scholarly journals 21 EFFECTS OF HETEROLOGOUS SEMEN PLASMA AND SEMEN EXTENDERS ON PROGRESSIVE MOTILITY OF FROZEN - THAWED RAM SPERM

2005 ◽  
Vol 17 (2) ◽  
pp. 160
Author(s):  
G. Mataveia ◽  
S.J. Terblanche ◽  
J.O. Nöthling ◽  
D. Gerber
Keyword(s):  
Repeated Measures ◽  
Fixed Effects ◽  
Skim Milk ◽  
Random Order ◽  
Progressive Motility ◽  
Heat Treated ◽  
Ram Sperm ◽  
Bovine Oocytes ◽  
Better Than

Frozen-thawed ram semen crosses the cervix poorly, necessitating laparoscopic insemination. Acceptable fertility can be achieved with frozen-thawed ram semen deposited at the external cervical opening if ram semen plasma (SP) is added (McPhie et al. 2000 14th ICAR 2, 78 abst). Homologous SP improves the fertility of frozen-thawed sperm of boars and dogs. Heterologous SP may have effects as well; the addition of bovine SP increased the ability of buffalo sperm (Syncerus caffer) to fertilize bovine oocytes in vitro (de Haas et al. 2003 Theriogenology 59, 392). The aim of the current study was to compare the effects of SP of rams (SPR), bulls (SPB), and dogs (SPD), protein-free TALP, Triladyl (Minitüb, Tiefenbach, Germany), and skim milk upon longevity and percentage of progressively motile frozen-thawed ram sperm. Three ejaculates from each of six rams (2 Dorpers, 2 Döhne merinos, and 2 merinos), aged 2–4 years, were extended in Triladyl, pooled and frozen as a single batch per ram at 200 × 106/mL in 0.25-mL straws. SPR was obtained from the same rams and SPB from 5 bulls by centrifugation, while the post-sperm fractions were collected from 5 dogs (SPD). Within a species, the SP from different donors was pooled and frozen in aliquots at −18°C. The 108 straws (6 rams, 6 diluents, 3 replicates) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 mL of the appropriate fluid at 37°C and kept for 6 h. Percentage of progressively motile sperm was estimated at ×200 magnification immediately and 2, 4 and 6 h after thawing. One person thawed the semen and prepared motility specimens, while another performed all motility evaluations. Data were evaluated by means of repeated-measures ANOVA, with rams as subjects and time and fluid as fixed effects. Non-significant interactions were removed from the model. Means were compared by means of Bonferroni's test (P < 0.05). The model included ram, time, fluid, and ram × fluid, and time × fluid interactions, which were all significant (P < 0.01). Mean motility decreased from each time to the next and were 39.0% (0 h), 26.0% (2 h), 19.6% (4 h) and 12.6% (6 h), SEM 1.38%, n = 108. Mean motility was higher for skim milk (39.9%) than for all other fluids except Triladyl (27.7%), which was better than SPB (13.0%), whereas TALP (20.5%) and SPR (21.9%) were similar to Triladyl and SPB (n = 72, SEM 2.85%). The interactions (ram × fluid or time × fluid) were mainly due to SPD, SPR, Triladyl, and TALP, while milk resulted in the best and SPB in the lowest motility. This study shows that heat-treated skim milk maintains progressive motility of frozen-thawed ram sperm better than the SP of various species and protein-free TALP. In contrast to SPR, skim milk is known to result in poor fertility of frozen-thawed ram semen after cervical insemination. It would thus appear that maintenance of progressive motility in vitro may be a poor indicator of fertility after cervical insemination.

scholarly journals Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa

2010 ◽  
Vol 81 (3) ◽  
Author(s):  
G.A. Mataveia ◽  
S.J. Terblanche ◽  
J.O. Nothling ◽  
D. Gerber
Keyword(s):  
Beneficial Effect ◽  
Seminal Plasma ◽  
Skim Milk ◽  
Random Order ◽  
The Other ◽  
Progressive Motility ◽  
Time Zero ◽  
Prostatic Fluid ◽  
Heat Treated ◽  
Single Batch

Ram seminal plasma increases the fertility of frozen-thawed ram spermatozoa deposited into the cervix. The aim of the current study was to compare the effect of ram seminal plasma to that of bull seminal plasma, dog prostatic fluid, protein-free TALP, TrilEq (Triladyl with 0.5 mℓ of Equex STM paste added to each 100 mℓ) and heat-treated skim milk on longevity and percentages of progressively motile and aberrantly motile frozen-thawed ram spermatozoa. Three ejaculates from each of 6 rams were extended in TrilEq, pooled and frozen in straws as a single batch per ram. One hundred and eight straws (3 straws from each ram for each fluid) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85mℓ of the appropriate fluid at 37 °C and kept at that temperature for 6 h. Motility was assessed at x200 magnification immediately (time zero) and 2, 4 and 6 h after thawing. Progressive motility decreased from each time to the next (P < 0.05) and was 39.0% (0 h), 26.0% (2 h), 19.6% (4 h) and 12.6% (6 h); SEM 1.24, n=108 for each group. Ram seminal plasma resulted in higher progressive motility than bull seminal plasma, lower than milk, and similar to the other fluids. Ram seminal plasma resulted in lower aberrant motility than protein-free TALP and similar aberrant motility to other fluids. The effect of ram seminal plasma and dog prostatic fluid was very similar. The effect of ram seminal plasma on the fertility of frozen-thawed ram spermatozoa deposited into the cervix is not due an exceptionally beneficial effect on the motility of spermatozoa.

scholarly journals 150PREGNANCY AND PARTURITION FOLLOWING TRANSFER OF BOVINE EMBRYOS CULTURED IN TWO MEDIA UNDER TWO OXYGEN CONCENTRATIONS

2004 ◽  
Vol 16 (2) ◽  
pp. 197 ◽  
Author(s):  
A. Fischer-Brown ◽  
R. Monson ◽  
D. Northey ◽  
T. Kuhlka ◽  
J. Rutledge
Keyword(s):  
Birth Weight ◽  
Pregnancy Rate ◽  
Repeated Measures ◽  
Fixed Effects ◽  
Linear Models ◽  
Statistical Significance ◽  
Bovine Embryos ◽  
Crown Rump Length ◽  
Oxygen Treatment

Developmental aberrations following transfer of in vitro-produced bovine embryos can result in early gestational losses and offspring abnormalities. An ongoing study tests the hypothesis that such aberrations occur with equal frequency among commonly employed culture systems. In year 1, embryos were produced using oocytes from abattoir-derived ovaries (breed unspecified) and a proven Angus bull selected for low birth weight. IVC treatments were 2×2 factorial for medium (KSOMaa or SOFaa) and oxygen concentration (5% or 20%). Angus recipients (n=61; 32 cows, 29 heifers) were randomly allotted to treatments for Day 7 transfers. Pregnancy was diagnosed with ultrasound several times during gestation (Table 1). At parturition calf weight, shoulder height, chest circumference, crown-rump length, and humeral and femoral length data were collected. Statistical analyses (Statistical Analysis System, Cary, NC) were logistic regression with a binomial distribution for pregnancy rate, and the general linear models procedure for calf measurements; included were fixed effects of medium, oxygen, and their interaction, with additional fixed effects of dam parity and calf sex where appropriate. No significant effects of medium or oxygen were found for pregnancy rate or calf measurements other than birth weight. Mean birth weight was higher in the KSOM, 20% oxygen treatment (Table 1), and medium-oxygen interaction for calf weight was also significant (P&lt;0.01). In year 2 embryos were produced using the same Angus bull and Angus oocytes. Angus recipients (n=38; 32 cows, 6 heifers) were randomly allotted to treatments. Fetal crown-rump lengths were measured by ultrasound weekly from Days 33 to 54 and were analyzed as repeated measures using the mixed procedure. Pregnancy outcome and LS means for crown-rump lengths are included in Table 1. Though insufficient recipient numbers preclude determination of statistical significance, of interest is the relatively small fetal size in early gestation and large birth weights in the KSOM, 20% oxygen treatment. This treatment also contained a Day 33 pregnancy, subsequently lost by Day 40, in which the fetus was too small to obtain an accurate measurement. Fetal growth will continue to be monitored throughout gestation. Data will be collected at parturition as in year 1, and pooled analyses will be done. Table 1

137 Assessment of fertilizing ability of Merino ram semen cold stored up to 48h by heterologous IVF of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 194 ◽  
Author(s):  
D. A. Galarza ◽  
M. Ladrón de Guevara ◽  
P. Beltrán-Breña ◽  
M. J. Sánchez-Calabuig ◽  
A. López-Sebastián ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Skim Milk ◽  
Semen Parameters ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Straight Line ◽  
Fertilizing Ability ◽  
Pronuclear Formation ◽  
Bovine Oocytes

The use of cold-stored ram semen has been applied in sheep AI programs, because it preserves its fertilizing ability similar to fresh. Besides, the heterologous IVF has been successfully employed to assess semen fertilizing ability in several species. Hence, we aimed to evaluate the fertilizing ability of ram semen cold stored up to 48h at 5°C by assessing heterologous IVF using bovine oocytes. Fifteen pools of 3 normospermic Merino ram (2-7 years) ejaculates were collected using artificial vagina, diluted to 200×106 spermatozoa mL−1 with ultra-heat-treatment-based extender (skim milk-6% egg yolk) and cold stored up to 48h. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF using fresh semen (FS, n=707), semen cold stored to 24h (CS24, n=832) or semen cold stored to 48h (CS48, n=611). In parallel, homologous IVF (control, n=1356) and parthenogenesis (parth control non-fertilized oocytes, n=334) were performed. Ram non-selected and selected (BoviPure, Nidacon International, Mölndal, Sweden) semen parameters were evaluated by computer-assisted semen analysis. Sperm-oocyte interaction was assessed at 2.5h post-insemination (hpi) by evaluating the number of bound spermatozoa, whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using phase contrast and confocal microscopy. Cleavage rate was evaluated in all groups at 48 hpi. Data obtained from 5 replicates were analysed using one-way ANOVA. Data was expressed as mean±standard error of the mean. In terms of sperm storage time, non-selected semen showed a significant decrease (P&lt;0.05) for CS24 and CS48 compared with FS on progressive motility [SPM (%): 52.30±4.1 and 36.9±5.5v. 71.3±1.6] and straight-line velocity (mm s−1: 132.2±6.1 and 109.7±6.3v. 176.7±4.3), respectively. However, selected semen showed a decrease (P&lt;0.05) only for CS48 when compared with CS24 or FS on SPM (35.6±3.9v. 56.1±6.91 and 59.3±2.6) and straight-line velocity (83.5±4.4v. 105.3±6.5 and 110±2.0), respectively. No differences were observed between heterologous IVF groups in all parameters evaluated. Homologous IVF showed a higher percentage of penetration only when compared with heterologous FS group (44.4±6.8v.12.5±4.5%; P&lt;0.01). The polyspermy was higher in heterologous CS24 group when compared with homologous IVF (11.4±3.4v. 3.8±2.2; P&lt;0.05). The homologous IVF group, as expected, showed the higher percentage of pronuclear formation at 18 hpi compared with heterologous IVF with FS (67.3±5.8v. 35.2±5.6%), CS24 (72.1±4.5 v. 37.2±5.7%) and CS48 (63.0±6.0 v. 27.0±5.6%), respectively (P&lt;0.001). Likewise, cleavage rate was higher in homologous group compared with heterologous IVF and parthenogenetic groups for FS (78.3±2.6.8v. 46.3±3.2 and 7.0±2.3%), CS24 (78.4±2.6 v. 48.3±3.2 and 4.9±2.0%), and CS48 (78.4±3.3 v. 43.3±3.5 and 4.3±1.2%), respectively (P&lt;0.001). In conclusion, Merino ram semen cold stored up to 48h maintains its fertilization ability to the same extend as fresh and can be used for sheep crossbreeding programs.

Effect of nutrient composition on the in vitro growth of urogenital lactobacilli and uropathogens

1998 ◽  
Vol 44 (9) ◽  
pp. 866-871 ◽  
Author(s):  
Gregor Reid ◽  
Foaud Soboh ◽  
Andrew W Bruce ◽  
Marc Mittelman
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Skim Milk ◽  
In Vitro Study ◽  
Defined Medium ◽  
Nutrient Composition ◽  
Vaginal Fluid ◽  
Natural Substances ◽  
Uropathogenic Bacteria ◽  
Better Than

Previous clinical studies have shown that nutrients and probiotic agents can alter the composition of the vaginal flora. The present in vitro study has shown that uropathogens have a growth advantage over lactobacilli, but potentially there are natural substances that could be applied vaginally to stimulate lactobacilli growth to the detriment of the pathogens. When chemically defined medium representative of vaginal fluid at pH 5.5 was supplemented with skim milk, it acted as a better substrate for Lactobacillus rhamnosus GR-1 than for uropathogenic bacteria and Candida albicans. Lactobacillus MRS medium, even at pH 4.5, supports the growth of pathogens, but when supplemented with ascorbic acid or EDTA, Lactobacillus growth was significantly higher. When L. rhamnosus GR-1 was coincubated in a combined nutrient composition of vitamins and lactose, it survived better than Escherichia coli and Enterococcus faecalis. These in vitro results provide a basis for testing nutritional supplements to alter the urogenital flora in an attempt to enhance restoration and maintenance of a normal disease-free state.Key words: nutrients, lactobacilli, uropathogens, growth.

21 EVALUATION OF SEMEN EXTENDERS FOR SHORT-TERM STORAGE OF RAM SEMEN AT 4°C

2017 ◽  
Vol 29 (1) ◽  
pp. 118
Author(s):  
M. Acharya ◽  
J. M. Burke ◽  
C. Hansen ◽  
R. W. Rorie
Keyword(s):  
Ethylene Glycol ◽  
Repeated Measures ◽  
Mixed Model ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Dilution Factor ◽  
Sperm Analysis ◽  
Progressive Motility ◽  
Ram Sperm ◽  
Over Time

Preliminary studies found that progressive motility of ram sperm declined ~75% when stored at 4°C for 24 h, and continued to decline over time when using extenders supplemented with 5% egg yolk. The current study evaluated the effects of different combinations of extenders, ethylene glycol (EG), egg yolk, and penicillamine, hypotaurine, and epinephrine on ram sperm progressive motility during storage. Semen collected from 3 Katahdin and 2 Suffolk rams by electroejaculation was distributed across treatment combinations consisting of either TRIS citrate or milk extender supplemented with 5 or 20% (v/v) egg yolk, ± 1% ethylene glycol (EG) and ± 20 µM penicillamine, 10 µM hypotaurine and 2 µM epinephrine (PHE). For each semen collection, TRIS citrate extender was prepared from a 4× solution so that the TRIS, citric acid and fructose concentration were constant at 300, 94.7, 27.8 mM, respectively, regardless of semen dilution factor. A 4× milk extender was also used so that the extender contained 10% (w/v) milk powder, regardless of semen dilution factor. Both extenders were supplemented with 50 µg mL−1 of gentamicin. Semen was diluted in extender to a final concentration of 300 million sperm/mL in 1.5-mL tubes, and cooled to 4°C over a 2- to 3-h period. Semen was evaluated initially and daily for 3 days, using computer-assisted sperm analysis. Repeated-measures data were analysed using the mixed model (JMP 12.0 software; SAS Institute Inc., Cary, NC, USA) for main effects of extender, supplements, and their interactions. Nonsignificant interactions were removed from the model before reanalysis. Data are presented as LSMeans ± standard errors. Initially, sperm progressive motility averaged 41 ± 6.2% across treatments. After an initial decline, overall progressive motility did not change (P > 0.05) significantly (mean of 22.3 ± 1.6 and 23.05 ± 1.3% at 48 and 72 h, respectively). Over time and across treatment combinations, mean progressive motility was maintained to a greater extent (P < 0.01) by milk than TRIS-based extender (28.2 ± 1.1 v. 18.9 ± 1.1%, respectively). Across extenders, progressive motility of sperm was similar (P = 0.50) for 5 and 20% egg yolk (22.2 ± 1.4 v. 24.4 ± 1.4). Addition of 1% EG increased (P < 0.01) progressive motility (25.8 ± 1.05 v. 21.3 ± 1.05). Addition of PHE also increased (P < 0.01) progressive motility from 20.9 ± 1.04 to 26.3 ± 1.04%. There was an interaction between EG and % egg yolk, primarily due to an effect on sperm stored in TRIS citrate extender. Addition of 1% EG to extender containing 5% egg yolk improved (P < 0.01) progressive motility from 18.5 ± 1.5 to 26.9 ± 1.5%). Addition of 1% EG to TRIS citrate extender also increased (P < 0.05) progressive motility, from 14.6 ± 1.5 to 23.2 ± 1.5%. Results indicate that milk extender supplemented with 1% EG, PHE, and either 5 or 20% egg yolk is capable of maintaining progressive motility of ram semen at ~60% of its initial value when stored at 4°C for up to 72 h. Additional studies are needed to evaluate pregnancy rate after insemination of ewes with stored semen.

Evaluation of the function of fresh and frozen - thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay

2010 ◽  
Vol 22 (4) ◽  
pp. 710 ◽  
Author(s):  
J. R. Clulow ◽  
G. Evans ◽  
W. M. C. Maxwell ◽  
L. H. A. Morris
Keyword(s):  
Zona Pellucida ◽  
Binding Assay ◽  
Binding Ability ◽  
Treatment Groups ◽  
Functional Integrity ◽  
Sperm Binding ◽  
Heterologous Sperm ◽  
Bovine Oocytes ◽  
Better Than

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen–thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen–thawed non-sorted spermatozoa; and (4) frozen–thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed ‘attached’ or ‘bound’ depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen–thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen–thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen–thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status.

“In vitro” and in Animal Model Studies on a Double Virus- Inactivated Factor VIII Concentrate

1995 ◽  
Vol 74 (03) ◽  
pp. 868-873 ◽  
Author(s):  
Silvana Arrighi ◽  
Roberta Rossi ◽  
Maria Giuseppina Borri ◽  
Vladimir Lesnikov ◽  
Marina Lesnikov ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Animal Model ◽  
Factor Viii ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Inactivation ◽  
Enveloped Viruses ◽  
Model Studies ◽  
Heat Treated

SummaryTo improve the safety of plasma derived factor VIII (FVIII) concentrate, we introduced a final super heat treatment (100° C for 30 min) as additional virus inactivation step applied to a lyophilized, highly purified FVIII concentrate (100 IU/mg of proteins) already virus inactivated using the solvent/detergent (SID) method during the manufacturing process.The efficiency of the super heat treatment was demonstrated in inactivating two non-lipid enveloped viruses (Hepatitis A virus and Poliovirus 1). The loss of FVIII procoagulant activity during the super heat treatment was of about 15%, estimated both by clotting and chromogenic assays. No substantial changes were observed in physical, biochemical and immunological characteristics of the heat treated FVIII concentrate in comparison with those of the FVIII before heat treatment.

MICROPROPAGATION OF CHICKPEA FROM SHOOT TIP AND NODE SEGMENT

2018 ◽  
Vol 24 (2) ◽  
Author(s):  
J. D. BARSHILE
Keyword(s):  
Shoot Tip ◽  
Root Induction ◽  
Multiple Response ◽  
Ms Medium ◽  
Growth Responses ◽  
Rapid Multiplication ◽  
Micro Propagation ◽  
G 12 ◽  
Better Than

Present investigation was undertaken to standardize technique for in vitro micro-propagation of chickpea( Cicer arietinum ) cultivar Vishwas (Phule G 12). Micropropagation method for chickpea was established and this method enabled much more efficient propagation of plants. The present work was aimed at evolving a protocol for rapid multiplication of chickpea using micropropagation technique. Explants from shoot tip and node segment were cultured on MS medium supplemented with different concentrations of BAP and Kinetin (1.0 to 2.5 mg/l) and their growth responses like shooting were elucidated. The maximum multiple response was observed with 2 mg/l concentration of BAP from both types of explant. The highest number of shoots (12.5 ± 0.3) was achieved on MS medium with 2 mg/l BAP using node segments. The medium supplemented with 2 mg/l of BAP was found better than all other concentrations. Individual shoots were transferred to IBA and IAA (1.0-1.5 mg/l) for root induction. MS medium supplemented with 2 mg/l of IBA proved better for rooting. Rooted plantlets were successfully hardened in greenhouse and established in the pot.

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