Skim Milk-glucose Research Articles

Effects of Percoll Washing and Glycosaminoglycans on the Lysophosphatidylcholin Induced Acrosomal Reaction in Stallion.

The present study was conducted to investigate the effects of skim milk glucose (SMG), percoll and Brackett and Oliphant medium supplemented with BSA (BO-BSA) washing and glycosaminoglycans treatments (GAG) on lysophosphatidylcholin (LPC)-induced acrosome reaction (AR) in stallion spermatozoa. Freshly collected semen was washed with SMG (300 g for 10 min), percoll (700 g for 30 min) and BO-BSA (300 g for 10 min). Part of the semen after each washing step was treated with LPC to asses the rate of AR. After SMG, percoll and BO-BSA washing, the spermatozoa were cultured with in 5 different concentrations of GAG (chondroitin sulfate; CS and heparin; 0, 3, 10, 30, 100 μg/ml) for 0, 1.5, 3 and 6 h at 38.5°C in 5% CO2 in air. The LPC-induced AR in fresh spermatozoa after washing with SMG, percoll and BO-BSA were 0.8%, 3.0%, 13.4% and 18.0%, respectively. A significant difference in the rate of AR was observed before and after percoll washing (P<0.05). The LPC-induced AR rates after CS and heparin treatments were 14.7-20.8% and 15.3-19.5%, respectively. No relationship was observed either with the concentration of GAG or culture time on the percentage of AR spermatozoa. Results of the present study indicate that the percoll and BO-BSA washing can potentiate the LPC induced AR in stallion spermatozoa. Moreover, CS or heparin may not be useful in inducing AR in stallion spermatozoa.

Effect of seminal extenders containing egg yolk and glycerol on motion characteristics and fertility of stallion spermatozoa

Three experiments were conducted to evaluate the effects of egg yolk and(or) glycerol added to a nonfat dried skim milk-glucose (NDSMG) extender on motion characteristics and fertility of stallion spermatozoa. In Experiment 1, ejaculates from each of 8 stallions were exposed to each of 4 extender treatments: 1) NDSMG, 2) NDSMG + 4% egg yolk (EY), 3) NDSMG + 4% glycerol (GL), and 4) NDSMG + 4% egg yolk + 4% glycerol (EY + GL). Samples were cooled at −0.7 °C/min from 37 to 20 °C; subsamples were then cooled at −0.05 or −0.5 °C/min from 20 to 5 °C. Percentages of motile spermatozoa (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h after initiation of cooling. There was no overall effect (P > 0.05) of cooling rate. PMOT was highest (P < 0.05) for spermatozoa extended in NDSMG + GL at 48 h. At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in NDSMG + EY. In Experiment 2, ejaculates from 8 stallions were exposed to each of 4 treatments: 1) NDSMG, 2) NDSMG + EY, 3) semen centrifuged in NDSMG and resuspended in NDSMG, and 4) semen centrifuged in NDSMG and resuspended in NDSMG + EY. Samples were cooled from 20 to 5 °C at each of 2 rates (−0.05, −0.5 °C/min). A detrimental interaction between seminal plasma and egg yolk was noted for PMOT at 6 h and for both MOT and PMOT at ≥24 h postcooling. Experiment 3 determined if egg yolk or glycerol affected fertility. The seminal treatments were 1) NDSMG, 2) ndsmg+ey with previous removal of seminal plasma, and 3) NDSMG + GL. All samples were cooled to 5 °C and stored 24 h before insemination. Embryo recovery rates 7 d after ovulation were lower for mares inseminated with spermatozoa cooled in NDSMG + EY (17%, 4 24 ) or NDSMG + GL (13%, 3 24 ) extenders, than semen cooled in NDSMG (50%, 12 24 ). We concluded that egg yolk (with seminal plasma removal) or glycerol added to NDSMG extender did not depress MOT or PMOT of cooled stallion spermatozoa but adversely affected fertility.

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.

Sperm concentration influences recovery of progressively motile spermatozoa and number of inseminations shipped in conventional containers

Summary Three ejaculates from each of 4 stallions were split into aliquots and diluted with skim milk-glucose extender to final concentrations of 25, 50, 100, 150, or 200 million progressively motile spermatozoa/ml. One aliquot per dilution was evaluated for the percentage progressive motility (PMS) and rate of forward movement (RFM) after 15 and 45 minutes of culture at 37°C. One aliquot was stored at 5°C for 24 hours and another for 48 hours at 5°C. The aliquots stored at 5°C were evaluated for PMS and RFM at 15 and 45 minutes of warming (37°C). For spermatozoa stored at 37°C following collection, concentration did not (P 6 progressively motile spermatozoa/ml provided higher (P 6 progressively motile spermatozoa/ml. However, in order to minimize insemination volume and maximize the number of inseminations available per shipment, a concentration of 100 x 10 6 progressively motile spermatozoa may be more appropriate than lower concentrations.

Assay of capacitated, freeze-damaged and extended stallion spermatozoa by filtration

Results of stallion semen assay with glass wool/Sephadex filters are highly correlated with fertility. A hypothesis was tested that mechanically damaged spermatozoa are trapped in glass wool while capacitated spermatozoa are trapped in Sephadex. Another objective was to assay stallion semen in extenders known to vary in their ability to maintain sperm fertility with these filters, and for sperm motility. In Experiment 1, ejaculates were split so that a portion was used for fresh semen filtration and another portion plunged into liquid nitrogen repeatedly to maximize the number of broken acrosomes. The remainder of the ejaculate was inseminated into the uterus of an estrous mare which was flushed 6–8 hours later. Semen from each treatment was filtered through cotton with and without Sephadex, and through glass wool with and without Sephadex. Ejaculates were also washed and incubated in a capacitation medium. Aliquotes were filtered every 2 hours for 10 hours through glass wool and Sephadex. In Experiment 2, stallion semen diluted in egg yolk-lactose, skim milk-glucose and Triscitrate-yolk was evaluated for motility and with glass wool and Sephadex filters daily for 4 days. In Experiment 1, more frozen-thawed spermatozoa were trapped in glass wool than in Sephadex filters. Filters containing Sephadex trapped all uterine-incubated cells, while cotton did not affect the trapping of spermatozoa. In the capacitation medium, the passage of cells through all filters decreased with time, and glass wool and Sephadex had additive effects. In Experiment 2, fewer spermatozoa passed Sephadex filters than glass wool filters after storage in the Tris-citrate extender, which is known to be detrimental to the fertility of stallion semen although it did maintain sperm motility. We suggest that Sephadex traps spermatozoa with capacitation-like changes.

Effect of serum on in vitro maturation of equine oocytes

This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38°C in either air or 5% CO2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-mL borosilicate tubes filled with 6 mL (topped) of extended spermatozoa, 35-mm Petri dishes filled with 3 mL of extended spermatozoa, and 35-mm Petri dishes with 200-μL microdroplets of extended spermatozoa under sterile mineral oil. For all treatments, individual samples were removed at 2, 4, 6 and 12 h of incubation to determine the percentage of motile cells. Overall, spermatozoa incubated in Petri dishes in both 3-mL and microdroplet treatments had significantly higher motility than those incubated in glass tubes (P<0.01). At 6 and 12 h of incubation in Petri dishes, progressive motility was significantly higher for spermatozoa extended in the Hank's salts solution than in the other media. Both the medium and container used significantly affected the longevity of motility of spermatozoa incubated at 38°C.

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20°C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20°C or 3) insemination with semen stored for 24 h at 5°C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 × 10 6 progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20°C or from semen stored for 24 h at 5°C were the same ( 11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 ± 2.9, 19.6 ± 2.6 and 20.5 ± 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 ± 2, semen stored for 24 h at 20°C was 57 ± 11 and semen stored for 24 h at 5°C was 80 ± 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5°C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20°C than that stored at 5°C.

Vanadium Toxicity and Distribution in Chicks and Rats

Studies of the effect of dietary vanadium revealed that 25 ppm vanadium added to a dry skim milk-glucose monohydrate diet was toxic to chicks as indicated by both depression of growth and mortality. Ammonium metavanadate and vanadyl sulfate were equally toxic. The addition of the essential trace elements, iron, copper, molybdenum, cobalt, zinc, and manganese to the basal diet did not affect the toxicity of vanadium. Scandium, titanium, and niobium, elements which resemble vanadium in many of their chemical and physical parameters, were not toxic when fed at 200 ppm nor did they influence the toxicity of vanadium. Radioisotope studies revealed that scandium and niobium were poorly absorbed or poorly retained compared to vanadium. EDTA completely prevented the toxicity of vanadium apparently by preventing its absorption from the intestinal tract. Ingested V48 was found to be mainly concentrated in the bone and kidney of young chicks and in the kidney of adult rats.