Yolk Citrate Extender Research Articles

Seminal plasma addition attenuates the dilution effect in bovine sperm

Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 × 10 6 sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 × 10 6 sperm/mL in EYC extender with (+SP) and without (−SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 × 10 6 sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 × 10 6/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the −SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the −SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR-14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the −SP samples at a concentration of 10 × 10 6 sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls.

The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.

Liner Collection Cone and pH Effects on Postthaw Motility, Staining, and Acrosomes of Bovine Spermatozoa

Sixteen ejaculates were collected, four each from four bulls, using artificial vaginas with polyethylene or rubber liner collection cones in a crossover design experiment. The ejaculates were diluted with egg yolk-citrate extender at pH 6.4 or 7.2, cooled, glycerolated, equilibrated, packaged in .5-ml French straws, frozen in nitrogen vapor, and stored in liquid nitrogen. Thirty frozen straws from each ejaculate were thawed rapidly (46.5°C for 12s), pooled, and then incubated at 46.5°C for periodic evaluation of progressive motility, differential staining, and acrosome morphology under thermal stress conditions. The postthaw motility of spermatozoa and percentage of unstained cells were higher both when collected in polyethylene than in rubber and when extended at pH 7.2 vs. 6.4, but no interaction was found between liner collection cone composition and pH for postthaw motility. Retention of spermatozoan motility during incubation under thermal stress was greater for cells collected in polyethylene, but not different due to pH. Neither pH nor composition of liner collection cone had an effect on postthaw acrosomal scores, but the time required for a 50% increase in severely damaged acrosomes was greater for spermatozoa collected in polyethylene than in rubber liner collection cones.

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 × 10 6 sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4°C). Three cooling rates (−1.0, −0.3 and −0.1°C/min) were also investigated at the lowest storage temperature (4°C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4°C than at 35 or 22°C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4°C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (−1.0 and −0.3°C) than at the slow cooling rate (−0.1°C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4°C. Additional studies are needed to evaluate the fertility of semen stored in this manner.

Effects of Extender and Insemination Dose on Postthaw Quality and Fertility of Bovine Sperm

Quality and fertility of sperm extended in egg yolk-Tes-Tris were compared with those of sperm in egg yolk-citrate and homogenized milk extenders. Extended semen was frozen in .5-ml plastic straws at 11, 15, 17, or 22×106 sperm per insemination dose. Laboratory evaluations at 0, 1, 2, and 4h after thawing semen utilized four tests of spermatozoal quality. Use of egg-yolk citrate extender resulted in a higher percentage of progressively motile sperm as determined visually at 0h after thawing than use of egg yolk-Tes-Tris or homogenized milk extenders. Sperm extended in egg yolk-citrate had 18% lower activity of bound amidase at 0h than sperm extended in egg yolk-Tes-Tris. The 75-d nonreturn rates were affected by insemination dose but not be extender or the interaction of extender and insemination dose. Fertility was lower after insemination of 11×106 sperm than for pooled data for the three higher insemination doses (64 vs. 68%). Based on all data, postthaw quality of sperm processed in the one-step egg yolk-Tes-Tris extender was similar to that for sperm extended in egg yolk-citrate or homogenized milk.

The concentration of estradiol-17 beta in bovine semen.

This study was conducted to evaluate the influence of age, breed, epididymectomy and semen processing on the concentration of estradiol-17 beta (E2) in bovine semen. Semen was collected either by electroejaculation or with an artificial vagina. Neat semen samples were stored at -20 C until analysis. Processed, frozen semen and an egg yolk-citrate semen extender were obtained from a commercial semen processing firm and stored in liquid nitrogen at -196 C. The concentration of E2 in semen was determined by radioimmunoassay. Semen from mature (greater than 24 mo), fertile Brahman (n = 19), Brangus (n = 16), Charolais (n = 29), Holstein (n = 15) and Santa Gertrudis (n = 25) bulls was analyzed for E2 concentration, and no difference (P greater than .10) between breeds was found. There was no difference (P greater than .10) in seminal E2 concentration between mature, fertile bulls (n = 104) and epididymectomized bulls (n = 22). In semen collected from prepuberal (12 to 16 mo, n = 21), peripuberal (17 to 20 mo, n = 17) and mature (greater than 24 mo, n = 19), Brahman bulls, the mature bulls had a lower (P less than .01) semen E2 concentration than peripuberal and prepuberal bulls. There were no differences (P greater than .10) in seminal E2 concentration among peripuberal Angus (n = 8), Hereford (n = 8) and Brahman (n = 17) bulls.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Sugars with Glycerol on Post-thaw Motility of Bovine Spermatozoa in Straws

Semen from each of seven bulls was diluted in egg yolk-citrate extender containing 0, 60, 120, or 180mM fructose (Experiment 1), lactose (Experiment 2), or raffinose (Experiment 3) in every possible combination with 0, 3, 6, 9, and 12% (vol/vol) glycerol, to establish if these sugars were cryoprotective for spermatozoa in straws. Fructose was not beneficial to post-thaw motility in the absence of glycerol and became increasingly detrimental as glycerol concentration was increased. Although lactose was beneficial when glycerol was absent or present at low concentration, no combination of glycerol and lactose was superior to glycerol alone. The response to raffinose and to lactose was similar. In all three experiments, post-thaw motility was as good or better with 6% glycerol than with any of the other glycerol percents. Also, motility was generally greater for spermatozoa frozen in French straws than for those in Continental straws frozen by the same procedure. Cryoprotective effects of lactose and raffinose, used singly and in combinations with each other, were examined further in a fourth experiment. All extenders contained a final 7% glycerol. Motility of spermatozoa frozen in the absence of lactose and raffinose did not differ from that with any combination of these sugars. Motility averaged 22% and 18% for spermatozoa in French or Continental straws and declined more rapidly during post-thaw incubation for spermatozoa in Continental straws.

Effects of Package, Extender, and Light on Stored Frozen Bull Spermatozoa

Spermatozoa were analyzed when fresh, freshly frozen, and stored at −196 C for 6 and 18 mo after processing in egg yolk-citrate and Promine-D extender with varying exposure to light and packaging in straws or glass ampules. Over all treatments there were decreases with time in spermatozoal motility, oxygen uptake, and cellular glutamic oxaloacetic transaminase due to freezing-thawing. Percent progressive motility decreased from spermatozoa freshly frozen (36.5%) to those stored for 6 mo (33.6%). Oxygen uptake in freshly frozen samples was 8.4 μl per 108 cells per h. In frozen semen stored for 6 and 18 mo corresponding values were 12.2 and 8.3 μl. Packaging methods did not have a significant effect on oxygen uptake, but package interacted with storage. Egg yolk-citrate extender supported greater progressive spermatoal motility than did Promine-D. Extender interacted with storage for all criteria. Visible light radiation reduced progressive spermatozoal motility from 40.2 to 38.0% and oxygen uptake from 11.8 to 10.8 μl per 108 cells per h. Light interacted with storage time. Correlations between all criteria and storage times were not important practically.

Influence of incubation in seminal plasma on subsequent metabolic and morphological characteristics of bovine spermatozoa

Incubation of bovine spermatozoa at 25° C for up to 8 hours in seminal plasma had little influence on oxygen uptake as compared to preincubation values but increased both the number of dead spermatozoa and proportion of spermatozoa with detached acrosomes. Washing spermatozoa and resuspension in either saline or seminal plasma followed by incubation for 4 or 8 hours decreased oxygen uptake and at 8 hours decreased the proportion of cells with deteched acrosomes as compared to 8 hour control values. When spermatozoa were not washed but were extended in egg yolk citrate extender or seminal plasma, oxygen uptake was greater for cells extended with seminal plasma. Storage of spermatozoa for 14 days at −196° C following incubation in seminal plasma showed little influence of incubation time in seminal plasma on postfreeze metabolic or morphological characteristics. These experiments indicate that incubation of spermatozoa in seminal plasma prior to storage has little beneficial effect and, on the contrary, may cause an increase in acrosomal loss.

Antibodies to egg yolk in blood serum of rabbits and cattle and cervical mucus of cattle inseminated artificially

Serum titers to egg yolk were induced in 6 rabbits by intravaginal deposition of an egg-yolk citrate extender used for artificial insemination of cattle. There was no effect of the low serum titers to egg yolk on fertility of the inseminated rabbits. Titers to egg-yolk semen extender were found in 3% of 59 cows of normal fertility compared to 29% of 14 repeat breeder cows of low fertility, all previously inseminated with semen diluted with egg yolk-citrate extender. Four of 6 cervical mucus samples (67%) from the repeat breeder cows had high titers to egg yolk, but only one also had a positive titer in blood serum.

Influence of Freezing Methods, Extenders, and Storage Temperatures on Motility and pH of Frozen Bovine Semen1,2

Summary Fourteen ejaculates, two from each of seven bulls, were extended in heated, whole homogenized milk (milk) and egg yolk-citrate (EYC), frozen with either dry ice-alcohol (DI) or liquid nitrogen (LN), then divided for storage at −79C (DI), −92C (Mechanical), and −196C (LN). Samples from each ejaculate and each treatment were evaluated for pH and per cent progressive motility at prefreeze, post-freeze, one day, ten days, one, three, six, and nine months. EYC extender maintained significantly higher (P There was little variation in mean pH values within extenders due to freezing methods or storage temperatures. The correlation coefficients between pH and motility and between change in pH and change in motility were low and in most instances nonsignificant.

Motility and Fertility during Post-Thawing Storage of Bovine Spermatozoa Frozen Concentrated, Thawed, and Re-Extended

Summary Twenty ejaculates of semen from 17 dairy bulls were extended to contain 200×10 6 and 20×10 6 sperm per milliliter in yolk citrate and whole milk extenders and frozen with 7% glycerol. The semen frozen with 200×10 6 sperm per milliliter was re-extended, after thawing, to 20×10 6 sperm per milliliter in yolk citrate, whole milk, skimmilk, and CUE, and the motility of these semen samples wTas estimated during 72 hr of storage at 5C. Freezing semen with 200×10 6 sperm per milliliter resulted in significantly improved motilities (P The average 60- to 90-day nonreturn percentages for semen frozen with 200×10 6 sperm per milliliter and re-extended after thawing to 15×10 6 motile sperm per milliliter in yolk-citrate and CUE was 51.8 and 63.0%, respectively, as measured with a total of 2,062 inseminations made between 24 and 60 hr after thawing. The 63.0% for CUE did not significantly differ from the average of 66.5% for control frozen semen thawed immediately before 675 cows were inseminated (P>.05), but both of these values were significantly higher than the 51.8% for the semen re-extended in yolk citrate (P

Survival and fertility of bovine sperm kept at variable temperatures in coconut milk extender

Bovine spermatozoa aged for 6–7 days at room temperatures (18–30° C.) in a coconut milk extender retained their fertilizing capacity. However, their fertilizing ability appeared to be impaired by environmental hazards in the form of oxygen damage, exposure to visible light, and temperature shock. Protective measures developed and used in the preparation and in the storage of the sperm suspensions to minimize premature loss of sperm function involved the addition of catalase and egg yolk to the extender and the maintenance of cell suspensions in the dark in vials containing a minimal air space. The experimental evidence obtained with this improved coconut milk extender established its ability to support and sustain the fertilizing capacity of bovine sperm for 6–7 days at room temperatures at a level comparable to the fertilizing capacity of refrigerated sperm in milk-glycerol or egg yolk-citrate-glycine extenders. The economic importance of this room temperature diluent is suggested by the fact that it eliminates the costly refrigeration required to maintain stored and shipped bovine semen at 5° C.Coconut milk extender modified by the addition of 5% egg yolk also proved to be a satisfactory storage medium for sperm at low temperatures. It was found to be superior to both egg yolk-citrate and skim-milk extenders in conserving motility at 5° C. The fertilizing capacity of Ayrshire and Brown Swiss sperm stored at 5° C. in coconut milk extender which contained 15% coconut milk and 5% egg yolk compared favourably with the fertilizing capacity reported for sperm stored at 5° C. in other more widely used diluents. The number of motile sperm recovered from this extender after freezing to −79° C. was comparable to the number of motile sperm recovered from the egg yolk-citrate and skim-milk test diluents. The results of this study indicate that coconut milk extender is a versatile, easily prepared medium which can be used to preserve functional sperm over a wide temperature range.

Preservation of Bovine Spermatozoa at −79 and −196° C

Summary Studies were conducted primarily to determine the effect of storage temperatures of −79 and −196° C. on fertility of bovine spermatozoa. A total of 145 ejaculates from 43 bulls of the Holstein, Jersey, Guernsey, Angus, Ayrshire, and Brown Swiss breeds were frozen in an egg yolk-citrate extender. The 60- to 90-day per cent nonreturn (NR) rate from 3,214 first-service inseminations with liquid nitrogen (LN) stored semen was 70.6, compared to 3,091 first services with a NR rate of 67.8% from DI storage. The weighted mean difference in per cent NR of 2.9 was statistically significant (P

Factors Affecting the Fertility of Frozen Bovine Spermatozoa

Summary Three separate studies were conducted in an attempt to determine the most suitable extender, the spermatozoan concentration necessary, and the method of filling and sealing glass ampules, to obtain optimal nonreturn rates with frozen bovine spermatozoa. In the first experiment, a total of 934 first-service cows were bred with semen extended and frozen in yolk-citrate extender, 945 cows with milk, 928 cows with yolk-glucose-glycine, and 902 cows with skimmilk. The 75-day nonreturn rates were 64.7, 66.2, 41.3, and 65.3%, respectively. Yolk-citrate, milk, and skimmilk, on the basis of this experiment, were acceptable semen extenders for frozen semen, whereas yolk-glucose-glycine was not suitable. For the second experiment, 469 first-service cows were bred with frozen semen containing 10 million live spermatoza per insemination at time of initial dilution, 479 cows with frozen semen containing 20 million spermatozoa, and 472 cows with semen containing 30 million spermatozoa per insemination. The 75-day nonreturn rates for the respective levels were 63.1, 68.9, and 72.7%. The difference between the 10- and 30- million level was highly significant (P In the third experiment, 294 first-service cows were inseminated with frozen semen for which the glass ampule had been gravity-filled-tip-sealed, 275 cows inseminated with frozen semen in ampules that had been gravity-filled-cut, pull-sealed; 313 cows inseminated with frozen semen in ampules that had been forced-filled-tip-sealed, and 254 cows inseminated with frozen semen in ampules that had been force-filled-cut, pull-sealed. The 75-day nonreturn rates for the respective methods of filling and sealing were 69.0, 73.8, 63.9, and 70.5%. None of the differences was significant. However, a significant difference existed between tip-sealed and pull-sealed, irrespective of method of filling. The difference between gravity-filled and force-filled, irrespective of method of sealing, was not significant.