Effects of cold storage on plasma membrane, DNA integrity and fertilizing ability of feline testicular spermatozoa

This study examined the effects of cold storage on plasma membrane, DNA integrity, and fertilizing ability of domestic cat spermatozoa. Intact cat testes were stored at 4°C in Dulbecco's phosphate buffered saline (DPBS) for 7 days. Membrane integrity (experiment 1) and DNA integrity (experiment 2) of extracted spermatozoa were assessed over time during storage. Testicular spermatozoa were also tested for their fertilizing ability via intracytoplasmic sperm injection (ICSI) in term of gamete activation and early embryonic development at 18 h (experiment 3). The membrane integrity of testicular spermatozoa was well preserved in DPBS for 4 days compared to non-preserved control (Day 0) (P<0.05). The incidence of testicular sperm DNA fragmentation was <1% after 7 days of cold storage and was not significantly affected by the duration of cold storage (P>0.05). Finally, testicular spermatozoa could form pronuclei and sustain embryo development following ICSI regardless of the storage time (P>0.05). In conclusion, cat testicular spermatozoa can be preserved at 4°C for up to 7 days without severely compromising of plasma membrane and DNA integrity while retaining a normal fertilizing ability.     

Intra- and interboar variability in flow cytometric sperm sex sorting

To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome–bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome–bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs.     

Semen collection,characterization, and cryopreservation in a Magellanic penguin (Spheniscus magellanicus)

A cooperative method was developed for collecting semen from a Magellanic penguin. Ejaculate parameters and semen production during a breeding season were characterized. Experiments were performed to study the effect on penguin spermatozoa of two temperatures (4°C and 21°C) for short‐term storage, and two cryoprotectants (dimethylsulfoxide [DMSO] and ethylene glycol [EG]) for long‐term storage (cryopreservation). All dilutions were made using modified Beltsville Poultry Semen Extender. Sperm quality was assessed by evaluating motility and forward progression (sperm motility index [SMI]), viability, and morphology. A total of 39 ejaculates was collected over the 40‐day study period. Thirty‐eight ejaculates contained spermatozoa, but semen quality decreased toward the end of the study period. Varying levels of urate contamination were present in all ejaculates. Sperm quality parameters were similar for diluted samples held at 4°C and 21°C, and samples maintained high numbers of viable (77.8 ± 5.4%) and morphologically normal (67.9 ± 2.5%) spermatozoa at 3 hr. SMI and percentage of viable sperm decreased (P < 0.05) and the number of spermatozoa with a bent head or midpiece increased (P < 0.05) for both temperature groups over the 3‐hr storage interval. DMSO and EG were equally effective in maintaining penguin sperm quality parameters during the cryopreservation and thawing process. Frozen‐thawed semen maintained 69 ± 5 and 78 ± 3% of its pre‐freeze SMI and viability, respectively. SMI and viability decreased slightly during the cooling and equilibration phases but remained relatively stable during the 3‐hr storage interval post‐thaw. Frozen‐thawed semen also exhibited an increase (P < 0.05) in spermatozoa with a bent head or midpiece over time. The pre‐freeze SMI was higher (P < 0.05) for ejaculates with low levels of urates (clean ejaculates) compared with ejaculates with high levels of urate contamination, but sperm viability and morphology were similar (P > 0.05). Both SMI and viability of frozen‐thawed spermatozoa were higher (P < 0.05) for clean than for contaminated ejaculates. This is the first report on penguin ejaculate parameters, semen production, and preliminary methods for short‐ and long‐term semen storage. Zoo Biol 18:199–214, 1999. © 1999 Wiley‐Liss, Inc.     

Ultrastructural abnormalities of boar spermatozoa

Described here are the main ultrastructural malformations observed in spermatozoa of ejaculates collected from healthy, adult Landrace boars following 2 days of sexual abstinence. Previously semen had been collected 3 times per week. Sperm concentration in the cell-rich fraction of ejaculates was approximately 700,000 sperm/mm(3). The aberrant gamete forms did not exceed 2% of the total number of spermatozoa. Ultrastructural anomalies of spermatozoa were classified into 2 groups: head malformations and tail malformations. These consisted of: 1) spermatozoa with expanded and vacuolated acrosomes, 2) spermatozoa with myelin figures within the perinuclear space, 3) macrocephalic spermatozoa with 2 nuclei and a deformed acrosomal vesicle, 4) spermatozoa with an expanded acrosomal apex, 5) spermatozoa with nuclear vacuoles, 6) macrocephalic spermatozoa with a roundish head, 7) spermatozoa with swollen mitochondria, 8) spermatozoa with additional mitochondria over the mitochondrial sheath, 9) spermatozoa without the central microtubular pair, 10) spermatozoa without some peripheral doublets, 11) spermatozoa with 1 or 2 coiled tails, 12) spermatozoa with a folded tail and a disorganized connecting piece, 13) spermatozoa with a vesiculated tail, and 14) spermatozoa with 2 tails fused by their respective mitochondrial sheaths.     

Effects of polyamines upon capacitation and fertilization in the guinea pig

The effects of polyamines upon the ability of guinea pig spermatozoa to undergo the acrosome reaction and upon sperm-egg fusion were investigated. All of the polyamines (polyarginines and compound 48/80) stimulated the occurrence of the acrosome reaction, although differences in the duration of the preincubation period required were noted. The stimulatory effect of the polyarginines could not be reversed by washing, while that of 48/80 was abolished by its removal prior to induction of the acrosome reaction. Polyarginine-treated spermatozoa were fully capable of fertilizing intact guinea pig eggs. In contrast, 48/80 completely blocked sperm-egg fusion at only 5-10 micrograms/ml. This inhibition was reversible by washing either treated gamete. The ability of polyamines which may also be present at the time and site of fertilization, in vivo, to inhibit or stimulate gamete functions or interactions is discussed.     

Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg yolk concentration

The aim of this study was to evaluate the effects of cold storage and egg yolk concentration on the distribution of spermatozoa within the different subpopulations. Twenty ejaculates from 4 dogs were collected, diluted in either TRIS buffer containing 20% (TEY20) or 10% centrifuged egg yolk (TEY10) and cooled following a conventional protocol. The kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates and after 24 and 72 h of preservation at 5°C. A multivariate clustering procedure separated 54,261 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (19.80%), Subpopulation 2 consisting of slow and low-linear spermatozoa (25.21%), Subpopulation 3 consisting of high speed and progressive spermatozoa (23.88%), and Subpopulation 4 consisting of highly active but non-progressive spermatozoa (31.11%). Although, cold storage had a significant (P<0.05) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after cold storage. Subpopulations 1 and 2 significantly (P<0.001) decreased during cold storage (Subpopulation 1: 26.6, 16.9 and 18.4%; and Subpopulation 2: 33.6, 21.3 and 24.0%, respectively, for fresh, 24 and 72 h post-cooled), whereas Subpopulations 3 and 4 significantly (P<0.05) increased (Subpopulation 3: 16.7, 27.6 and 24.3%, and Subpopulation 4: 23.1, 34.1 and 33.4%, respectively, for fresh, 24 and 72 h post-cooled). Regarding the relative percentage of spermatozoa within each extender, Subpopulation 3 was more frequently observed in TEY20 after both 24 and 72 h of cold storage. Significant correlations (P<0.05) were found between the proportions of spermatozoa assigned to Subpopulation 3 in the fresh ejaculates and those in stored samples after 24 h (r=0.48498). In conclusion, cold storage significantly modified both the specific parameters and the distribution of spermatozoa within subpopulations. These changes did not affect the general motile sperm structure present in dog, which is conserved during cold storage. The analysis of the changes observed in structures of subpopulations also suggests that the TEY20 provide more effective preservation of dog semen during cold storage.     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

Sperm morphology is better in the second ejaculate than in the first in domestic cats electroejaculated twice during the same period of anesthesia

Several species produce ejaculates of inferior quality after a period of sexual abstinence, but the frequency of semen collection has thus far not been shown to affect sperm morphology in felids. The aim of this study was to determine whether sperm morphology and motility would differ between 2 ejaculates collected from the same cat within a short interval. Fifteen male domestic cats were anesthetized and then electroejaculated twice, with a 5- to 10-min interval between treatments. A standardized electroejaculation regimen was used with 80 stimuli, from 2 to 5 V, for each ejaculate. The first ejaculates contained significantly higher (P < 0.05) proportions of distal droplets, coiled tails and immotile spermatozoa than the second ejaculates, which contained significantly higher proportions of morphologically normal spermatozoa (40.9 vs 54.6%) but a lower sperm count (39.0 x 10(6) vs 5.2 x 10(6)). The higher proportions of defective spermatozoa and the lower motility in the first ejaculate than in the second were probably due to the aging of spermatozoa in the epididymis. These results show that the second ejaculate collected within a short interval has better sperm morphology and motility than the first and that this should be considered when evaluating semen quality in the domestic cat and when collecting cat semen to be used for artificial insemination or to be frozen for storage.     

Morphology of spermatozoa in the cauda epididymidis before and after electroejaculation and a comparison with ejaculated spermatozoa in the domestic cat

When 2 ejaculates are collected by electroejaculation from the domestic cat within a period of 10 min the first ejaculate has a higher proportion of abnormal spermatozoa than the second. The reason for this difference is not known for the domestic cat, but in other species long-term epididymal storage results in a higher proportion of abnormal spermatozoa. The aims of this study were to determine the proportions of abnormal spermatozoa in the cauda epididymidis and to ascertain if electroejaculation affects this proportion. Therefore the proportions of spermatozoa in the cauda epididymidis with different morphological abnormalities were compared before and after ejaculation. In addition, the proportion of morphologically abnormal spermatozoa in the epididymis was compared with that in the ejaculate. Nine privately-owned domestic cats were anesthetized, and one testicle was surgically removed. An ejaculate was collected by electroejaculation, after which the remaining testicle was ectomized. There were no significant differences in the proportions of different sperm abnormalities between the cauda epididymidis removed before ejaculation and the one removed after ejaculation. A significantly (P = 0.009) higher proportion of spermatozoa with tail abnormalities was found in the ejaculates compared with the cauda epididymides (11.1 and 1.6%, respectively), while, as expected, there was a lower proportion of spermatozoa with distal droplets in the ejaculates than in the cauda epididymides (35.1 and 75.9%, respectively). This new information contributes to the understanding of the etiology of sperm defects in the domestic cat, and is of importance when evaluating a semen sample in this species.     

Motility characteristics and membrane integrity of cryopreserved human spermatozoa.

The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.     

Isolation by using albumin columns of a cohort of fast-swimming human spermatozoa

Unwashed human spermatozoa enter columns of 20% albumin in cohorts and not in a continuous stream. Using normospermic ejaculates, a single cohort, representing approximately 10% of the total number of spermatozoa added, appeared within 1 h of addition. In contrast, when ejaculates from asthenozoospermic men were tested using the same incubation period (1 h), the cohorts contained less than 5% of the total. Exposure of ejaculates to 2-deoxyadenosine, a potent stimulator of sperm motility, doubled the number of spermatozoa present in the cohort, but not the average velocity of the group. We suggest that spermatozoa also enter the cervical mucus and pass through the female reproductive tract in cohesive groups.     

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.     

Functional activity of adenylyl and guanylyl cyclases in human spermatozoa with different motility

The most important functional characteristic of ejaculated spermatozoa is their ability to engage in directed sustained movement, which to a large extent determines their fertility. It is assumed that enzymes with cyclase activity—adenylyl cyclase (AC) and guanylyl cyclase (GC)—soluble and membrane-bound forms of which are found in human and mammalian sperm, play the key role in regulation of motility. However, the functional activity of the cyclases in ejaculated spermatozoa with different motilities and their contribution to the regulation of this process are virtually unexplored. The goal of this work was to determine the functional characteristics of AC and GC in ejaculates of human spermatozoa with different contents of motile forms and the study of regulation of these enzymes by hormones and nonhormonal agents. We found differences in the activity and regulatory properties of AC and GC in ejaculates differing in motile forms of spermatozoa. The basal AC activity and its sensitivity to bicarbonate anions and manganese cations, activators of cytosolic AC (cAC), were increased in ejaculates with a high proportion of motile spermatozoa. At the same time, the AC effects of forskolin, GppNHp, and adrenergic receptor agonists acting via membrane-bound AC (mAC) in this case were significantly reduced. Cytosolic GC in the ejaculates with a high proportion of motile spermatozoa was more sensitive to manganese cations, but the basal activity of GC was altered slightly. An increase in the content of motile spermatozoa in ejaculate led to a decrease in the sensitivity of CNP to receptor GC, while the sensitivity to ANP was maintained, which indicates a change in the pattern of enzyme regulation with natriuretic peptides in favor of ANP, an important regulator of sperm chemotaxis. Thus, we have concluded that the change in proportion of motile spermatozoa in ejaculate induces changes of functional activity and regulatory properties of soluble and membrane-bound forms of AC and GC, which can be used to control the motility, chemotaxis, acrosomal reaction, and other processes determining fertility of male germ cells.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Liquid storage of turkey semen: changes in quality parameters, lipid composition and susceptibility to induced in vitro peroxidation in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa

The study considered two major aims: (a) to measure the changes in quality parameters, lipid composition and antioxidant activity occurring in turkey spermatozoa during liquid storage; (b) to determine if the enrichment of sperm in n-3 fatty acids and alpha-tocopherol affect sperm survival during storage. Turkey breeders were fed a control diet or an Omega3 diet enriched with fish oil and alpha-tocopheryl-acetate. Ejaculates were pooled (5ejaculates/pool; 4pools/treatment) and stored in vitro for 48h at 4 degrees C. Viability, motility, susceptibility to induced peroxidation and alpha-tocopherol content were measured in spermatozoa; lipid and phospholipid fatty acid composition were measured in spermatozoa and seminal plasma. The proportion of motile and viable spermatozoa significantly decreased, and the proportion of dead spermatozoa significantly increased. The susceptibility of turkey spermatozoa to induced peroxidation also significantly increased during storage. The enrichment of turkey spermatozoa with n-3 long chain PUFA and vitamin E by dietary treatment did not prevent the negative effect of storage on sperm quality and sensitivity to induced in vitro peroxidation; however, it was efficient in partially prevent the increase of sperm death, therefore the proportion of dead spermatozoa was higher in control (37.4%) compared to treated spermatozoa (31.7%) after 48h liquid storage. Major changes were recorded in the lipid composition of turkey spermatozoa during liquid storage in both experimental dietary groups, whereas no significant changes were measured in seminal plasma. In spermatozoa, a great loss in the phospholipid and free cholesterol content was measured. Moreover, the loss in total sperm phospholipid was associated to a peculiar and selective decrease in the bounded fatty acids: saturates and monounsaturates were greatly reduced and polyunsaturates did not change. As a consequence, the polyunsaturated to saturated fatty acid ratio increased during 48h liquid storage. The observed changes in the lipid and phospholipid-bound fatty acid composition of turkey spermatozoa occurring during liquid storage might be related to different events and have been discussed.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.