Effect of controlled exercise on semen characteristics in two-year-old stallions

A two-phase study was conducted to evaluate the effect of controlled daily exercise on semen characteristics of 2-yr-old stallions. Eight nonexercised, sexually inexperienced 2-year-old Morgan stallions were randomly assigned to one of two groups. In Phase 1, four stallions were randomly assigned to receive daily controlled exercise for 16 wk. In Phase 2, these stallions were confined to box stalls for 16 wk. The remaining four stallions were confined to box stalls for 16 wk during Phase 1 and received daily controlled exercise for 16 wk during Phase 2. Individual semen collections were obtained from all stallions every 14 days. Daily sperm output (DSO) was determined by daily semen collections for seven consecutive days on Wk 0, 8, 16, 24, and 32. Mean DSO (10(9)) in Phase 1 was 3.52+/-.62 and 2.79+/-.75 for Wk O and 3.12+/-.39 and 2.61+/-.68 for Wk 16 for exercised and nonexercised stallions, respectively. Mean DSO (10(9)) in Phase 2 was 2.61+/-.68 and 3.12+/-.39 for Wk 16 and 2.48+/-.69 and 3.95+/-.51 for Wk 32 for exercised and nonexercised stallions, respectively. No effect (P>.05) of treatment was observed in either phase when parameters of spermatozoa concentration, total spermatozoa per ejaculate, DSO, total volume, gel-free volume, gel volume, percentage motility, percentage live, pH, and percentage normal were measured and analyzed.     

Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception

The present study was based on data obtained from artificial inseminations (AIs) performed with cryopreserved semen from elite bulls used in the Norwegian breeding program. Semen was diluted to standardize the number of spermatozoa to 18 million per AI dose. The aim of the study was to investigate whether the net sperm concentration at semen collection and the storage period in liquid nitrogen have any effect on probability of conception in dairy cattle. We demonstrated that the natural range of sperm concentration at semen collection within some of the bulls was associated with the probability of conception. However, no primary trend among bulls was found on the effect of sperm concentration at semen collection. This appears to be due to differences among bulls in their response to the dilution ratio of seminal plasma to extender. The effect of storage time was investigated in semen that had been stored between 1000 days and 2400 days in AI straws in liquid nitrogen at the AI center. Our findings showed that use of semen with the longest storage period, i.e. 1951-2400 days, resulted in a more than one percentage point lower probability of conception than semen with a shorter storage period. In conclusion, the net sperm concentration at semen collection, which affects the dilution ratio of seminal plasma to extender, should be considered individually among bulls to achieve optimal reproductive performance. Furthermore, this study gives support to the idea that a measurable degree of damage to the spermatozoa could occur during the preservation time in liquid nitrogen.     

Morphology of spermatozoa in semen from stallions of normal fertility.

Semen samples were collected from 3 fertile stallions by means of an 'open' artificial vagina and examined under scanning and transmission electron microscopy. The stallion spermatozoon has many features in common with that of other mammals but differs specifically in that it has an asymmetric head, an abaxial position of the tail and an acrosome of small volume. The presence of microtubules in the neck is also a characteristic of stallion spermatozoa.     

Relationship between sperm motility, morphology and the fertility of stallions

Sperm quality has an important role in determining fertility. Although there have been numerous studies to document the relationship between sperm quality and fertility, the methods of determining this association and conclusions vary. In the present study, computer-assisted sperm analysis (CASA) was used for evaluation of sperm motility, and differential interference contrast (DIC) microscopy was used for evaluating sperm morphologic features of breeding stallions. Fertility was measured using three endpoints: seasonal pregnancy rate (PR), percent pregnant/cycle (PC), and percent pregnant/first cycle (FCP). Increased total sperm motility (P = 0.08) and progressive path velocity (P = 0.06) tended to be associated with higher PR, whereas percent coiled tails (P = 0.02) was associated with a lower PR. Sperm motility variables associated with an increase in PC and FCP included total, progressive, and rapid sperm motility, and increased path and progressive velocity. Percent pregnant/first cycle was the only fertility measure able to discriminate among high, average, and low fertility groups, based on total and progressive sperm motility. Percent normal sperm was the only morphology variable associated with an increased PC and FCP, whereas increased levels of most sperm morphologic abnormalities (including abnormal and detached heads, proximal and distal droplets, general midpiece abnormality, and coiled tails) were associated with a decline in PC and FCP. Sperm quality variables most highly correlated with fertility included percent total sperm motility (PR, r = 0.37, P < 0.05; PC, r = 0.59, P < 0.05; and FCP, r = 0.64, P < 0.05), and percent morphologically normal sperm (PC, r = 0.42, P < 0.05; and FCP, r = 0.39, P < 0.05).     

Effect of storage time and temperature on stallion sperm DNA and fertility

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P < 0.05) rise in the SCSA measures (Mean(alpha1), S.D.(alpha(t)), and percent cells outside the main population-COMP(alpha(t))) overtime, with the degree of rise being more dramatic at 37 degrees C. Over all stallions, samples stored at 5 degrees C showed no significant (P > 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 degrees C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

Correlations between heterospermic fertility and assays of porcine semen quality before and after cryopreservation

The relative fertilizing potential of frozen-thawed semen from four black and four white boars was determined following heterospermic insemination. A heterospermic index (HI) was computed for each of the 16 possible pairs of black and white boars. Correlation coefficients were computed between the HI and several in vitro tests of semen quality before and afttr cryopreservation of the semen. For the in vitro tests before cryopreservation, the HI were negatively correlated (-0.57) with spermatozoal motility before cooling the semen, but they were not correlated with spermatozoal motility after cooling to 5 degrees C. After freezing and thawing, the HI were correlated with the following in vitro tests: spermatozoal motility (0.50), spermatozoa with either normal or damaged apical ridges (0.31), spermatozoa with missing apical ridges (-0.51), spermatozoa filtered through sephadex columns (0.32), spermatozoa with acrosin-activity (0.38), percentage of maximal releasable glutamic oxalacetic transaminase (GOT) present extracellularly (0.54), spermatozoal intracellular GOT (-0.57), spermatozoa bound per zona-free hamster oocyte (0.64), and percentage of zona-free hamster oocytes penetrated (0.75). The HI were not correlated with the following in vitro tests after freezing and thawing: spermatozoa with normal apical ridges, damaged apical ridges and loose acrosomal caps, extracellular and maximal releasable GOT, and the number of penetrations per zona-free hamster oocyte. The multiple regression correlation coefficient between the HI and four selected variables from three in vitro tests was 0.94. This high correlation indicated that the fertilizing potential of the semen could be accurately predicted with four variables that appeared to measure different properties of the spermatozoa.     

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5 degrees C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5 degrees C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5 degrees C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa.

Approximately 1 week was required to stabilize the extragonadal sperm reserves in stallions ejaculated daily for 10 weeks. The true daily sperm output of a stallion was equal to the mean daily sperm output of seven ejaculates +/- 1-35 X 10(9) spermatozoa. Mean concentrations of spermatozoa/ml and number of spermatozoa/ejaculate were higher (P less than 0-01) for X1 and X3/week ejaculation frequencies than for a X6/week frequency. Sperm output/week was nearly identical for a X6/week frequency. Sperm output/week was nearly identical for the X3 and X6 frequencies and higher (P less than 0-01) than the X1 frequency. Increase of ejaculation frequency from one to two ejaculates/day twice weekly significantly (P less than 0-01) raised the output of spermatozoa/week. Gel-free semen volume, spermatozoa/ml, and number of spermatozoa/ejaculate were higher (P less than 0-01) in the first, than in the second, ejaculate. Collection of semen on alternate days would be a practical ejaculation frequency for inseminating mares. Two ejaculates collected twice a week would be a practical ejaculation frequency for long-term storage of stallion semen.     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

Effect of dialysis on quality characteristics of turkey semen during liquid storage

Low molecular weight substances such as zinc and peroxides are present in seminal plasma and are responsible for deleterious effects in stored semen. On the contrary, molecules larger than 50 kDa are beneficial to in-vitro storage of spermatozoa. Since the effects of different seminal plasma fractions in turkey semen are not completely known, the purpose of the study was to determine the effects of turkey semen dialysis with a 12-14 kDa cut-off on viability, hypo-osmotic membrane integrity, or sperm motility of turkey spermatozoa stored up to 48 h at 5 degrees C. Twelve pools of semen, each pool originating from four toms, were used. Each pool was divided into two aliquots, one of which was dialyzed while the other represented the control. Each semen aliquot was evaluated for sperm viability, membrane integrity and motility after 6, 24 and 48 h of in-vitro storage. Cold storage of turkey semen for 48 h significantly worsened (P<0.01) sperm viability, hypo-osmotic membrane integrity, and sperm motility index of both control and dialyzed samples. After 24 and 48 h sperm viability, membrane integrity and sperm motility index were better (P<0.01) in dialyzed semen compared to the control.