Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT.

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Productive Performance, Physiological Variables, and Carcass Quality of Finishing Pigs Supplemented with Ferulic Acid and Grape Pomace under Heat Stress Conditions.

The effect of individual and combined supplementation of FA and GPM on physiological variables, productive performance, and carcass characteristics of finishing pigs under heat stress conditions were investigated. Forty Yorkshire × Duroc pigs (80.23 kg) were individually housed and randomly distributed into 4 groups under a 2 × 2 factorial arrangement (n = 10): Control (basal diet, BD); FA, BD + 25 mg FA; GPM, BD with 2.5% GPM; and MIX, BD with 25 mg FA and 2.5% GPM. Additives were supplemented for 31 days. The inclusion of FA or GPM did not modify rectal temperature and respiratory rate. There was an effect of the interaction on FI, which increased when only GPM was supplemented, with respect to Control and MIX (p < 0.05). Average daily gain (ADG) and feed conversion (FC) were not affected by treatments (p > 0.05). The inclusion of FA improved hot and cold carcass weight, while the addition of GPM decreased the marbling (p < 0.05) and tended to increase loin area (p < 0.10). GPM increased liver weight (p < 0.05). The addition of GPM and FA can improve some carcass characteristics under heat stress conditions. It is necessary to continue investigating different levels of inclusion of GPM and FA in finishing pigs' diets.

Chromium Methionine and Ractopamine Supplementation in Summer Diets for Grower-Finisher Pigs Reared under Heat Stress.

This study aimed to determine the effects of the dietary supplementation of chromium methionine (CrMet) and ractopamine (RAC) on pigs in the growing-finishing stage under heat stress. The parameters evaluated included productive behavior, blood components, carcass characteristics, organ weight, and meat quality. This study was conducted during the summer season in Sonora, Mexico. The treatments included: (1) control diet (CON), a base diet (BD) formulated to satisfy the nutritional requirements of pigs; (2) RAC, BD plus 10 ppm RAC supplemented during the last 34 days of the study; (3) CrMet-S, BD supplemented with 0.8 ppm of Cr from CrMet during the last 34 days; and (4) CrMet-L, BD supplemented with 0.8 ppm of Cr from CrMet for an 81 d period. RAC supplementation improved the productive behavior and main carcass characteristics of the pigs compared with CON. However, RAC and CrMet supplementation during the last 34 days showed similar results in terms of weight gain, carcass quality, blood components, organ weight, and meat quality. The addition of CrMet-S had a moderate (although not significant) increase in productive performance and carcass weight. These findings are encouraging, as they suggest that CrMet may be a potential alternative for growth promotion. However, more research is needed.

Addition of a Mixture of Plant Extracts to Diets for Growing-Finishing Pigs on Growth Performance, Blood Metabolites, Carcass Traits, Organ Weight as a Percentage of Live Weight, Quality and Sensorial Analysis of Meat.

The effect of plant extracts (PE; artichoke, celery, beet, onion, garlic, spinach, avocado, oats, and parsley) in the diet of growing pigs under heat stress was investigated. Parameters included growth performance, blood constituents, carcass characteristics, organ percentage, quality and sensory appraisal of the pork. The study was performed during the Mexican summer, using 60 pigs. Treatments included the control, to which 0.1% PE, and 0.15% PE were added. The use of PE (0.1 and 0.15%) generated an increase in the average daily gain (ADG, by 10.0% for both treatments), and final live weight (LW, by 6.3% and 6.8%) (p < 0.05). The level of blood albumin at 95 kg was higher when supplementing with 0.1% PE (p < 0.05). At 120 kg LW, creatine kinase values showed a tendency to be different (p = 0.07). Carcass weight increased (p < 0.05) when adding PE. Supplementation with 0.1% PE decreased (p < 0.05) the red/green (a *) hue of the meat, whereas supplementation with 0.1% and 0.15% PE increased the yellow/blue (b *) hue (p < 0.05). The addition of PE improves pig growth performance, and carcass weight by reducing the negative effects of heat stress, without markedly modifying blood constituents, meat quality, and sensory attributes of the pork.

Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O.

The ICSI-sperm mediated gene transfer (ICSI-SMGT) has been used to produce transgenic mice with high efficiency; however, the efficiency of this technique in farm animals is still less than desirable. Pretreatment of sperm with membrane destabilizing agents can improve the efficiency of ICSI in cattle. The objective of the present study was to evaluate streptolysin-O (SLO) as a novel treatment to permeabilize the bovine sperm membrane and assess its effect on efficiency of generating transgenic embryos by ICSI-SMGT. First, there was evaluation of the plasma membrane integrity (SYBR/PI), acrosome membrane integrity (PNA/FITC), DNA damage (TUNEL) and binding capacity of exogenous DNA (Nick Translation) in bull sperm treated with SLO. Subsequently, there was assessment of embryonic development and the efficiency in generating transgenic embryos with enhanced expression of the gene for green fluorescent protein (EGFP). Results indicate that SLO efficiently permeabilizes the plasma and acrosome membranes of bull spermatozoa and increases binding of exogenous DNA mostly to the post-acrosomal region and tail without greatly affecting the integrity of the DNA. Furthermore, treatment of bull spermatozoa with SLO prior to the injection of oocytes by ICSI-SMGT significantly increased the rate of embryo expression of the EGFP gene. Future experiments are still needed to determine the effect of this treatment on the development and transgene expression in fetuses and animals produced by ICSI-SMGT.