insemination to produce lambs, so finding methods that preserve ram sperm can be useful. OBJECTIVE: To determine the protective effects of different concentrations of laminarin on ram sperm motility, viability, abnormalities, membrane, and DNA integrity, superoxide dismutase enzyme (SOD) activity, and malondialdehyde (MDA) production after freeze-thawing. MATERIALS AND METHODS: The ejaculates of four rams were collected and stored at 35 degree C. Semen samples were diluted with a tris-base extender containing 100, 200, 400, and 800 ug/mL of laminarin and a control extender containing no laminarin, then frozen in liquid nitrogen after 4 h in the refrigerator. RESULTS: In the treatment of frozen-thawed spermatozoa with 800 ug/mL laminarin, motility, viability, membrane integrity, and DNA integrity were significantly higher than in the control. In spermatozoa that were exposed to 800 ug/mL laminarin after thawing, MDA production was significantly lower than in the control group. The percentage of abnormal spermatozoa in 800 ug/mL laminarin was significantly lower than that in the control. CONCLUSION: The addition of 800 ug/mL laminarin to the freezing extender increases motility, viability, SOD activity, and plasma membrane integrity, while reducing abnormality and MDA production in freeze-thawed ram semen. https://doi.org/10.54680/fr24110110812.

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle.

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26µM of BCB for 90min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB-: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB-: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB-). After fertilization, the zygotes produced from OBCB+ and OBCB- oocytes were further stained with BCB for 10min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB-, OBCB-/ZBCB++, OBCB-/ZBCB+ and OBCB-/ZBCB-). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P<0.05) than ZBCB treatment (ZBCB-; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB-: 28.8%) was less (P<0.01) than single application of BCB test (ZBCB-: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB- zygotes (36.6%) were greater (P<0.05) than that from C oocytes (25.7%), OBCB- oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P>0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB- zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB-: 48.0%) was greater (P<0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB-: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.

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

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.

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.