Enhanced freezability of goat spermatozoa collected into tubes containing extender supplemented with bovine serum albumin (BSA)

The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.     

Characteristics of bovine spermatozoa after migration through a bovine serum albumin gradient

Experiments were conducted to determine the efficiency with which viable, morphologically normal bovine spermatozoa could be isolated using a discontinuous bovine serum albumin (BSA) gradient. In the first experiment, extended semen was layered on top of a BSA gradient (4% BSA over 10% BSA) contained in a 500-ml separatory funnel. When comparing 1, 3, 5, 7, 14 or 21 X 10(9) spermatozoa applied to the gradient, the percentage of spermatozoa recovered from the lower third of the 10% BSA ranged from 2.9 to 18.5%. The greatest recovery was achieved when 1 X 10(9) sperm cells were applied. Increasing the number of spermatozoa applied to the gradient increased the percentage of spermatozoa remaining in the upper portions of the gradient. Motility of spermatozoa immediately after collection from the 10% BSA layer of the gradient was greater than 90%, regardless of the number of spermatozoa applied. In a second experiment with freeze-thawed separated or unseparated spermatozoa, post-thaw motility (greater than 60%) and acrosomal integrity (greater than 85%) of separated spermatozoa (4 or 10% BSA layer) was greater (P less than .05) than that of unseparated spermatozoa (38 and 66%, respectively). The discontinuous gradient excluded decapitated spermatozoa and spermatozoa with mid-piece and principal piece abnormalities from entering the lower layers. Sperm cells with head abnormalities were not separated. These data indicate that a population of spermatozoa with a high frequency of viable, motile, morphologically-normal bovine spermatozoa can be isolated using a discontinuous BSA gradient.     

Sex ratio after insemination of bovine spermatozoa isolated using a bovine serum albumin gradient

This experiment was undertaken to determine if a method reported to successfully enrich the proportion of Y-chromosome-bearing spermatozoa in human semen could be adapted for separation of bovine spermatozoa. Semen was collected from four Angus bulls and aliquots were either separated on discontinuous gradients of bovine serum albumin (BSA) or untreated before processing for cryopreservation. Two hundred seventy-one cows or heifers were assigned randomly to be artificially inseminated (20 X 10(6) sperm/insemination) with separated or unseparated spermatozoa. The proportions of male offspring were 45 and 54% after inseminations with separated or unseparated spermatozoa, respectively. In a second phase of the experiment, pooled semen from three Holstein bulls was either extended and frozen without separation or frozen after separation using the discontinuous BSA gradient. Separated and unseparated spermatozoa were analyzed by flow cytometry to determine the ratio of X- and Y-chromosome-bearing spermatozoa based on differences in DNA content. The ratios of X- and Y-bearing spermatozoa in separated or unseparated samples were indistinguishable. We concluded that the separation method did not enrich the proportion of Y-bearing bovine spermatozoa.     

Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa

This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4 degrees C (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4 degrees C (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 x 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.     

Effects of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram spermatozoa

In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 +/- 2.9 and 58.3 +/- 6.7%, respectively) than in the positive control (F) group (41.7 +/- 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 +/- 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 +/- 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 +/- 1.3 and 6.3 +/- 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 +/- 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 +/- 2.4 and 25.0 +/- 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 +/- 2.4 and 18.8 +/- 1.3%, respectively) and F (28.8 +/- 3.8 and 18.8 +/- 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing.     

Long-term liquid storage of porcine spermatozoa separated using a discontinuous bovine serum albumin gradient

Two experiments were conducted to determine efficacy of a discontinuous bovine serum albumin (BSA) gradient for isolating viable porcine spermatozoa more tolerant to 5-d liquid storage in Beltsville Thawing Solution (BTS) at 15 degrees C. The gradient, contained in a 500-ml separatory funnel, consisted of 4% BSA (60 ml) over 10% BSA (60 ml). Spermatozoa were extended in 26 ml of BTS, layered on top of the gradient, and allowed to migrate through the BSA. The quality of spermatozoa separated by the gradient varied among boars. However, populations of spermatozoa isolated from the bottom 30 ml of the gradient (Fraction 4) consistently contained a high percentage of spermatozoa with acrosomes possessing normal apical ridges (NAR; 89.6%) and progressively motile spermatozoa (MOT; 84.0%), as well as spermatozoa with high velocity (VEL; 336.5 mu/s). Increasing sperm migration time, but not gradient temperature, increased the number of spermatozoa recovered in Fraction 4, but it did not reduce quality of the separated spermatozoa. Spermatozoa isolated in Fraction 4 had greater NAR, MOT and VEL after 5-d storage in BTS than did unseparated spermatozoa. Boar spermatozoa isolated on a discontinuous BSA gradient were more tolerant to storage at 15 degrees C than were unseparated spermatozoa. Such a population may be desirable for use in artificial insemination programs.     

Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition

The reproductive performance of dairy cows may be improved by feeding conjugated linoleic acid (CLA) supplements during early lactation. The mechanism of action of t10,c12 CLA is not clearly known. Our objective was to investigate the effect of t10,c12 CLA on oocyte maturation and lipid composition of cumulus oocyte complexes (COC). The developmental potential of oocytes incubated in in vitro maturation (IVM) medium supplemented with t10,c12 CLA to the blastocyst stage and embryo quality were also assessed. In experiment 1, abattoir-derived oocytes were matured in TCM199 + 10% serum supplemented with 100 μM t10,c12 CLA (t10,c12 CLA n = 672) or without it (control n = 672). Mature oocytes were either stained for chromatin configuration or inseminated and cultured for embryo development assessment. In experiment 2, COC and IVM culture media were subjected to fatty acid (FA) analysis prior and after maturation with t10,c12 CLA or without it (control). Total lipids and FA profiles in oocytes, cumulus cells and culture media were determined by gas chromatography. t10,c12 CLA supplementation to IVM medium improved (p = 0.05) embryo quality evaluated morphologically. This effect was associated with t10,c12 CLA presence (3.1 ± 0.7%, p = 0.04) and lower levels of arachidonic acid in FA profile of t10,c12 CLA mature oocytes (immature oocytes = 4.4 ± 1.9%, t10,c12 CLA mature oocytes = 1.0 ± 0.7%, p = 0.05). Differences in myristic and eicotrienoic acids, saturated and unsaturated FA concentrations between oocytes and cumulus cells were detected (p ≤ 0.05). In conclusion, the presence of t10,c12 CLA during maturation interfered on lipid metabolism improving bovine oocyte competence to develop into higher quality embryos.     

The positive effects of seminal plasma during the freezing process on cryosurvival of sperm with poor freezability in the rhesus macaque (Macaca mulatta)

The objective was to examine the effect of seminal plasma on cryopreservation of sperm from rhesus macaques. Sperm cryosurvival was evaluated by sperm motility and acrosomal integrity. Compared with slow cooling (-0.4 C/min) from 37 C (body temperature) to 4 C, rapid cooling (-16 C/min) caused cold shock in rhesus macaque sperm. The cryosurvival of sperm was decreased regardless of the presence or absence of seminal plasma (P<0.05). However, the presence of seminal plasma during cold shock at a rapid cooling rate improved sperm motility and acrosomal integrity in individual monkeys. Male-to-male variation in sperm cryosurvival was observed after cryopreservation (P<0.05), and the presence of seminal plasma during sperm cryopreservation improved sperm motility and acrosomal integrity in individual monkeys (P<0.05). Furthermore, by adding seminal plasma from monkeys with good sperm cryosurvival to sperm freezing extender, the frozen-thawed motility and acrosomal integrity of sperm from monkey with poor cryosurvival were improved (P<0.05). The present study indicated that seminal fluid is beneficial to sperm undergoing cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus macaques with poor sperm freezability could be improved by the presence of seminal plasma from males with good sperm cryosurvival. This finding provides a useful method for genetic preservation in this important species.     

Cryopreservation of canine spermatozoa using a skim milk-based extender and a short equilibration time

Although useful spermatozoa cryopreservation techniques have been established, long-term equilibration seems to be required before freezing the spermatozoa of many species, including dogs. The fertility of cryopreserved dog spermatozoa from five males for a reduced equilibration period (0, 30, 60, 120 and 180 min) in a skim milk (SM)-based extender containing raffinose was evaluated in the present study. When the sperm was diluted with the extender at room temperature (RT) and cryopreserved without equilibration, the proportion of total motile spermatozoa (TMS) after thawing was lower (27%) than when the sperm was equilibrated for 30 min (33%), 60 min (32%), 120 min (44%; p < .05) or 180 min (29%). The proportion of TMS increased as the equilibration time increased and peaked at 120 min. Acrosome integrity was significantly lower in the cryopreserved spermatozoa that had not undergone the initial equilibration than in the equilibrated spermatozoa (p < .05). The normal rate of acrosomes increased with the extension of the first equilibration and peaked at 120 min. When frozen–thawed spermatozoa that had been diluted at RT and subjected to an initial equilibration lasting 60 or 180 min were transcervically inseminated into recipients, there were no differences in the delivery rate, litter size or breeding efficiency. In the cryopreservation of canine spermatozoa using a SM-based extender, even if the initial equilibration time was shortened to 60 min, the results were comparable to those obtained when the conventional method (with an initial equilibration time of 180 min) was used.     

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.     

Effect of insulin and linoleic acid on satellite cell differentiation

Differentiation of rat skeletal muscle satellite cells was studied in vitro. Linoleic acid and insulin, two unrelated compounds that reportedly stimulate differentiation of other types of myogenic cells, were used to examine the regulation of differentiation in satellite cell cultures. As in cultures of chick embryo muscle cells, linoleic acid stimulated fusion but only at low serum concentrations or in defined medium without fibroblast growth factor (FGF). The effects of insulin on differentiation were quite variable, however; at very low cell densities no stimulatory effect was observed. In intermediate and, to a lesser extent, high density satellite cell cultures, the addition of insulin at concentrations between .01 and 1.0 microM stimulated satellite cell fusion. Whenever increases in fusion were observed, however, a parallel increase in cell number was also found. A closer examination of the relationship between differentiation and the presence or absence of mitogenic agents in the medium suggested that a mitogenic signal and the resultant proliferation of cells prevented differentiation. Subsequent experiments indicated that fusion could be induced by lower serum concentration or by removal of FGF, as long as linoleic acid was present in the medium. Therefore, proliferation and differentiation appear to be antagonistic processes in cultured satellite cells. If the rate of proliferation is depressed, either by mitogen removal or by increasing cell density, differentiation is favored. Differentiation can, therefore, be regulated and applied to in vitro studies of satellite cell activity.     

Effect of a rumen-protected conjugated linoleic acid mixture on hepatic lipid metabolism in heifers

This study was performed to assess the effects of rumen-protected conjugated linoleic acid (CLA) on hepatic lipid metabolism in heifers. In particular, it was of interest whether feeding CLA causes development of fatty liver as observed recently in mice. Thirty-six growing heifers with an initial body weight of 185 kg were allotted to three treatment groups and fed daily 250 g of different rumen-protected fats for 16 weeks: The control group received 250 g of a CLA-free control fat, the CLA100 group received 100 g of a CLA fat containing 2.4% of cis-9, trans-11 CLA and 2.1% of trans-10, cis-12 CLA and 150 g control fat and the CLA250 group received 250 g of the CLA fat. CLA supplementation had no effect on animal performance parameters, liver weight and hepatic triglyceride concentration. Moreover, mRNA expression of hepatic genes involved in lipogenesis, β-oxidation and fatty acid transport was not influenced by dietary CLA. The fatty acid composition of hepatic total lipids, with particular consideration of ratios of fatty acids indicative of Δ9-, Δ6- and Δ5-desaturation, was also less influenced by dietary CLA. In conclusion, the study shows that dietary rumen-protected CLA has less effect on hepatic lipid metabolism in young heifers and does not induce the development of a fatty liver such as in mice.