Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Aversive conditioning of periodic spontaneous erection adversely affects sexual behavior and semen in stallions

Periodic spontaneous erection and penile movements known as masturbation (SEAM) occur normally at approximately 90 min intervals in awake equids. SEAM in horses has traditionally been misunderstood by many horsemen as aberrant behavior that should be eliminated. Accordingly, it is not uncommon for trainers of performance stallions or managers of breeding stallions to punish SEAM in an attempt to eliminate the behavior. Previous clinical observations and preliminary unsystematic trials had suggested that attempts to stop stallion SEAM may lead to an increase rather than a decrease in SEAM, and at the same time may suppress sexual behavior (SB) in a breeding stallion. The present work evaluated the effects of aversive conditioning of SEAM on SEAM, SB, and semen. In Experiment 1, four mature pony stallions were subjected to aversive conditioning of SEAM in a within- and between-subjects half cross-over design. The SEAM erection interval tended to be less after aversive conditioning, suggesting an increase in SEAM frequency. Eleven other SEAM measures were each similar before and after aversive conditioning of SEAM. In standard sexual behavior trials with a stimulus mare and dummy mount, erection latency, ejaculation latency, mount readiness latency, and number of mounts to ejaculation increased after aversive conditioning of SEAM; erection rigidity score, number of ejaculatory pulses, and vocalization rate decreased. Number of thrusts to ejaculation was similar before and after aversive conditioning of SEAM. All affected SB measures indicated suppressed sexual arousal and breeding efficiency after SEAM. In Experiment 1, ejaculated semen was not evaluated. Because in Experiment 1, the number of ejaculatory urethral pulses was less after aversive conditioning, Experiment 2 was similarly designed, but included evaluation of semen, both immediately and again 1 week after aversive conditioning was completed. Experiment 2 included 12 aversively conditioned stallions, and 4 yoked controls. In Experiment 2, masturbation episode duration tended to be less after aversive conditioning, while the remaining 11 SEAM measures were unaffected by aversive conditioning of SEAM. Of SB measures, erection latency, mount readiness latency, thrusts to ejaculation, and ejaculation latency were significantly greater after aversive conditioning. Erection rigidity score and number of ejaculatory pulses were less after aversive conditioning. These differences are consistent with suppressed sexual arousal and reduced breeding efficiency. Semen volume and total number of sperm per ejaculate were significantly less after aversive conditioning. These findings are consistent with clinical anecdotes and preliminary trials indicating that aversive conditioning of SEAM in stallions suppresses sexual arousal and breeding behavior. Of considerable interest both clinically and theoretically, is the finding that aversive conditioning target behavior of SEAM was not suppressed by aversive conditioning, while SB and semen during semen collection trials were both adversely affected.     

Comparison of ticarcillin and piperacillin in Kenney's semen extender

Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial concentration. All three solutions were stored at 4 degrees C and aliquots were obtained at 24 and 48 h to determine sperm motility and microbial concentration. Mean percentages of motile and progressively motile sperm did not differ significantly among control and antibiotic-containing solutions after storage. Control-extended semen samples from ejaculates of stallions (n=11) were contaminated with aerobic gram-positive and gram-negative bacteria. In solutions that contained either antibiotic, growth of these microbes was inhibited after 1, 24, and 48 h at 4 degrees C. Semen samples from stallions (n=5) were extended with Kenney's glucose skim milk extender containing no antibiotic, ticarcillin or piperacillin and then inoculated with approximately 5 x 10(2)CFU/mL Klebsiella pneumoniae or Pseudomonas aeruginosa; there was no significant difference between antibiotics in the inhibition of microbial growth. In conclusion, piperacillin was an appropriate alternative to ticarcillin in extenders for equine semen.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Prefreezing and post-thaw semen characteristics of five ram breeds collected by electroejaculation

Rams representing five breeds were electroejaculated twice weekly, during a three-week collection period. Ejaculates were evaluated for volume and concentration before freezing and for rate of motility and percentages of motile and abnormal cells both before and after freezing. Interactions between breed and collection period were evident (P<0.05) for semen volume and post-thaw values for rate of motility and percentage motile cells. Breeds differed (P<0.05) in these traits during some periods. In contrast, pre-freezing observations of rate of motility, percentage motile and abnormal cells and post-thaw percentage abnormal cells did not differ (P>0.15) among breeds. Sperm concentration per ejaculate tended to vary (P=0.11) among breeds. Semen characteristics frequently varied across collection periods. Rams within a breed differed (P<0.01) in all semen traits except post-thaw rate of motility and percentage motile cells. Semen was negatively affected by the freezing and thawing procedure. Ram within a breed and ejaculate within ram should be considered when selecting electroejaculated semen for freezing and subsequent use in artificial insemination.     

Prostaglandins in stallion semen

The purpose of the experiment was to obtain preparatory information about the presence of prostaglandins in semen collected from various types of horses after different periods of sexual rest. Semen was collected with an artificial vagina. Prostaglandin-like activity was estimated by the bioassay procedure described by Vane (1). Results are expressed in ng/ml PGE(2) of seminal plasma. The total concentration of prostaglandins in the full ejaculate averaged 43.73 +/- 4.93 ng/ml of plasma while the total amount of prostaglandins in the ejaculate was 1076 ng. Taking into consideration the period of sexual rest in the stallion, statistically significant differences were found in the prostaglandin level in the semen of all the stallions.     

Semen parameters and level of microsatellite heterozygosity in Noriker draught horse stallions

It was the aim of the present study to determine physiological values for different semen parameters in an endangered draught horse breed, the Austrian Noriker. Because small population size is often believed to cause a decrease in fertility and/or semen quality through inbreeding and a reduction in genetic variation, the general genomic heterogeneity of the breed was estimated on the basis of microsatellite variation and correlated to semen parameters. Semen could be collected from 104 of 139 stallions with semen collection being more often successful in younger stallions. Mean volume of ejaculates was 90.8+/-55.1 ml, density 243+/-114 x 10(6)ml(-1), total sperm count 21.0+/-23.7 x 10(9), percentage of morphologically normal spermatozoa 38+/-18% and total motility 50+/-23%. Total sperm count and semen motility were significantly affected by age. Blood samples of 134 stallions were analysed for 12 microsatellite DNA markers. Genotypes of 110 stallions with at least 11 successfully typed markers were used for calculation of heterozygosity. A total of 82 alleles was identified with a mean of 6.8 alleles per marker. Heterozygosity varied between 35 and 76% for the different markers, mean heterozygosity was calculated to 63%. No correlation between heterozygosity and semen parameters was found.     

Stallion ejaculation induced by manual stimulation of the penis

This paper reports the use of a procedure for collection of semen from stallions by manual stimulation of the penis while the stallion is standing. Our use of this method with 18 stallions of various ages and types of semen collection experience indicates that this method may be an efficient alternative to traditional semen collection techniques using an artificial vagina and stimulus mare or dummy mount mare. Our observations, together with those of others who have tried the manual technique, suggest that both animals and handlers can be readily trained to use this method. Limited data suggests that semen samples obtained by manual stimulation are similar to those obtained using an artificial vagina.     

Reproductive parameters of miniature stallions

Breeding soundness evaluation (BSE) of stallions is a routine component of stud farm practice. Guidelines for assessing satisfactory breeding potential have been developed using data derived from stallions of full-size breeds. In view of the increasing popularity of miniature stallions, knowledge of normal semen parameters of these stallions is important. Therefore, testicular measurements and semen parameters from 216 sexually rested miniature stallions were obtained. Semen was collected twice, 1.5 to 3 h apart, using an artificial vagina. Values were averaged over the 2 collections because of the sexual inexperience of the stallions. The smaller stallions (Group A, 72 to 86 cm; Group B, 87 to 96 cm) had smaller testicles (P<0.05), and Group A stallions had the lowest ejaculate volume (P<0.05) compared with Group C (97 to 104 cm) stallions. Thus, although there was no difference in the concentration of spermatozoa per milliliter between groups of stallions, Group A stallions had fewer total spermatozoa in their ejaculate than Group C stallions (4.31+/-0.47x10(9) vs. 5.41+/-0.30x10(9), P<0.05). Moreover, miniature stallions had smaller testicles and fewer total spermatozoa in their ejaculate than is commonly accepted as normal in full-size stallions. Average total scrotal width of miniature stallions was found to be 7.13, 7.38 and 7.95 cm for Groups A, B and C, respectively. The average total number of spermatozoa in the ejaculates of miniature stallions in this study was 4.94+/-0.22x10(9) cells, with 1.75+/-0.09x10(9) total normal, motile spermatozoa. When only stallions <96.5 cm in height were considered (conforming to requirements of the American Miniature Horse Association Registry), the average total number of spermatozoa in the ejaculates was 4.59+/-0.30x10(9) cells, with 1.70+/-0.11x 10(9) total normal, motile spermatozoa. Based on these findings, different criteria should be used to evaluate the potential breeding soundness of miniature stallions than are commonly applied to full-size stallions.     

Recent advances in cooled-semen technology

The majority of horse registries approve the use of artificial insemination, and horse breeding has widely taken benefit from the use of cooled-stored semen. New insights into cooled-semen technology open possibilities to reduce problems such as impaired semen quality after cooled-storage in individual stallions. The stallion itself has major impacts on quality and fertility of cooled-stored semen. Dietary supplementation of antioxidants and polyunsaturated fatty acids improves semen quality in a variety of species, but only few studies on this topic exist in the horse. Proper semen collection and handling is the main key to the maintenance of semen quality during cooled-storage. Semen collection should be achieved by minimal sexual stimulation with a single mount; this results in high sperm concentration, low content of seminal plasma and minimal contamination with bacteria. Milk-based semen extenders are most popular for semen processing and storage. The development of more defined extenders containing only the beneficial milk ingredients has made extender quality more constant and reliable. Semen is often centrifuged to decrease the seminal plasma content. Centrifugation results in a recovery rate of only 75% of spermatozoa in the semen pellet. Recovery rates after centrifugation may be improved with use of a "cushion technique" allowing higher centrifugation force and duration. However, this is not routinely used in cooled-semen technology. After slow-cooling, semen-storage and shipping is best performed at 5 degrees C, maintaining semen motility, membrane integrity and DNA integrity for up to 40 h after collection. Shipping containers created from Styrofoam boxes provide maintenance of semen quality at low cost.     

Evaluation of fresh and frozen-thawed semen of individual ganders by assessment of spermatozoa motility and morphology

Individual differences in gander Anser anser L. reaction to semen collection procedure, quality and quantity of fresh semen and its susceptibility to the freezing process are discussed. Semen was collected individually by dorso-abdominal massage, from 1-year old White Koluda ganders (n = 12) every 2-3 days. Ganders' reactions to massage were observed during the entire reproductive cycle (from 11 February to 13 June, from every male 40 semen collections were performed). For individual evaluation and freezing purpose semen was collected 13 times from every male. In the fresh semen, the following parameters were evaluated: ejaculate volume, color, density, blood or fecal contamination, motility, concentration and morphology of spermatozoa. Motility and spermatozoa morphology were evaluated in the frozen-thawed semen. Semen diluted in 2:1 ratio with EK diluent was frozen with 6% of dimethyl-formamide (DMF) to -140 degrees C at a rate 60 degrees C/min. Semen was thawed by placing the straws in a 60 degrees C water-bath for 4-5 s. Ten out of 12 ganders had from 67.5 to 100.0% positive reactions resulting in semen ejaculation. Significant (P < or = 0.01) differences in fresh semen quality of particular ganders were observed for all evaluated traits. In 1-year-old gander semen morphologically intact spermatozoa constitute only 27.8-45.2% of all cells. Therefore, the sperm quality factor (SQF), proposed by the authors, which includes ejaculate volume, sperm concentration and the percentage of live normal spermatozoa, seems to be a good predictor of gander semen fertilizing ability. The SQF of individual ganders varied from 7.7 to 11.5. The percentage of live normal spermatozoa in the frozen-thawed semen depended mainly on fresh semen quality. In relation to the fresh semen average from 57.2 to 63.2% of spermatozoa survived freezing process and from 23.9 to 38.5% remained morphologically intact.     

Freezability and Fertility Results with Uncentrifuged Stallion Semen

The first (1 to 3) sperm-rich fractions of the ejaculate were collected from 4 stallions using an open-ended vagina. The volume of the collected fractions was 12 ± 8 ml with a density of 475 ± 200 million spermatozoa/ml. Before freezing, the semen was diluted with a skim-milk based extender 1:1 to 1: 8 (volume of semen: volume of extender), depending on the initial sperm concentration to achieve a final concentration of 100 million/ml. The total number of spermatozoa in an insemination dose ranged from 0.7 to 1 billion spermatozoa. Within 12 h after ovulation, 48 mares were inseminated in 70 cycles. The total single-cycle pregnancy rate at day 21 was 24%, but varied from 10% to 33% per cycle among the stallions.     

Effect of precocious collection on semen output and quality in young Holstein bulls

Semen production units compete heavily with each other, so they tend to select and collect bulls at the earliest possible age, even before puberty, in order to reduce the interval between generations. This study is a retrospective analysis of the effect of precocious collection on semen quality in Holstein bulls. The semen parameters of early- and late-maturing bulls collected before and after 410 days of age, respectively, were compared over two periods, 1991-1995 and 1997-1999. These periods were defined in relation to the collection rhythms (three collections of two ejaculates at 15 days interval before 1996 and adaptation of the collection rhythms to individual physiological capacity after 1996) and the collection conditions. The effects of age, precocious collection and the interaction between age and precocious collection on mean semen parameters (volume of the ejaculate, sperm motility, percent of motile spermatozoa per ejaculate, total sperm concentration and mobile sperm concentration) measured on collections 1-6 (n = 358 for 1991-1995 and n = 121 for 1997-1999), 7-12 (n = 255 for 1991-1995 and n = 80 for 1997-1999) and 13-18 (n = 92 for 1991-1995 and n = 36 for 1997-1999) were studied by covariance analysis. The semen quality of bulls collected at the early age differed from that of bulls collected after 410 days of age for the first period when the collection rhythm was intense. No effect of precocious collection was evidenced for the second period, suggesting the importance of individual adaptation of the collection rhythm to sexual maturation in young bulls. Early collections at a semen production unit reduced the time needed to obtain the number of insemination straws required for the progeny-testing program by 40 days. Early sperm collection is, thus, of economic and technical interest in well managed semen production units.     

Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina

A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri artificial vagina than by the Equidame phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame phantom had lower mean volumes (22.1 +/- 2.3 mL [+/- SEM] versus 101.6 +/- 9.3 mL) and significantly higher mean sperm concentrations (218.0 +/- 25.8 x 10(6) vs 86.2 +/- 8.1 x 10(6) cells/mL; P<0.05) than the total ejaculates collected by the Missouri device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri artificial vagina.     

Sexual behavior and serum concentrations of reproductive hormones in impotent stallions

In an attempt to ascertain the cause of abnormal sexual behavior, serum concentrations of hormones were examined in normal (n=7) and impotent (n=7) stallions. Normal stallions achieved an erection (63 ± 19 sec) when exposed to a teaser mare, and mounted (17 ± 6 sec) and ejaculated (38 ± 5 sec) upon subsequent exposure to an estrous mare. In general, impotent stallions did not achieve an erection, mount or ejaculate. Serum concentrations of testosterone in normal and impotent stallions were similar; however, concentrations of luteinizing hormone (LH) were lower (P < 0.05) in impotent than in normal stallions. Further, the serum concentrations of estradiol-17β in impotent stallions were also lower (P < 0.05) than those in normal stallions. Apparently, there is a physiological basis for impotence in stallions involving decreased serum concentrations of LH and estradiol, but not testosterone; however, whether the reduced concentrations of LH and estradiol occur prior to, or result from, behaviorial impotence is not clear.     

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.     

Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 degrees C

Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the concentration of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation in native semen and after addition of extender, cooling and storage. Semen was collected from fertile Shetland stallions. In experiment 1, activity of antioxidative enzymes was determined immediately after semen collection and after 24 h storage at 5 degrees C. Enzyme activities were measured in native semen, semen diluted with semen extender, spermatozoa resuspended after centrifugation in extender and 0.9% NaCl as well as in undiluted and extender-diluted seminal plasma. In experiment 2, TBARS concentrations were analysed during storage of semen at 5 degrees C for 24 h. Semen storage for 24 h at 5 degrees C did not change activity of the examined enzymes. Antioxidative activity was significantly higher in extended than in native semen as well as in extended plasma than in undiluted plasma. In conclusion, the addition of semen extender increases the antioxidative activity in seminal plasma of stallions. Basal antioxidative activity in native semen as well as increased activity in extended semen are maintained over 24 h storage at 5 degrees C. TBARS content did not increase during semen storage. In conclusion, lipid peroxidation does not increase substantially during semen storage. The enzymatic antioxidative activity in semen apparently prevents ROS formation and is further increased by addition of semen extender.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

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.